Information

How is the side-polarity of the myosin filament in myofibril maintained?

How is the side-polarity of the myosin filament in myofibril maintained?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

If myosin molecules are the properly oriented relative to their position in the the myosin filaments, the sarcomere is not functional. But how is the orientation of the myosin molecules determined? Why is there a symmetry in the arrangement of myosin molecules in the myosin filament? And are there any diseases associated with the improper arrangement of myosin molecules?


Locomotion and Movement Important Extra Questions Very Short Answer Type

Question 1.
What is a tendon?
Answer:
The dense connective tissue joins bone and skeletal muscle.

Question 2.
What are antagonistic muscles?
Answer:
The. pair of muscles which at a joint produce opposite movements.

Question 3.
What is tetanus?
Answer:
The continued state of muscular contraction is called tetanus.

Question 4.
What is threshold stimulus?
Answer:
The stimulus of minimum strength which is required to bring about muscular contraction is called the threshold stimulus.

Question 5.
What is a muscle twitch?
Answer:
The single contraction of muscle upon receiving the stimulus is called muscle twitch. (Contraction is followed by relaxation).

Question 6.
What is sarcomere?
Answer:
The functional unit of myofibril contracts and causes the shortening of muscle fibre.

Question 7.
How many bones are present in the human skeleton?
Answer:
The human skeleton contains 206 bones.

Question 8.
What are synovial joints?
Answer:
These are freely movable joints due to the presence of synovial fluid in the synovial cavity.

Question 9.
What is locomotion?
Answer:
The bodily movement in animals from one place to the other is called locomotion.

Question 10.
What is rigour mortis?
Answer:
Stiffening of muscle after death.

Question 11.
Name the proteins which help in muscle contraction.
Answer:
Myosin and actin.

Question 12.
What is the function of synovial fluid?
Answer:
Synovial fluid acts as a lubricant.

Question 13.
What is a pivot joint?
Answer:
The joint allows the turning or rotational movements, e.g., between atlas and axis vertebra.

Question 14.
Which of the movable joint makes the hip joint?
Answer:
Ball and socket joint.

Question 15.
Which muscle contracts to make your palm face upwards?
Answer:
Supinator.

Question 16.
How many bones are there in the human skull?
Answer:
29

Question 17.
Which type of movable joint is the knee joint?
Answer:
Hinge joint.

Question 18.
Name the band of the skeletal joint which permits movements in a single plane only.
Answer:
Hinge joint.

Question 19.
Differentiate between A-band and I-band.
Answer:

  • A-band is a dark band having myosin filaments.
  • I-band is a light band having thin filaments.

Question 20.
What is the total number of bones in our body?
Answer:
206.

Question 21.
Name the five different categories of vertebrae in your backbone.
Answer:

Question 20.
Where inside the bones are blood cells produced?
Answer:
The bone marrow of long bones.

Question 23.
Give one example of a ball and socket joint.
Answer:
Shoulder joint.

Locomotion and Movement Important Extra Questions Short Answer Type

Question 1.
List the mechanical function of the skeleton.
Answer:

  1. It provides a rigid framework of the body and definite shape to organs.
  2. It supports the weight of the body.
  3. It protects the internal organs.
  4. Its long bones function as a lever.
  5. Skeletal muscles with flexible connective tissue bands called tendons in association with endoskeleton and joints give locomotion and movements to different body parts.

Question 2.
List some biological function of the skeleton.
Answer:

  1. Provides attachment surface to muscles.
  2. Serves as storage depot of calcium and phosphate minerals.
  3. Act in erythropoiesis.
  4. Ear ossicles help in sound wave propagation.
  5. Redbone marrow present inside the marrow cavity of long bones such as femur, humerus and in interstices of spongy bones of vertebrae, sternum, scapula etc. help in the formation of RBCs, WBCs and platelets of the blood. This process is known as Haemopoiesis.

Question 3.
List different modes of locomotion and movement in hydra.
Answer:

  1. Contraction and expansion
  2. Bending and swaying
  3. Looping
  4. Somersaulting.
  5. Floating
  6. Gliding
  7. Swimming
  8. Walking.

Question 4.
What are the different molecules present in muscles?
Answer:

  1. Contractile proteins viz. actin, myosin and tropomyosin.
  2. Enzymes and other proteins like troponin.
  3. Carbohydrate as a substrate for energy.
  4. Energy carries viz. ATP, ADP, AMP and CP.
  5. Ions viz. Na + , K + , Mg ++ , Ca + , CI + .

Question 5.
Differentiate between isotonic and isometric contraction
Answer:

Isotonic contraction Isometric contraction
1. There is a change in the shape of muscles. 1. There is no change in the shape of muscles
2. Muscles maintains tension. 2. Muscles maintains length.
3. Muscles contracts and the load is lifted 3. Muscles contracts against a load that can’t be lifted.

Question 6.
A red muscles fibre works for a prolonged period, whereas a white muscle fibre gets fatigued, why?
Answer:
Red muscle fibres contain oxygen storing pigment myoglobin and a large number of mitochondria, so they can have O2 supply for aerobic respiration and release of energy for a longer period.

White muscles fibres do not have myoglobin pigment. They face a short supply of O2 and much depends on anaerobic respiration, so they get fatigued soon.

Question 7.
What are the main types of joints present in the human body?
Answer:
The types of joints present in the body of man are:
1. Fixed or fibrous joints: There is no movement at all in articulating joints, because of the presence of tough, inextensible, white fibrous tissue, e.g., skull bones.

2. Slightly movable or cartilaginous joints: A limited movement is possible in articulating bones. A dense disc of white cartilage joins the articulating surfaces, e.g., vertebrae and public symphysis.


Different types of joints

3. Freely movable or synovial joints: Free movement is possible due to the presence of synovial fluid in the synovial cavity, between the articulating bones, e.g., hinge joint, ball and socket joint.

Question 8.
What are the advantages of the movement of body parts?
Answer:
The movement has the following advantages:

  1. With change in body posture and limb movement, equilibrium of the body is maintained.
  2. Limb movement causes locomotion.
  3. Food is captured by movement of tentacles, limbs, jaw, tongue etc. in different animals.
  4. Changes in environment surrounding can be sensed by the movement of the eyeball, pinna etc.
  5. Blood circulation is possible by heart movement.
  6. Movement of the diaphragm causes inhales and exhale (breathing).

Question 9.
What are the advantages of locomotion?
Answer:
The bodily movements or locomotion has the following advantages:

  1. It enables the body to shift it entirely from one place to the other.
  2. It protects the organism from predation.
  3. It helps the animals to make the search for their food and other nutritional requirements.
  4. It helps the animal to seek a mate for reproduction.

Question 10.
Draw a labelled diagram of the joint found between the pelvic girdle and femur. Also, write the type of this joint.
Answer:
Type of the joint: The joint between pelvic and femur bones is a ball and socket synovial joint.

Synovial ball and socket joint between pelvic and femur

Question 11.
Why movement and locomotion are necessary among animals?
Answer:
Movement and locomotion are necessary among animals for the survival of them. It enables, them to procure food, search for shelter, find mates, protect themselves from predatory and perform many other life activities.

Question 12.
Elucidate the types of movements found among the animals.
Answer:
Movements among animals vary greatly. Movement involves three basic mechanisms.
These are:

Amoeboid movement is typical of Amoeba. Amoeba moves with the help of pseudopodia. Amoeboid movement helps in the food capture and change of place as well.

The same method of movement is also employed by the leucocytes, like phagocytes and macrophages of the human lymphatic system for engulfment of antigen and migration of circulatory fluid. In protozoan ciliary movement is seen. Muscular movement is the basic mechanism used in the majority of vertebrates including humans. Most multicellular animals possess muscle fibres for the movement of different organs and at¬taining locomotion.

Question 13.
What is muscle? Write the names of different types of muscles?
Answer:
Locomotion in humans depends on the movements of muscle fibres (muscle cells). Muscles are made up of contractile fibres which in turn formed of myofibrils. In humans, muscles constitute nearly 40 – 50 per cent of the total body weight.

Muscles are broadly classified into three categories.

  1. Skeletal muscles: These are attached to the bones by tendons and help in the movement of the part of the skeleton. These muscles are under the control of the conscious mind and can be moved to the wall.
    Skeletal muscles are termed voluntary muscles.
  2. Cardiac muscles: These are also striated and occur exclusively on the heart.
  3. Smooth muscles: These are involuntary and non-striated muscles and are innervated by the autonomic nervous system.

Question 14.
How the skeletal muscle contracts?
Answer:
During contraction, the actin and myosin filaments slide past each other to reduce the length of the sarcomeres. The actin filaments move inwards towards the centre of the sarcomere. The heads of the myosin filaments operate as ‘hooks’, attaching to the F-actin they form cross-bridges, then change their relative configuration and pull the actin filaments.

As 3 result, the Z-lines limiting the sarcomeres are drawn closer together, but the length of A-bands remain unchanged. The I-bands reduce in length.

However, the net result is the shortening of the sarcomere. The actin filament slides out from the A-band resulting in the lengthening of the sarcomere.

Question 15.
What is arthritis? How is it caused?
Answer:
It is a disorder of bones in which fibrous tissues are attached with bones- and become ossified, making the joints immovable.

It is caused by the inflammation of the joints. It is of several types, e.g., rheumatoid arthritis, osteoarthritis ‘and gouty arthritis.

Question 16.
Write the names of the factors which are responsible for osteoporosis.
Answer:
Imbalances of hormones like thyrocalcitonin, parathyroid and sex hormones, deficiencies of calcium and vitamin D are the major Causativetors.

Question 17.
How do the joints help in the movement? Explain.
Answer:
A synovial or movable joint is a joint which allows the movement of collating bones such that they can move extensively upon each other. In joints, there is a space called a synovial cavity. This cavity remains filled in a fluid called synovial fluid.

The movement of an organ occurs due to the pulling of bones. Movement takes place along the joints which act as the fulcrum of the liver. In fact, the joints function as a lever. Due to the presence of a number of joints movement of the different body parts and the whole body is possible.

Question 18.
How calcium affects the process of muscle contraction?
Answer:
Muscle fibres are excitable. Normally, a nerve impulse arriving at the neuromuscular junction initiates a contractile response. A neurotransmitter released at the neuromuscular junction enters into the sarcomere through its membrane channel. The opening of the channel also results in the inflow of Na + inside the sarcomere and generates an action potential in the muscle fibre.

The sarcoplasmic reticulum releases the stored Ca ++ , which binds with the specific sites present on the troponin component of the thin filament. As a result, the active sites present on the F-actin molecules are exposed. These sites are specific to the myosin head, which exhibits Mg ++ dependent ATP as activity.

During relaxation of the muscle, the Ca ++ is pumped back into the sarcoplasmic reticulum. As a result, the troponin component becomes free. The cross-bridge breaks and the thin filament occupies its normal position. The muscle relaxes.

Question 19.
Write the difference between movable and immovable joints.
Answer:

Movable joints Immovable joints
1. The articulating surfaces are kept in close contact by a fibrous capsule and a slippery synovial fluid occurs in the space between the articulating surfaces of the bone. 1. The articulating bones at this joint are firmly held together by dense bands of tough inextensible white fibrous tissue.
2. It permits considerable movement of the articulating bones. 2. It does not permit any movement of the articulating bones.

Question 20.
Fill in the blanks:
Answer:

  1. Troponin is a part of Myosin filament.
  2. The Head of the myosin has AT passive activity.
  3. Humerus Radius and Ulna bones are found in the forearm.
  4. The acetabulum is present in the Pelvic girdle.
  5. The ball and socket joint is a Movable girdle.

Question 21.
Match column I with column II

Column I Column- II
(a) Smooth muscle (i) Myoglobin
(b) Tropomyosin (ii) Third class lever
(c) Red muscle (iii) Thin filament
(d) Skull (iv) Sutures
(e) Forearm (v) Involuntary

Column I Column- II
(a) Smooth muscle (v) Involuntary
(b) Tropomyosin (iii) Thin filament
(c) Red muscle (i) Myoglobin
(d) Skull (iv) Sutures
(e) Forearm (ii) Third class lever

Question 22.
What is a joint? Write its type with an example.
Answer:
Joints are the place of articulation between two or more bones or between a bone and cartilage. Due to the presence of a number of joints, the movement of the different body parts and the whole body is possible.

There are three types of joints:

  1. Fixed or immovable joints: There is no space between the bones. They are attached very tightly with the help of white fibrous connective tissue.
  2. Slightly movable or cartilaginous: It is an articulation between the bones that allow a very little movement.
  3. Movable joints or synovial: It is a joint which allows the movement of articulating bones such that they can move extensively upon each other. In such joints a synovial vanity is present.

Question 23.
What is the role of the girdle in the skeleton?
Answer:
Girdle bones provide a connection between the axial skeleton and limbs. The two girdles are named pectoral and pelvic girdles. Each girdle is formed of two halves.

Locomotion and Movement Important Extra Questions Long Answer Type

Question 1.
(a) During muscular contraction what are the chemical changes that take place. Describe in a listed form.
Answer:
The main chemical events that happen during muscular contraction described by Albert Szent Gyorgi are
1. Acetylcholine is released from vesicles at the neuromuscular junction. It stimulates the muscle.

2. Hydrolysis of ATP in the presence of Ca ++ and Mg ++ Energy used up in muscle contraction.

3. ADP is charged again by taking phosphate from creatine phosphate (CP).

4. During relaxation, creatine is phosphorylated, energy being provided by anaerobic conversion of muscle glycogen into lactic acid.
Creatine + ATP— Creating-phosphate + ADP

5. Energy released by hydrolysis of ATP causes rotation of myosin heads and bring near the actin filaments, actomyosin complex is formed, eventually, sarcomere shortens.

6. Ca ++ are actively transported to the sarcoplasmic reticulum, no more Ca ++ available for ATP breakdown, no further energy available for further contraction of the sarcomere.

7. Part of the energy is utilized by breaking of cross-bridges and the muscle relaxes.

(b) What are the main groups of vertebrae in the vertebral column of man?
Answer:
There are 5 groups of vertebrae namely cervical, thoracic, lumbar, sacral and coccygeal vertebral.
(The vertebral formula is C7, T12, L5, C3-5 = 32 – 34).

Question 2.
(a) What purposes does movement of external body parts in relation to body axis serve in animals?
Answer:

  1. The movement of limbs, appendages, head and trunk serves to change the body posture to maintain equilibrium against gravity.
  2. Limb movements are prerequisites for carrying out locomotion.
  3. Prehension of food involves movement of tongue, jaws, snout, tentacles, limbs and appendages in different animals.
  4. Movement of eyeballs and pinna of ear help to collect information from the external environment.

(b) What are fibrous joint and cartilaginous joints and their biological function?
Answer:

  1. Fibrous joint: The articulating bones are firmly held together by the dense bands of tough, inextensible white fibrous tissues. They provide strength and support for the body or protection of delicate structures which cannot withstand any kind of deformation.
  2. Cartilaginous joints: In cartilaginous joints, a dense disc of white fibrocartilage joins the opposing surfaces of the articulating bones to each other. This allows a limited movement at the joints.

(c) Explain Antagonistic muscles.
Answer:
Antagonistic muscles: Antagonistic muscles are those which contract to produce opposite movements at the same joint. When a muscle contract to produce a movement, its antagonistic must relax to allow that movement to take place, e.g., the bicep is a FLEXER for the elbow joint and the tricep is it’s antagonistic and an EXTENSOR for that joint.

During flexion at the elbow, the biceps contract and the tricep relax, during extension at the same joint the tricep contracts and the biceps relaxes.

(d) Distinguish between muscles twitch and tetanus or explain muscle twitch and tetanus.
Answer:
A single isolated contraction caused by a single nerve impulse or electric shock is called a muscle twitch. Immediately after the brief twitch, the muscle fibres relax.

Tetanus is a continued state of concentration caused by many repeated stimuli. Much higher tension is developed in tetanus than in an isolated twitch. Almost all our daily activities are carried out by tetanic contractions of muscles.

Question 3.
How thick and thin filaments are arranged in a muscle fibre?

relationship between actin and myosin filaments in stretched and contracted states
Answer:
Each striated muscle contains thin actin and thick myosin filaments. These filaments are longitudinally arranged inside light I bands and dark A bands respectively. The actin and myosin filaments remain cross-linked with each other in the myofibril. Sarcomeres are the rows of functional unit in each myofibril, each extending from the dark Z- line of the next I band. Each sarcomere thus comprises of A band in the middle with 2 half I band on its two sides.

From each Z line, the actin filaments through half of the I band intermingles with the ends of myosin filaments in the A band. The myofibril is surrounded at each I band by the tubules and cisternae of sarcoplasmic reticulum and at each junction of A and I bands by a TI tubule communicating with the cell exterior, which is shown in the figure. The relationship between actin (thin filament) and myosin (thick filament).


What steps are involved in the myosin powerstroke?

Each myosin motor protein possesses ATPase activity and functions in a cyclical manner that couples ATP binding and hydrolysis to a conformational change in the protein. This process is known as the &lsquopowerstroke cycle&rsquo (reviewed in [1] [2] [3] ) and is outlined in the steps below using myosin II as an example. T

The &ldquopower stroke&rdquo mechanism for myosin movement along actin filaments:

The direction in which the actin filament will be moved is dictated by the structural orientation of myosin in relation to the filament. A complete round of ATP hydrolysis produces a single &lsquostep&rsquo or movement of myosin along the actin filament. This process is regulated by changes in the concentration of intracellular free calcium (reviewed in [4] ). The steps involved are detailed below:

Step 1: At the end of the previous round of movement and the start of the next cycle, the myosin head lacks a bound ATP and it is attached to the actin filament in a very short-lived conformation known as the &lsquorigor conformation&rsquo.

Step 2: ATP binding to the myosin head domain induces a small conformational shift in the actin-binding site that reduces its affinity for actin and causes the myosin head to release the actin filament.

Step 3: ATP binding also causes a large conformational shift in the &lsquolever arm&rsquo of myosin that bends the myosin head into a position further along the filament. ATP is then hydrolysed, leaving the inorganic phosphate and ADP bound to myosin.

Step 4: The myosin head makes weak contact with the actin filament and a slight conformational change occurs on myosin that promotes the release of the inorganic phosphate.

Step 5: The release of inorganic phosphate reinforces the binding interaction between myosin and actin and subsequently triggers the &lsquopower stroke&rsquo. The power stroke is the key force-generating step used by myosin motor proteins. Forces are generated on the actin filament as the myosin protein reverts back to its original conformation.

Step 6: As myosin regains its original conformation, the ADP is released, but the myosin head remains tightly bound to the filament at a new position from where it started, thereby bringing the cycle back to the beginning.


Materials and Methods

Construction of a GFP::myosin heavy chain expression vector

The myosin heavy chain was encoded by an embryonic chicken myosin cDNA that had been epitope-tagged as previously described (Kinose et al., 1996 Molina et al., 1987). The GFP::myosin heavy chain fusion was constructed in the GFP expression vector pS65T-C1 (CLONTECH Laboratories, Inc., Palo Alto). The 5′ end of the GFP coding region through a unique PmlI restriction site was derived from the pS65T-C1 vector. The 3′ end of the GFP coding region was derived from a thermally stable, fast folding GFP variant, GFP5 (Siemering et al., 1996). This segment was amplified by PCR from the vector pcGFP5 and modified to include a six base linker sequence with a unique KpnI site for fusion to the myosin coding sequence using the 5′ PCR primer CAA GGA CGA CGG GAA CTA CAA GAC and the 3′ primer CAT GGG TAC CTT GTA TAG TTC ATC CAT GCC. The 436 bp PCR product was cut with PmlI and KpnI and cloned together with a KpnI-BamHI insert containing the full-length myosin heavy chain sequence into PmlI and BamHI cut pS65T-C1. The resulting 10.6 bp vector, pGFP5-MHC (myosin heavy chain), was used as the base for all transfections and mutagenesis. The FHC mutations were introduced in the myosin coding region by PCR mutagenesis. These segments were cloned into unique restriction sites of the base expression vector then confirmed by sequencing.

Preparation and transfection of embryonic cardiomyocytes

Primary cultures of chicken cardiomyocytes were prepared from stage 26-30 white leghorn chicken embryos (Hamburger and Hamilton, 1992). The hearts were excised from 1-2 dozen embryos, the pericardium and atria were removed, and ventricular cardiomyocytes were prepared using brief trypsin treatment followed by collagenase digestion as described previously (Moncman and Wang, 1995). The top layer of the cell pellet was enriched in cardiomyocytes and was re-suspended in 5% FBS/DMEM, counted and plated at 1×10 4 cardiomyocytes/cm 2 on glass coverslips or glass-bottom 35 mm tissue culture dishes that were pretreated with 10 μg/ml of mouse EHS cell laminin (Colognato et al., 1999). Cells are maintained in 5% FBS, DMEM without glutamine at 37°C and 5% CO2 and allowed to recover for 24 hours before transfection. For fluorescent observation, cardiomyocytes were shifted to 5% FBS in Hepes buffered DMEM/F12 medium without phenol red (Life Sciences, Gaitherburg, MD).

Expression vector DNA used in transfection assays was purified on CsCl gradients (Moncman et al., 1993) before transfection with Fugene 6 reagent (Boehringer Mannheim, Germany). The transfection mixture contained 2 μg vector DNA and 4 μl Fugene per 35 mm dish. Cells were incubated for 18 hours with this mixture then transferred to fresh medium.

Immunofluorescence microscopy and antibodies

Cells were processed for immunofluorescence 48-120 hours post-transfection essentially as described previously (Kinose et al., 1996). The cells were incubated with primary antibodies at 2 μg/μl for 1 hour at room temperature or overnight at 4°C. Rhodamine-labeled secondary antibodies were diluted 1:600 in 1% BSA, 0.05% Tween in PBS. Coverslips were washed extensively with PBS before sealing the surface to a glass slide with FITC guard mounting medium (Molecular Probes, Eugene, OR). Monoclonal antibodies (mAb) 12C5.3 and 10F12.3 react specifically with chicken skeletal muscle myosin (Winkelmann et al., 1995 Winkelmann et al., 1993). mAb F18 reacts with striated muscle myosin heavy chains (Miller et al., 1989). mAb RT11 reacts with the PEVK repeat region of titin (Moncman and Wang, 1996) and mAb N114 reacts with the cardiac specific form of nebulin (Moncman and Wang, 1995). The cardiomyocytes were sub-confluent at 72 hours post-transfection when they were fixed and stained for scoring.

Microscopy

Digital images were collected on an Olympus IX70 inverted fluorescence microscope (Olympus America Inc., Melville, NY) with a MicroMax cooled CCD camera (Roper Scientific, Princeton, NJ) using IpLab image analysis software (Scanalytics Inc., Fairfax, VA). Live cell imaging and time-lapse experiments were done with a PDMI-2 micro-incubator and perfusion pump (Harvard Apparatus Inc., Holliston, MA). The cells were plated on laminin-coated 12 mm glass coverslip inserts in 35 mm culture dishes. Differential interference contrast (DIC) images of contracting cardiomyocytes were recorded with a 100× 1.3 NA Plan Apo objective and a Hamamatsu C2400 CCD camera with an Argus 20 image processor (Hamamatsu USA Inc., Bridgewater, NJ). Digitized video sequences were collected at 30 frames/second and analyzed with IpLab image analysis software using a script for detection and tracking of z-lines.

Adenovirus vectors for expression of GFP-myosin

The complete coding regions of the WT and mutant GFP-myosin cDNAs were excised from the pGFP5-MHC vectors at unique flanking NheI and BamHI restriction sites and cloned between an enhanced CMV promoter and a SV40 polyadenylation signal sequence in a modified AdEasy shuttle vector, pCMVShuttle (He et al., 1998). Recombinant adenovirus DNA was prepared by homologous recombination of shuttle vectors with the pAdEasy1 vector in E. coli strain BJ5183 essentially as described previously (Chow et al., 2002 He et al., 1998). Colonies were selected for kanamycin resistance and plasmid DNA was characterized by restriction digestion. The recombinant vector DNA was subcloned into E. coli DH10B cells and the 38 kb pAdGFP-MHC plasmid was purified by CsCl density gradient. Human 293 cells werere transfected with linearlized pAdGFP-MHC DNA as previously described (Chow et al., 2002). The recombinant adenovirus was harvested once virus plaques were evident and about 50% of the cells were rounded and detaching from the surface. Virus stocks were amplified and virus titer was determined by infection of confluent 293. Virus titers of 10 10 -10 11 plaque forming units (PFU)/ml were routinely achieved.

Adenovirus infection of C2C12 myotubes and preparation of GFP-myosin

Maintenance of the mouse myogenic cell line, C2C12 (CRL 1772 American Type Culture Collection, Rockville, MD), has been described in detail elsewhere (Chow et al., 2002 Kinose et al., 1996). For isolation of GFP-myosin, myoblasts were plated on 100 mm tissue culture dishes that had been pretreated with 10 μg/ml of mouse laminin. The cells were seeded at an initial density of 7.5×10 4 cells/cm 2 . Cells at 60-70% confluence were induced to differentiate by switching them to fusion medium. The cells were infected with AdGFP-MHC ∼48 hours later at a multiplicity of infection (MOI) of 1000-3000 in fresh medium, and incubation was continued for an additional 24-36 hours. The medium was changed daily after infection. Myosin was isolated from 10-20 100 mm dishes of infected C2C12 myotubes as previously described (Kinose et al., 1996). Further purification by ion-exchange chromatography on a Mono Q HR5/5 column (Pharmacia, Piscataway, NJ) was done in 40 mM sodium pyrophosphate pH 7.5, 1 mM DTT, and myosin eluted with a linear 0-0.5 M NaCl gradient.

Motility assays and data analysis


BDM (2,3-butanedione monoxime), an inhibitor of myosin-actin interaction, suppresses myofibrillogenesis in skeletal muscle cells in culture

During the initial phase of myofibrillogenesis in developing muscle cells, the majority of thin filaments lie parallel to, and exhibit correct polarity and spatial position with thick filaments, as in mature myofibrils. Since myosin is known to function as an accelerator of actin polymerization in vitro, it has been postulated that myosin-actin interaction is important in the initial phase of myofibrillogenesis. To clarify further the role of actin-myosin interaction in myofibril formation during development, BDM (2, 3-butanedione 2-monoxime), an inhibitor of myosin ATPase, was applied to primary cultures of skeletal muscle to inhibit myosin activity during myofibrillogenesis, and myofibril formation was examined. When 10 mM BDM was added to the myotubes just after fusion and the cultures were maintained for a further 4 days, cross-striated myofibrils were scarcely observed by fluorescence microscopy when examined by staining with antibodies to actin, myosin, troponin and alpha-actinin, whereas in the control myotubes not exposed to BDM, typical sarcomeric structures were detected. Electron microscopy revealed a disorganized arrangement of myofilaments and incomplete sarcomeric structures in the BDM-treated myotubes. Thus, formation of cross-striated myofibrils was remarkably suppressed in the BDM-treated myotubes. When the myotubes cultured in BDM-containing media were transferred to control media, sarcomeric structures were formed in 2-3 days, suggesting that the inhibitory effect of BDM on myotubes is reversible. These results suggest that actin-myosin interaction plays a critical role in the early process of myofibrillogenesis.


Discussion

A growing number of proteins are being identified in the sarcomere, which do not fit to a more traditional view of the sarcomere as a static structure of great regularity (Sanger and Sanger,2001). Some of these new proteins combine a structural role with signalling functions, like the differentially spliced giant G-protein regulator obscurin, which localises to the sarcomeric Z-disk in early developing hearts but is later found at the M-band(Young et al., 2001).

MURF2 belongs to the MURF family of muscle-specific RING/B-box zinc-finger proteins (Centner et al., 2001)first identified by Spencer et al.(Spencer et al., 2000) in a search for ligands of the serum response factor (SRF). A second MURF member,MURF1, was identified as a ligand of titin close to the M-band, and about 12 nm N-terminal to the kinase domain. It was hence speculated that MURFs might be regulators of the titin kinase domain(Centner et al., 2001). In adult cardiac muscle, MURF1 was found both at the Z-disk and the M-band MURF3 was assigned to the Z-disk. MURF1 was recently proposed to play a role in thick filament assembly (McElhinny et al.,2002). However, a MURF1 knockout mouse shows no defects in primary myofibrillogenesis (Bodine et al.,2001), but rather a resistance to atrophy in agreement with the up-regulation of MURF1 under these conditions. These observations suggest that the functions of MURF1 in myofibril assembly are partly redundant or dispensable. To understand MURF functions, a detailed analysis of the role in myofibril assembly is clearly required. The previous studies on MURF1 and MURF3 have focussed on cardiac myocytes, where primary myofibril assembly cannot be studied due to the preformed myofibrils. In this study, we therefore present the first detailed temporal analysis of a MURF-protein, MURF2, during myogenic differentiation in skeletal muscle.

MURF2 was isolated on affinity beads of an A-band titin fragment(Iakovenko and Gautel, 2000)containing the binding site for MURF1, possibly due to its ability to form heterodimers with MURF1 and MURF3 (Centner et al., 2001) Spencer et al.,2000). However, we demonstrate here for the first time that MURF2 also displays intrinsic titin-binding properties which may contribute to the cellular localisation near the M-band of mature cardiac sarcomeres. Whereas MURF1 appears to remain expressed in all striated muscles at all stages of differentiation, we find that MURF2 is down-regulated in mature skeletal myotubes and is excluded from mature skeletal sarcomeres. These results are in good agreement with northern blot analysis which suggested that MURF2 is expressed at best weakly in adult cardiac and skeletal tissues(Centner et al., 2001), and that MURF1 is strongly expressed in fetal heart muscle(Dai and Liew, 2001). Our data together with those of Spencer et al.(Spencer et al., 2000) show that both MURF3 and MURF2 proteins are microtubule associated proteins but that expression and localisation remain distinct, indicating that MURF2 and MURF3 could fulfil different tasks during differentiation. Whereas MURF3 was detected in proliferating myoblasts and in adult skeletal and cardiac muscle(Spencer et al., 2000), MURF2 shows temporal dynamics in its expression in skeletal muscle cells with highest levels early after the onset of differentiation(Fig. 2A,B).

During differentiation, MURF2 distribution follows the morphological reorganisation undergone by the microtubule cytoskeleton(Fischman, 1970Gundersen et al., 1989Okazaki and Holtzer, 1965). At early stages of differentiation, the MURF2 antibody stains distinct microtubules all along the length of the nascent myotube, while at later stages, segmented rod-like and dotty structures were observed. These various morphologies reflect the reorganization of the microtubule network(Cartwright and Goldstein,1982 Gundersen et al.,1989 Warren,1974), and/or specific intracellular relocalisation of MURF2. This association is also evident in cells treated with nocodazole where MURF2 adopts a diffuse distribution whereas titin on SFLS is not impaired (data not shown).

The closest association between MURF2 and sarcomeric proteins is observed for sarcomeric myosin, well before the integration of myosin into nascent myofibrils. Although sarcomeric myosin appears as one of the first myofibrillar proteins, the striated A-band arrangement is observed only at a very late stage (Person et al.,2000 Rudy et al.,2001 Van der Ven et al.,1999). MURF2 is the first MURF protein known to show morphological myosin association. In agreement with this, we also detected MURF2 in preparations of sarcomeric myosin (Fig. 2E). According to our immunofluorescence data, MURF2 and myosin association take place at the beginning of differentiation, when the microtubule network is still preserved on large scale. Interestingly, myosin filament assembly is independent of actin filaments(Guo et al., 1986Holtzer et al., 1997LoRusso et al., 1997Rhee et al., 1994Sanger et al., 1986Schultheiss et al., 1990Wang and Wright, 1988), but requires the microtubule cytoskeleton for the formation and the organization of the thick filaments in A-bands (Antin et al., 1981 Toyama et al.,1982).

In contrast, actin and Z-disk titin co-assemble early on and initiate the formation of a Z-disk-titin-M-band scaffold into which myosin is finally integrated. Our observations suggest that the Z-disk/titin/M-band scaffold only transiently co-aligns with MURF2-containing microtubules during sarcomere formation. However, both myosin and A-band titin colocalise with MURF2 in non-striated and nascent striated myofibrils, parallel and closely apposed to striated myofibrils. Many observations showed that the organisation of the N-terminal titin domains precede those of the C-terminal regions, revealing the sequential and structural order of titin molecule unravelling(Ehler et al., 1999Fürst et al., 1989bKomiyama et al., 1993Mayans et al., 1998Schultheiss et al., 1990Soeno et al., 1999Van der Loop et al., 1996Van der Ven et al., 1999). The temporal order of the spatial relationship of MURF2 with respect to Z-disk,I-band and A-band titin are in good agreement with an involvement in the straightening and stretching of the giant titin molecule during sarcomere formation. Co-alignment of MURF2 and Z-disk titin takes place at the beginning of differentiation, as indicated by the integrity of the microtubule network staining revealed with MURF2 antibody (Fig. 5D). Since overlap of MURF2 and I-band titin is only observed on irregular cross-striated sarcomeres (Fig. 5B), MURF2 seems to align with I-band titin only briefly. As expected due to the biochemical association with titin, MURF2 localises with A-band titin. Since definite striations of A-band titin epitopes are formed at a late stage of differentiation, one could hypothesise that MURF2 interacts with the folded-up titin molecule when their A-band portion starts to stretch out from the Z-disk portion.

The early and persistent association of MURF2 with both sarcomeric myosin and microtubules, and the parallel alignment of microtubules and their associated proteins with nascent striated myofibrils suggest that microtubules are indeed involved in translocating myosin filaments to the sites of final sarcomere assembly. MURFs appear to act as a transient adaptor between sarcomeric proteins, especially myosin and titin, and the microtubule network. At the beginning of differentiation, the MURF2/myosin complex interacts with microtubules, allowing the dispersal of myosin throughout the myotube along the microtubule network. During differentiation, MURF2 thus brings myosin filaments at the vicinity of maturing titin filaments. MURF2 homo- or hetero-multimeres could then coordinate binding sites on myosin and titin. MURF1, which interacts most strongly with A-band titin(McElhinny et al., 2002), may provide the link to the titin filament, although we show here that titin binding is not an exclusive property of MURF1. This may explain why a knockout of MURF1 is without consequences for primary sarcomere formation(Bodine et al., 2001). This proposed role of MURFs provides an explanation how A-band titin and myosin are finally brought in tight register, summarised synoptically in the sketch inFig. 9. The processes of active transport of myosin, and the stretching out and aligning of nascent myofibrils, must require force and hence the activity of molecular motors. It will be interesting to see whether these are microtubule-based and whether such motors associate with MURFs.

MURF2 in relation to titin, myosin and the filamentous systems of actin and microtubules during sarcomere formation. (A) At initial stages of myofibril assembly, MURF2 (red ovals) is microtubule associated. Myosin (dark green rods) colocalises early with MURF-decorated microtubules (light-green). (B)Titin (red chains) localises in dot-like aggregates on actin SFLS (blue) with a spacing of around 1 μm. Black links, α-actinin crosslinks. These structures co-align with MURF-decorated microtubules. (C) MURF2 and myosin colocalise strictly in nascent striated myofibrils, when the cross-striated pattern of myosin begins to form and titin Z-Z staining increases to the mature approximately 2 μm pattern. Transient colocalisation with A-band titin is observed at this stage. MURF-MURF heteromultimeres could link different sarcomeric components at this stage and result in the exact alignment of titin and myosin. (D) In mature myofibrils, myosin and actin are arranged in highly ordered cross-striated patterns with ordered polarity(actin pointed ends marked by arrows), and the titin molecule is extended,with the N-terminal portion remaining in the Z-disk and the C-terminus integrated into the M-band. Depending on differentiation state and/or muscle type, MURF2 can be present in the M-band or the nucleus. Bar, 1 μm.

MURF2 in relation to titin, myosin and the filamentous systems of actin and microtubules during sarcomere formation. (A) At initial stages of myofibril assembly, MURF2 (red ovals) is microtubule associated. Myosin (dark green rods) colocalises early with MURF-decorated microtubules (light-green). (B)Titin (red chains) localises in dot-like aggregates on actin SFLS (blue) with a spacing of around 1 μm. Black links, α-actinin crosslinks. These structures co-align with MURF-decorated microtubules. (C) MURF2 and myosin colocalise strictly in nascent striated myofibrils, when the cross-striated pattern of myosin begins to form and titin Z-Z staining increases to the mature approximately 2 μm pattern. Transient colocalisation with A-band titin is observed at this stage. MURF-MURF heteromultimeres could link different sarcomeric components at this stage and result in the exact alignment of titin and myosin. (D) In mature myofibrils, myosin and actin are arranged in highly ordered cross-striated patterns with ordered polarity(actin pointed ends marked by arrows), and the titin molecule is extended,with the N-terminal portion remaining in the Z-disk and the C-terminus integrated into the M-band. Depending on differentiation state and/or muscle type, MURF2 can be present in the M-band or the nucleus. Bar, 1 μm.

We found that MURF2 is expressed in multiple isoforms, some of which are tissue-specific. MURF2 p60B is generated by alternative reading frame use, the first description of this novel splice mechanism(Klemke et al., 2001) in a muscle protein. Apart from creating the potential for innumerable permutations in complex formation during sarcomere assembly, the differentially expressed MURFs may have other, muscle-type-specific functions. The ablation of MURF3 by antisense RNA dramatically suppresses myogenic differentiation on the transcriptional level (Spencer et al.,2000) apart from impairing myofibril formation. This may suggest a role in the control muscle differentiation apart from that of a transient structural adaptor during myofibril formation. A role for MURF3 in muscle gene transcription could be inferred from the putative interaction with SRF(Spencer et al., 2000). MURF1,also known as SMRZ (Dai and Liew,2001), was found to localise to the nucleus when transfected into C2C12 myoblasts, as well as to translocate in cardiac myocytes(McElhinny et al., 2002). MURF1 can also interact via the highly conserved RING domain with the ubiquitin-like SUMO-2/SMT3b (Dai and Liew,2001), linking MURFs to potential roles in nuclear transport,transcription regulation and signal transduction(Müller et al., 2001). Our data provide the first evidence that endogenous MURF2 is translocated to the nucleus and the nuclear lamina in response to stimulation of serum-starved cardiac myocytes. MURF2 can thus shuttle between three cellular compartments:microtubules, M-bands and the nucleus. However, MURF2 is detectable in the nucleus of neonatal rat cardiomyocytes only briefly after shifting serum-starved cells from low to high-serum conditions, indicating an involvement in nuclear signalling in a narrow time window and related to the stress of serum-withdrawal. Although we could not observe MURF2 in skeletal myotube nuclei, we are currently investigating the localisation in skeletal muscle tissues. Whether MURFs act as transcriptional co-activators or co-repressors in the nucleus in addition to a more structural involvement in sarcomere assembly will now need to be elucidated. Our observations suggest that the sarcomere not only receives input from many signal transduction pathways, but may also relay information to the transcriptional machinery and could thus regulate muscle-specific gene expression as first proposed by Iakovenko and Gautel (Iakovenko and Gautel, 2000). MURFs emerge as novel components of this crosstalk,and the various signals resulting in stress-induced MURF translocation now need to be identified.


MATERIALS AND METHODS

Cell culture and transfection.

Experimental animals were cared for as outlined in the Guide for the Care and Use of Experimental Animals (Animal Care Committees of the Hokkaido University and the NARO Institute of Livestock and Grassland Science), which the committee accepted. Primary chick skeletal muscle cells were prepared as previously described (37), with slight modifications. Briefly, skeletal muscle cells were isolated from day 11 chick embryonic pectoral muscles and cultured on Matrigel-coated (BD Bioscience) Labtech chamber slides (Nalge Nunc International) for immunofluorescence staining, or on collagen-coated glass bottom dishes (Matsunami Glass Ind.) for FRAP experiments. The growth medium [10% chick embryo extract and 10% horse serum in minimum essential medium (all from Life Technologies)] was shifted to differentiation medium (1.5% chick embryo extract and 5% horse serum in minimum essential medium) to induce muscle differentiation on the next day of transfection. Growth and differentiation media were supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin (Life Technologies). Transfection was performed by using Lipofectamine LTX and Plus reagents on the day after cell plating (Life Technologies).

To block the polymerization of microtubules, nocodazole (Sigma) was added to the culture medium at a final concentration of 2 μM (34). The actomyosin interaction was inhibited by treating the cells with N-benzyl-p-toluene sulphonamide (BTS Tokyo Chemical Industry) at a final concentration of 30 μM (26), and protein synthesis was prevented by treating the cells with cycloheximide (CX Wako Pure Chemical Industries) at a final concentration of 10 μM (14). Chemical agents were added to the medium at 1 h (nocodazole, BTS, and CX) or 10 h (CX) before the initiation of experiments and, in the case of the FRAP assays, maintained in the medium throughout the experiments. Dimethyl sulfoxide (DMSO) was added to the medium as vehicle controls.

CDNA constructs.

Full-length mouse Myh3 cDNA was cloned by a polymerase chain reaction-based method, as described previously (39). In brief, cDNA corresponding to mouse Myh3 (45–5868 in NM_001099635) contained in the pcDNA3.1/N-FLAG vector was subcloned into a peGFP vector (Clontech) or a humanized monomeric Kikume green-red 1 vector (KikGR1 Medical and Biological Laboratories). The cDNAs corresponding to mouse Mybpc1 (228–3611 in NM_001252372) and mouse Myom3 (111–4430 in NM_001085509) were cloned from first-strand cDNA, which was prepared from mouse myotube mRNA on day 8 of culture, and then subcloned into a pmCherry vector (Clontech). Polymerase chain reaction primers for Mybpc1 and Myom3 were as follows: Mybpc1, 5′-tttgaattct ATGCCAGAACCCACTAAG-3′ and 5′-tttggtacc CTACGACTGTTGCTGCCCC-3′ and Myom3, 5′-tttgaattct ATGACTCTGCCCCACAGCCC-3′ and 5′-tttggtacc TTACTTCTTCTTCAGCTCC-3′. All generated fluorescent proteins were tagged at the NH2-terminus of the target proteins, and all constructs were verified by sequencing with a 3730 DNA Analyzer (Applied Biosystems).

FRAP assays and photoconversion experiments.

At 7 days postdifferentiation, skeletal muscle cells transfected with an expression vector encoding an eGFP + Myh3 fusion protein were subjected to FRAP analysis with a TCS-SP5 Confocal Laser Scanning Microscope (Leica). Doubly transfected skeletal muscle cells carrying the eGFP + Myh3 expression vector and a vector encoding either a mCherry + Myom3 or mCherry + Mybpc1 fusion protein were also used for FRAP assays. The regions of interest were bleached for 30 s, and the FRAP data were recorded at 1-h intervals by using Leica Confocal Software. The relative fluorescence intensity was calculated as the ratio of the fluorescence intensity of the bleached area to the fluorescence intensity of the unbleached area at each time point. For the calculations, a curve-fitting method was used to extract the mobile fraction (Mf) and the half-life (t1/2) from the FRAP curve, as previously reported (55). Image-J1.46r (National Institutes of Health) was used to fit the data to the following equation: FI = Mf [1 − e (1 − bt) ] + c, where FI is the relative fluorescence intensity of the bleached area at time t, Mf is of the exponential process with rate constant b, and c is relative fluorescence intensity of the bleached area at time t0.

KikGR1 is green fluorescent protein that can be irreversibly converted to a red color following irradiation with ultraviolet (UV) light (53). KikGR1 + Myh3 fusion protein was utilized for a green-to-red photoconversion analysis with a TCS-SP5 Confocal Microscope. The regions of interest were photoactivated after UV irradiation, and images were recorded at 1-h intervals. The relative green fluorescence intensity was calculated as the ratio of the green fluorescence intensity at each time point relative to that of the green fluorescence intensity pre-photoconversion. To correct these intensities, the relative green fluorescence intensity pre-photoconversion was defined as 1, and the relative green fluorescence intensity just after photoconversion was defined as 0. The relative red fluorescence intensity was similarly calculated as the ratio of the red fluorescence intensity at each time point relative to that of the red fluorescence intensity immediately after photoconversion. To correct these intensities, the relative red fluorescence intensity just after photoconversion was defined as 1, and the relative red fluorescence intensity just after photoconversion in the non-photoconverted area was defined as 0. The Mf and t1/2 for photoconversion were calculated as described above. All FRAP and photoconversion analyses were carried out by using a microscope stage-top incubator (Tokai Hit) to control the temperature, humidity, and CO2 concentration.

Immunofluorescence staining.

The staining protocol has been described in detail by Ojima et al. (40). The antibodies used in the immunofluorescence assays were as follows: primary mouse anti-sarcomeric α-actinin antibody (1:1,000 dilution clone EA53 Sigma), primary mouse anti-Myh antibody (1:100 dilution clone F59 Developmental Studies Hybridoma Bank, University of Iowa) (33), primary mouse anti-Myom antibody (1:50 dilution clone B4 Developmental Studies Hybridoma Bank, University of Iowa) (16), and Alexa-555-conjugated secondary antibodies (Life Technologies). Specimens were stored with mounting media (Vector Laboratories), and subsequently analyzed by using a LSM 700 Confocal Laser Scanning Microscope (Carl Zeiss,) equipped with a Plan-Apochromat ×63 (numerical aperture 1.4) lens. Images were manipulated by using Zen 2012 imaging software (Carl Zeiss,).

Preparation of cytosolic myosin.

The cytosolic fraction was isolated as described by Isaacs and Fulton (23), with slight modifications. Briefly, the cytosolic fraction from cultured skeletal muscle cells was collected as follows. First, day 6–7 cultured chick muscle cells were immersed in lysis buffer [10 mM Tris·HCl (pH 7.5), 0.15 M CsCl, 1 mM EDTA-Cs, and 0.5% (vol/vol) Triton X-100] containing protease inhibitors (28 μM E64, 1.5 μM aprotinin, 50 μM leupeptin, 40 μM bestatin, 0.7 μM calpastatin, and 2 mM phenylmethanesulfonyl fluoride) for 30 min at 4°C. The lysate was centrifuged at 1,500 g for 10 min to remove cellular debris. The supernatant fraction was further centrifuged at 100,000 g for 1 h. The protein concentration of the centrifuged supernatant was quantified with a Bradford protein assay kit (Bio-Rad Laboratories). Specimens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then stained with SyproRuby (Bio-Rad Laboratories). Gels were scanned by using an Ettan DIGE Imager (GE Healthcare UK).

Immunoblot analysis.

Specimens were subjected to SDS-PAGE, as described above, followed by immunoblot analysis. After gels were transferred to Immobilon-P transfer membranes (Millipore), the membranes were blocked with 5% nonfat skim milk (Nacalai Tesque) and incubated with anti-myosin antibody (1:1,000 dilution clone MF20 Developmental Studies Hybridoma Bank, University of Iowa) (48), anti-GFP antibody (1:2,000 dilution clone GF090R Nacalai Tesque), anti-GAPDH antibody (1:2,000 dilution clone 3H12, Medical and Biological Laboratories) or anti-red fluorescence protein antibody cocktail (1:1,000 dilution mixture of clones 1G9 and 3B5 Medical and Biological Laboratories). Next, the membranes were incubated with peroxidase-conjugated secondary antibodies (1:10 dilution Nichirei), and the immunoreactive bands were visualized by using the Pierce enhanced chemiluminescence-Plus Western Blotting Substrate (Thermo Fisher Scientific). Band intensities were measured with Image-J1.46r (National Institutes of Health).


ISC Biology Question Paper 2011 Solved for Class 12

Part-I
(Attempt All Questions)

Question 1.
(a) Mention one significant difference between each of the following : [5]
(i) Growth and Development
(ii) Muscle twitch and Tetanus
(iii) Heartwood and Sapwood
(iv) Leghaemoglobin and Haemoglobin
(v) Collateral vascular bundle and Concentric vascular bundle.

(b) Give reasons for the following : [5]
(i) Adrenaline is referred to as emergency hormone.
(ii) Despite availability of plenty of water, leaves of certain plants wilt during the day and recover in the evening.
(iii) Hybrid seeds should be raised every year.
(iv) The wing of a bat is said to be homologous to the wing of a bird and analogous to the wing of an insect.
(v) Symptoms of deficiency of certain nutrients appear in the old leaves first.

(c) Give scientific terms for each of the following : [5]
(i) The development of more than one embryo in a seed.
(ii) The process of growing old.
(iii) Method of inducing early flowering in plants by pre-treatment of their seeds at lower temperatures.
(iv) Determination of the age of a tree by counting the number of annual rings.
(v) The type of growth in which the volume of the body increases without the increase in the number of body cells.
(vi) A condition when the muscles deteriorate and the person becomes invalid.

(d) Mention the most significant function of each of the following : [3]
(i) Fovea centralis
(ii) Lymphocytes
(iii) Bundle of His
(iv) Calyptra
(v) Bulliform cells
(vi) Quiescent centre

(e) State the best-known contribution of the following scientists : [2]
(i) Ernst Haeckel
(ii) Carl Landsteiner
(iii) Robert Koch

(f) Expand the following :
(i) Hensen [2]
(ii) G-6PD
(ii) DPD
(iii) MRI
(iv) SAN
Answer:
(a) (i)

Growth Development
Growth is the irreversible increase in dry weight, mass or volume of a cell, organ or organism. Development is the sequence of events that occur in the life history of a cell, organ or organism which includes growth, differentiation, maturation and senescence.

Muscle twitch Tetanus
A muscle twitch is the single isolated contraction of the muscle libre by a single nerve impulse or by a single electric shock of adequate strength, followed by immediate relaxation. Tetanus is the continued state of contraction of a muscle fibre stimulated by many nerve impulses or electric shocks. Contraction remains until the stimulation continues.

Heartwood Sapwood
Heartwood (duramen) is the inner dark coloured, non-functional secondary xylem. Sapwood (alburnum) is the outer, light-coloured, functional secondary xylem in most of the old dicot trees.

Leghaemoglobin Haemoglobin
Leghaemoglobin is the pinkish pigment present in the cells of root nodules of leguminous plants and acts as an oxygen scavenger to protect the nitrogen-fixing enzyme nitrogenase of the bacteroids. Haemoglobin is the blood pigment meant for transport of oxygen and regulates other things as carbon dioxide, nitric oxide, etc.

Collateral vascular bundle Concentric vascular bundle
Collateral vascular bundles are those conjoint bundles (contain both xylem and phloem) in which both phloem and xylem lie on same radius, with phloem on the outer side and xylem towards the inner side. Concentric vascular bundles have one type of vascular tissue (xylem or phloem) forms a solid core while the other surrounds it completely on all sides.

(b)
(i) Adrenaline is referred to as emergency hormone because it prepares the animal to meet any emergency condition by flight or fight reactions i.e., by running away (flight) to safety, or by giving a tough fight to the enemy (fight reaction). It increases the heart rate, blood pressure, blood sugar level, blood supply to muscles and brain etc.

(ii) Leaves of certain plants wilt during the day because the rate of transpiration is much higher than the rate of water absorption by the roots. As in the evening, the rate of transpiration decreases the plants regain their turgidity in the evening and recover.

(iii) The plants raised from the hybrid seeds show segregation of characters and do not maintain hybrid character necessitating the need to produce hybrid seeds every year.

(iv) The wing of a bat is homologous to the wing of the birds because both have the same basic plan of organisation and are modified forelimbs having different shapes, adapted for different habitats whereas it is analogous to the wing of an insect. The basic structure of the wing of insect is different from the wing of a bat. However, their function is similar. The deficiency symptoms of certain nutrients appear in the old leaves first because they are mobile and required in large detectable quantities for the synthesis of organic molecules. Polyembryony

(c)
(i) Polyembryony
(ii) Ageing (Senescence)
(iii) Vernalisation
(iv) Dendrochronology
(v) Auxetic growth
(vi) Duchenne’s Muscular Dystrophy (t)MD)

(d)
(i) It has only cone cells and is the place of most distinct vision.
(ii) A type of white blood cells, meant for eliminating the antigen (microbes and their toxins) by releasing antibodies. These are B and T-lymphocytes.
(iii) It is a bundle of heart-muscles that fapidly transmit the cardiac impulse received from Ay node to all parts of the ventricles causing them to contract.
(iv) Calyptra is a cone shaped structure that covers the root-tips and develops as a result of cclidivision by the meristem called calyptrogen in monocot roots.
(v) Bulliform cells are large thin walled protruding epidermal cells present on the upper epidermis of leaves of many grasses. They lose water and become flaccid and help to roll up leaves to reduce the exposed surface in case of water deficiency.
(vi) Quiescent centre is present in the centre of root-apex and functions as reserve meristem. Here divisions are very few. They can survive stress and provide cells to a regenerating meristem and helps in the recovery of roots after irradiation.

(e)
(1) Ernst Haeckel proposed biogenetic law which states that ‘Ontogeny repeats phylogeny’.
(ii) Karl Landsteiner discovered ABO blood groups in human beings.
(iii) Robert Koch (1843-1910) German physician contributed much to the field of microbiology and infectious diseases. He was the first to discover tuberculosis bacterium. He proposed Koch’s postulates which state certain requirements should be fulfilled if the disease causing character of any organism is to be proved.
(iv) Hensen discovered the disease leprosy, proposed the sliding theory of muscle contraction.

(f)
(i) Glucose – 6 phosphate dehydrogenase.
(ii) Diffusion Pressure Deficit
(iii) Magnetic Resonance Imaging
(iv) Sino-Atrial Node

Part-II
Section – A
(Attempt Three Questions)

Question 2.
(a) What are guard cells ? Explain their role in regulating transpiration. [4]
(b) Explain tunica corpus theory of origin of shoot apex. [3]
(c) Give one function and one deficiency symptom of each of the following in plants : [3]
(i) Magnesium
(ii) Calcium
(iii) Molybdenum
Answer:
(a) The guard cells are two small, specialised green epidermal cells surrounding a stomata. They are kidney-shaped in dicot plants and dumb-bell shaped in cereals/monocot plants. The expansion and contraction of thin-walled sides/ends of guard cells regulate the opening and closure move-ments.

Mechanism of Stomatal Movement: Stomata function as turgor operated valves because their opening and closing movement is governed by turgor changes of the guard cells. Whenever, the guard cells swell up due to increased turgor, a pore is created between them. With the loss of turgor the stomatal pores are closed. Stomata generally open during the day and close during the night with a few exceptions. The important factors which govern the sotmatal opening are light, high pH or reduced CO2 and availability of water. The opposite factors govern stomatal closure, viz., darkness, lowpR or high CO2 and dehydration.

(b) According to tunica-corpus theory of Schmidt (1924), the shoot apex has two parts, outer mantle like tunica and inner cellular mass known as corpus (Fig). Cells of tunica are small. They undergo anticlinal divisions and, therefore, take part in surface growth. Cells derived from tunica give rise to epidermis of both stem as well as leaves. If tunica is more than one layer in thickness, the outer layer differentiates into epidermis while ‘ the inner layers contribute to the formation of leaf interior and cortial tissues.

Cells of corpus are comparatively larger. They divide in different planes. Cells derived from corpus form procambium and ground meristem. Procambium is slow to differentiate. Initially its cells are narrow, elongated and densely cytoplasmic. They occur in parallel files. Procambium gives rise to primary phloem, primary xylem and intrafascicular cambium between the two (in case of dicots and gymnosperms). Ground meristem differentiates into pith in the centre and pericycle, endodermis, cortex and hypodermis respectively towards the outer side.

Element Function Deficiency Symptom
(i) Magnesium Chlorophyll formation Interveinal chlorosis with anthocyanin pigmentation
(ii) Calcium Meristematic activity i.e., cell divisions connected with chromosome formation Stunted growth, degeneration of meristem
(iii) Molybdenum Nitrogen metabolism Mottled chlorosis with marginal necrosis, the upper half of lamina fall down (whiptail disease)

Question 3.
(a) Describe the development of female gametophyte in Angiosperms.
(b) Explain the mass flow hypothesis of transport of food.
(c) Differentiate between cyclic and non-cyclic photophosphorylation
Answer:
(a) One hypodermal nucellar cell of the micropylar region differentiates as the sporogenous cell. It forms a diploid megaspore mother cell or megasporocyte. The megaspore mother cell undergoes meiosis (Megasporogenesis) and forms a row of four haploid megaspores. Only the chalazal megaspore remains functional while the other three degenerate. The functional megaspore enlarges and gives rise to female gametophyte, also called embryo-sac. It lies in the micropylar area of the nucellus.

Micropylar end Chalazal end Functional megaspore divides by three mitotic divisions to form 8-nucleate, or 7-celled embryo- sac (= female gametophyte). The development of female gametophyte from megaspore is called Megagametogenesis. The development of embryo-sac in angiosperms is generally Monosporic i. e., embryo-sac develops from a uninucleate megaspore.

Embryo sac is an oval multicellular structure. It is covered over by a thin membrane derived from the parent megaspore wall. The typical or Polygonum type of embryo sac (Fig.) contains 8 nuclei but 7 cells – 3 micropylar, 3 chalazal and one central. The three micropylar cells are collectively known as egg apparatus. They are pyriform in outline and are arranged in a triangular fashion. One cell is larger and is called egg or oosphere. The remaining two cells are called synergids or help cells. Each of them bears a filiform apparatus in the micropylar region. The egg or oosphere represents the single female gamete of the embryo sac. The synergids help in obtaining nourishment from the outer nucellar cells, guide the path of pollen tube by their secretion and function as shock absorbers during the penetration of the pollen tube into the embryo sac.

The three chalazal cells of the embryo sac are called antipodal cells.

The central cell is the largest cell of the embryo sac. It has a highly vacuolated cytoplasm and contains two polar nuclei which have large nucleoli. The polar nuclei often fuse to form a single diploid secondary or fusion nucleus. Thus, all the cells of the embryo sac are haploid except the central cell which becomes diploid due to fusion of two polar nuclei.

(b) Mass Flow or Pressure Flow Hypothesis. It was put forward by Munch (1927,1930). According to this hypothesis, organic substances move from the region of high osmotic pressure to the region of.low osmotic pressure in a mass flow due to the development of a gradient of turgor pressure. This can be proved by taking two interconnected osmometers, one with high solute concentration. The two osmometers of the apparatus are placed in water (Fig.). More water enters the osmometer having high solute concentration as compared to the other. It will, therefore, come to have high turgor pressure which forces the solution to pass into the second osmometer by a mass flow. If the solutes are replenished in the donor osmometer and immobilised in the recipient osmometer, the mass flow can be maintained indefinitely.

Sieve tube system is fully adapted to mass flow of solutes. Here the vacuoles are fully permeable because of the absence of tonoplast. A continuous high osmotic concentration is present in the mesophyll cells (due to photosynthesis) and storage cells (due to mobilization of reserve food). The organic substances present in them are passed into the sieve tubes (by means of transfer cells). A high osmotic concentration, therefore, develops in the sieve tubes of the source. The sieve tubes absorb water from the surrounding xylem and develop a high turgor pressure (Fig.). It causes the flow of organic solution toward the area of low turgor pressure. A low turgor is maintained in the sink region by converting soluble organic substances into insoluble form. Water passes back into xylem.

(c) Differences between Cyclic and Non-cyclic Photophosphorylation

  1. It is performed by photosystem I independently.
  2. An external source of electrons is not required.
  3. It is not connected with photolysis of water. Therefore, no oxygen is evolved.
  4. It synthesises only ATP.
  5. The system does not take part in photosynthesis except in certain bacteria.
  6. It occurs mostly in stromal or intergranal thylakoids.
  7. ATP synthesis is not affected by DCMU.
  8. It is helpful in photosynthesis only in some bacteria.
  1. It is performed by collaboration of both photosystems II and I.
  2. The process requires an external electron donor.
  3. It is connected with photolysis of water and liberation of oxygen.
  4. Noncyclic photophosphorylation is not only connected with ATP synthesis but also production of NADPH.
  5. The system is connected with C02 fixation.
  6. It occurs in the granal thylakoids.
  7. DCMU inhibits noncyclic photophosphorylation.
  8. It takes part in photosynthesis in all plants including blue-green algae.

Question 4.
(a) Explain in detail the digestion of carbohydrates, as the food passes through the alimentary canal. [4]
(b) Describe step by step what happens in the different phases of the cardiac cycle in human beings. [3]
(c) Write the effects of cytokinins on plants. [3]
Answer:
(a) Digestion of Carbohydrates:
Carbohydrates are of three kinds – polysaccharides, disaccharides, and monosaccharides. Polysaccharides and disaccharides are broken down to monosaccharides during the process of digestion. Starch and cellulose are polysaccharides that are present in cereal grains, potato, tubers and fruits. Sucrose (in cane sugar), maltose (in germinating grains), and lactose (in milk) are disaccharides. Enzymes which act on carbohydrates are called carbohydrases.

1. Digestion of Carbohydrates in the Oral Cavity Action of Saliva. In oral cavity, the food is mixed with saliva. The saliva contains an enzyme called salivary amylase (also called ptyalin) which converts starch into maltose, isomaltose and small dextrins called ‘limit’ dextrins.

The gastric juice in stomach does not contain carbohydrate-digesting enzyme.

2. Digestion of Carbohydrates in the small intestine

  • Action of Pancreatic Juice. The pancreatic juice contains starch digesting enzyme called pancreatic amylase which converts starch into maltose, isomaltose and ‘limit’ dextrins.
  • Action of Intestinal Juice. Intestinal juice contains maltase, isomaltase, sucrase, lactase and ‘Limit’ Dextrinase which act as follows :

Digestion of Cellulose. The cellulose is not digested by human beings but, however, it is digested by the microorganisms (bacteria and protozoans) in the alimentary canal of herbivorous mammals. These microorganisms ferment cellulose into short-chain fatty acids such as acetic and propionic acid. These acids are then absorbed and utilized by the animal. Cellulose form roughage and helps in digestion process in human beings.

(b) Cardiac Cycle
The cardiac cycle consists of one heartbeat or one cycle of contraction and relaxation of the cardiac muscle. During a heartbeat there is contraction and relaxation of atria and ventricles The , contraction phase is called the systole while the relaxation phase is called the diastole. When both the atria and ventricles are in diastolic or relaxed phase, this is referred to as a joint diastole. During this phase, the blood flows from the superior and inferior venae cavae into the atria and from the atria to the respective ventricles through auriculo-ventricular valves. But there is no flow of blood from the ventricles to the aorta and pulmonary trunk as the semilunar valves remain closed.

The successive stages of the cardiac cycle arc briefly described below.

  1. Atrial Systole. The atria contract due to a wave of contraction stimulated by the SA node. The blood is forced into the ventricles as the bicuspid and tricuspid valves are open.
  2. Beginning of Ventricular Systole. The ventricles contraction is stimulated by the AV node. The bicuspid and tricuspid valves close immediately producing part of the first heart sound.
  3. Period of Rising Pressure. The pressure in the ventricles rises. The semilunar valves remain closed. The blood does not flow into or out of the ventricles.
  4. Complete Ventricular Systole. When the ventricles complete their contraction, the blood flows into the pulmonary trunk and aorta as the semilunar valves open.
  5. Beginning of Ventricular diastole. The ventricles relax and the semilunar valves are closed. This causes the second heart sound.
  6. Period of Falling Pressure. The pressure within the ventricles continues to decrease. The bicuspid and tricuspid valves still remain closed. Blood flows from the veins into the relaxed atria.
  7. Complete Ventricular Diastole. The tricuspid and bicuspid valves open when the pressure in the ventricles falls and blood flows from the atria into the ventricles. Contraction of the heart does not cause this blood flow. It is due to the fact that the pressure within the relaxed ventricles is less than that in the atria and veins.
  1. Cell Division. Cytokinins are essential for cytokinesis though chromosome doubling can occur in their absence. In the presence of auxin, cytokinins bring about-division even in permanent cells. Cell division in callus (unorganised, undifferentiated irregular mass of dividing cells in tissue culture) is found to require both the hormones.
  2. Cell Elongation. Like auxin and gibberellins, cytokinins also cause cell elongation.
  3. Morphogenesis. Both auxin and cytokinins are essential for morphogenesis or differentiation of tissues and organs. Buds develop when cytokinins are in excess while roots are formed when their ratios are reversed (Skoog and Miller, 1957).
  4. Differentiation. Cytokinins induce plastid differentiation, lignification and differentiation of interfascicular cambium.
  5. Senescence (Richmond-Lang Effect). Cytokinins delay the senescence of leaves and other organs.
  6. Apical Dominance. Presence of cytokinin in an area causes preferential movement of nu-trients towards it. When applied to lateral buds, they help in their growth despite the pres-ence of apical bud. They thus act antagonistically to auxin which promotes apical dominance.
  7. Seed Dormancy. Like gibberellins, they overcome seed dormancy of various types, including red light requirement of Lettuce and Tobacco seeds.
  8. Resistance. Cytokinins increase resistance to high or low temperature and disease.
  9. Phloem Transport. They help in phloem transport.
  10. Accumulation of Salts. Cytokinins induce accumulation of salts inside the cells.
  11. Flowering. Cytokinins can replace photoperiodic requirement of flowering in certain cases.
  12. Sex Expression. Like auxins and ethylene, cytokinins promote femaleness in flowers.
  13. Parthenocarpy. Crane (1965) has reported induction of parthenocarpy through cytokinin.

Question 5.
(a) Write about the chemical changes which occur during contraction of skeletal muscles. [4]
(b) Draw a neat labelled diagram of the L. S. of a kidney. [4]
(c) What is chloride shift? , [2]
Answer:
(a) Mechanism of Muscle Contraction: The contraction of skeletal muscle includes ultrastructural and biochemical events.

1. Ultrastructural/Physical events (Biophysics of Muscle Contraction).
Myosin and actin are special type of proteins. Myosin forms the thick filaments and the actin forms the thin filaments of the myofibrils of the muscle fibres. The myosin and actin help in the contraction or shortening of muscles by the formation of cross bridges. The cross bridges are the portions of the myosin filaments overlapped by actin filaments.

H. E. Huxley and A. F. Huxley in 1954 proposed a theory to explain the process of muscular contraction. This theory is known as sliding filament theory, which is now generally accepted. This theory states that the actin (thin) filaments slide over the myosin (thick) filaments to penetrate deeper into the A bands in the contracting muscle fibre. The thin filaments meet in the centre of the sarcomere. As such the width of the A band remains constant. However, the I bands shorten and ultimately disappear. This shortens the sarcomere.

As all the sarcomeres of the myofibril shorten simultaneously the muscle fibre shortens. During relaxation the action filaments slide out of A band, thereby lengthening the sarcomere and these crossbridges disappear. This indicates the presence of active sites on the actin filaments into which the cross-bridges temporarily hook to pull the filaments a short distance and then release them. It means that contraction and relaxation of muslces are brought about by the repetitive formation and breakage of crossbridges respectively.

The proteins, troponin and tropomyosin, which are closely associated with actin, are also important in regulating the attachment of actin to the crossbridges.

2. Biochemical Events (Biochemistry of Muscle Contraction). Albert Szent Gyorgyi and other worked out the biochemical events associated with the muscle contraction. These biochemical events are summarised below.

  1. The nerve impulse stimulates a muscle fibre at the neuromuscular junction or motor end plate, producing acetylcholine.
  2. Acetylcholine brings out the release of calcium ions from the sarcoplasmic reticulum of the muscle into the interior of muscle fibre which becomes bound with specific sites on troponin of their filament.
  3. Myosin now binds with actin to form actomyosin in the presence of ATP and calcium ions.
  4. Energy for muscle contraction is provided by hydrolysis of ATP by myosin ATP- ase enzyme. This hydrolysis produces ADP, inorganic phosphate and energy (used in muscle contraction). Phosphocreatine donates its high energy and phosphate to ADP, producing ATP.

Phosphocreatine serves as an energy source for a few seconds for metabolic processes in the muscle cells to begin to produce greater quantities of ATP. Phosphocreatine is again formed in relaxing muscle by using ATP produced by carbohydrate oxidation.

5. At the end of muscle contraction, the conversion of ADP into ATP takes place. The muscle is rich in glycogen which is broken down into lactic acid through a series of reactions (glycolysis) and liberates energy. Some of this energy is used for the reformation of phosphocreatine and also for the conversion of 4/5th of lactic acid back into glycogen. The l/5th of lactic acid is oxidised to water and carbon dioxide. These reactions taking place in the muscle and liver, are proposed by Cori and Cori, hence known as Cori’s cycle.

(c) A small amount of bicarbonate ions is transported in the erythrocytes (RBCs), whereas most of them diffuse into the blood plasma to be carried by it.

Exit of bicarbonate ions, from RBCs considerably change ionic balance between the plasma and the erythrocytes. To restore this ionic balance, the chloride ions diffuse from the plasma into the erythrocytes. This movement of chloride ions is called chloride shift ( = Hamburger’s phenomenon). This process maintains an acid-base equilibrium of pH 7.4 for the blood and electrical balance between erythrocytes and plasma.

Question 6.
(a) Explain how the human ear helps in hearing. [4]
(b) Briefly describe the events that occur during the proliferative phase of the menstrual cycle. [3]
(c) Mention the site of secretion and function of the following : [3]
(i) Glucocorticoids
(ii) Calcitonin
(iii) Glucagon
Answer:
(a) Mechanism of hearing : The ear pinna collects and directs the sound waves travelling through air into the external auditory canal. They create vibrations in tympanic membrane (eardrum). These vibrations are transmitted through the chain of ossicles to the perilymph i.e., malleus transmits the message to the adjoining incus. The incus, in turn, transmits the vibrations to stapes. Stapes bone that fits into a membranous opening, the oval window, on the inner wall of the middle ear. Thus, in this process the force of vibration undergoes considerable amplification since the chain of ossicles acts as a lever and the area of the tympanic membrane is much greater than that of the footplate of stapes which increases the force per unit area.

The movement of stapes towards vestibuli, sets up pressure wave in perilymph. This wave passes from vestibule into scale vestibuli, and travels through it to the apex of cochlea. At this point, scala vestibuli is continuous with scala tympani. The pressure wave passes into scala tympani and again traverses the whole length of cochlea. In this way vibrations are set up in the perilymph and through it in the basilar membrane. The basilar membrane moves up and down, distorting hair cells of organ of corti. These distortions generate nerve impulses that travel through auditory nerve to the appropriate part of the brain where the sensation of hearing is felt (recognised).

Fig- Schematic representation of the conduction of sound vibrations in the ear.

(b) Proliferative phase : After menstruation, the proliferative phase starts with the growth and proliferation of tissues on the wall of the uterus, fallopian tubes and vagina.
1. Changes in uterus : At the onset of proliferative phase, the endometrium (the mucous membrance of the uterus) is thinnest (about 0.5-1 mm thick) as all superficial layers cast off during the menstrual bleeding. As the proliferative phase progresses, endometrium glands grow in length, epithelial cells of the endometrium proliferate, growth of the endometrium stroma occurs, and the blood vessels grow. Just before ovulation, the endometrium becomes about 3-5 mm thick. The myometrial contractions become more powerful and secretion of the glands of the uterine cervix becomes very thin at the time of ovulation to facilitate the entry of spermatozoa. The uterine changes are due to the rising concentration of estrogen.

2. Changes in the ovary: The ovarian cycle progresses side by side. During the proliferative phase an immature follicle ripens into a Graafian follicle. Since the proliferative phase is associated with a growing follicle in the ovary, this phase is also called follicular phase. The proliferative phase extends for 10 – 12 days and at its end the ovum is ejected (ovulation) from the Graafian follicle of the ovary.

Hormone Site of Secretion Function
(i) Glucocorticoids Adrenal Cortex Synthesis of carbohydrates from non-carbohydrates e.g., cortisol, degradation of proteins and fats.
(ii) Calcitonin bC cells in the thyroid gland Regulate calcium and phosphate level in the body.
(iii) Glucagon A-cells in islets of Langerhans in Pancreas Convert stored glycogen into glucose to maintain its blood level.

Section – B
(Answer any two questions)

Question 7.
(a) Explain the evolution of the long neck of giraffe according to Darwin and Lamarck. [4]
(b) Explain briefly : [4]
(i) Environmental resistance
(ii) Albinism
(iii) Plant introduction
(iv) Palaeontology

(c) Write two uses of each of the following : [2]
(i) Emblica officinalis
(ii) Adhatoda vesica
Answer:
(a) Lamarck explained that the ancestors of giraffe were bearing a small neck and fore-limbs and were like horses. But as they were living in places with no surface vegetation, they had to stretch their neck and fore-limbs to take the leaves from tall plants for food, which resulted in the slight elongation of these parts. Whatever they acquired in one generation was transmitted to the next generation with the result that a race of long-necked and long fore-limbed animal was developed. The long neck of giraffe, as is found today, can be explained on the basis of Darwinism in the following way:

The giraffes had originally a mixed population with short and long necks. As the leaves on the lower branches of trees became scarce the giraffes were forced to reach the leaves on higher branches of trees. The animals with comparatively longer neck were certainly more fit because they could reach the leaves on higher branches and, therefore, they had better chances of survival. Those with comparatively shorter neck could not reach the higher leaves and hence, die out. So, animals with longer neck were selected by nature. They fed comfortably and reproduced more offspring. Thus, in the course of time and generation after generation, the present-day long-necked giraffes originated by natural selection.

(b) (i) Environmental Resistance: The sum total of inhibitory environmental factors, both biotic end abiotic such as drought, high temperature, shortage of food, shelter, predation, pathogens, diseases etc. which regulate population size and do not permit unlimited growth of population is called environment resistance. Because of environmental resistance, the populations are unable to reach full biotic potential.

(ii) Albinism. The individuals suffering from albinism are called albino. They lack melanin pigment in their skin, hair, iris of eye etc. Such persons are susceptible to bright sun rays and develop eye disorders and skin diseases. Albinism results from inheritance of autosomal gene mutation. Hence the individual lacks the enzyme tyrosinase which is essential for synthesis of melanin. The gene for albinism is a recessive gene (a) which can be expressed only in homozygous (aa) condition. A person with its dominant allele (A) will be normal. The condition is known to affect mammals, fish, birds, reptiles and amphibians. Albinism is a genetic disorder, while the most common term for an organism affected by albinism is ‘albino’. Most organisms with albinism appear white or very pale.

(iii) Plant Introduction : Plant introduction means introducing a plant having desirable characters (e.g., vigorous growth, high yield, disease resistance, etc) from a region or a country where it grows naturally to a region or country where it did not occur earlier.

The adaptation of an individual to a changed environment, or the adjustment of a species or a population to a changed environment over a number of generations is called acclimatization (or acclimation).

Plant introduction has played a significant role in the development of agriculture throughout . the world. Some of the most important commercial crops cultivated extensively in India today are introductions from other countries. For example, Gossypium hirsutum, Cinchona was first introduced into the Nilgris from Peru in 1860. Potato (Solanum tuberosum), chilli (Capsicum annuum), tobacco (Nicotiana tobaccum), guava (Psidium guajava), custard apple (Annona squamosa), cashewnut (Anacardium occidentale) and Papaya (‘Carica papaya) are some of the other examples of crops successfully introduced in India.

Plant introduction can be useful in three different ways :
(a) the introduced material can be used directly by increasing it enmass.
(b) desirable strains can be selected from the introduced material.
(c) the introduced material can be used as a parent for hybridisation with adapted local varieties.
(iv) Palaeontology : It is the study of past life based on the fossil record. The fossils are petrified (turned in to stone) remains or impressions of ancient organisms preserved by natural means in the sedimentary rocks or other media such as amber, asphalt, volcanic – ash, ice, peat bogs, sand and mud. Palaeontology furnishes the most direct and reliable evidence for evolution, as it deals with the actual organisms that lived in the past.

(c)
(i) Emblica officinalis (Euphorbiaceae): Fruits rich in vitamin C, commonly pickled and used as a medicine.
(ii) Adhatoda vesica (Acanthaceae)Used as expectorant in cough, asthma.

Question 8.
(a) Give four applications of tissue culture in crop improvement. [4]
(b) What do you understand by the term population growth ? Give three ways of discouraging population growth. [4]
(c) Define : [2]
(i) Coacervates
(ii) Gene bank
Answer:
(a) Applications of tissue Culture (Micropropagation) :

  • It helps in rapid multiplication of plants.
  • A large number of plantlets are obtained within a short period and from a small space.
  • Plants are obtained throughout the year under controlled conditions, independent of seasons.
  • Sterile plants or plants which cannot maintain their characters by sexual reproduction are multiplied by this method.
  • It is an easy, safe and economical method for plant propagation.
  • In case of ornamentals, tissue culture plants give better growth, more flowers and less fall-out.
  • Genetically similar plants (soma clones) are formed by this method. Therefore, desirable characters (genotype) and desired sex of superior variety are kept constant for many generations.
  • The rare plant and endangered species are multiplied by this method and such plants are saved.

(b) Population growth refers to increase in the total number of organisms occupying a certain area.

The various methods to discourage population growth are :
(a) Education : People particularly those in reproductive age group, should be educated about the advantage of a small family, and the consequent benefits to the nation. People should be educated about the affects of overpopulation by the Government agencies mass media such as radio, television, newspaper, magazines, posters and educational institution can play an important role in this campaign. It will certainly help check population growth.

(b) Marriageable age : Raising the age of marriage will decrease reproductive span, can help in reducing population growth. At present, the marriageable age is 18 years for females and 21 years for males. Social change and the increasing aspiration for education and career in women, encourage them to delay marriage and postpone reproduction.

(c) Family planning : The family planning includes many birth control measures. Following family planning methods should be adopted :

  • Use of oral contraceptive pills by women.
  • Use of vaginal diaphragms.
  • Use of intrauterine contraceptive devices (IUCD) like copper-T and loop.
  • Surgical techniques of birth control like tubectomy in females and vasectomy in males.
  • Medical termination of pregnancy.
  • Natural family planning by having intercourse only during safe period or withdrawing penis before ejaculation.

(c) (i) A coacervate was a cluster of the membrane-bound macromolecules of prebiotic soup in the ocean. The macromolecules aggregated and formed small colloidal, masses in the form of insoluble droplets which finally precipitated and formed a larger and denser colloidal system called the coacervates. The coacervates had various macromolecules in different combinations and in specific proportions. Coacervates are considered to be the first living molecules which gave rise to life. Oparin has considered the coacervates as the sole living molecules which gave rise to life on the earth.

(ii) Gene Banks : They are the institutes that maintain stocks of viable seeds (seed banks), live growing plants (orchards), tissue culture and frozen germplasm with the whole range of genetic variability.

Question 9.
(a) Explain the role of Rh factor in blood incompatibility. [4]
(b) State the main morphological changes that occurred in the ancestors of modem man. [3]
(c) Describe briefly the functions of the following : [3]
(i) CT Scan
(ii) External prosthesis
(iii) Pacemaker
Answer:
(a) Rh blood group incompatibility : Rh factor is an antigen that is found at the surface of RBCs. It was first discovered on the RBCs of Rhesus monkey that is why it is named as Rh factor. Normally, there is no antibody for this antigen. About 85 to 99 per cent population possess this antigen. The persons with this antigen are called Rh +ve and those without it are Rh -ve. Rh factor is expressed by a dominant gene R so Rh +ve persons possess either RR or Rr genotype whereas Rh -ve persons possess rr.

Fig. Rh Factoc incompatibility. Rh positive foetus in Rh negative mother. A. First Pregnancy. B. Mothers body after first pregnancy. More production of AntiRh factors. C. Second pregnancy. RBCs of the foetus are destroyed by Anti Rh factors of the mother.

During inheritance, if both parents are Rh-ve then their offsprings, will also be Rh -ve. A Rh – ve mother having a Rh +ve husband may carry a Rh +ve child. In this case Rh factor produced by child’s blood may enter the stream of mother (at the time of delivery) and cause the production of anti – Rh antibodies in her blood but it results no ill effect. If the same mother conceives again a Rh +ve child second time, then the result may be disastrous. It is because, Rh +ve blood of foetus reacts with anti-Rh antibodies which are already present in her blood finally resulting into a condition called erythroblastosis foetalis.

In blood transfusion, Rh-ve blood can be given safely to a Rh+ve individual. When Rh+ve blood is transfused into Rh-ve individual the recipient develops anti-Rh antibodies in his blood. Usually no complications develops after one transfusion but, if more Rh+ve blood is transfused, the antibodies formed will destroy the RBCs of the Rh +ve blood. To avoid this, Rh factor is determined before such blood transfusions.

(b) The following main morphological changes occurred in the ancestors of modem man.

  • Narrowing and elevation of nose.
  • Formation of chin.
  • Reduction of brow ridges.
  • Flattening of face.
  • Reduction in body hair.
  • Development of curves in the vertebral column for erect posture.
  • Formation of bowel-like pelvic girdle with broad ilia (pi. of ilium) in support of viscera.
  • Increase in height.
  • Attainment of erect posture and bipedal locomotion.
  • Enlargement and rounding of cranium.
  • Increase in brain size and intelligence.
  • Broadening of the forehead and with vertical elevation.

(c)
(i) Computed Scanning (also called computed tomography – (CT): The technique was invented by Sir Godfrey Hounsfield who was awarded a Nobel prize in 1979 for this major achievement. During the last few years, advances in CT technology have led to fast scan times and improved image quality. As a result the scope of CT has widened enormously so that it is now applied to almost any anatomical site.

In CT scanning a computer is used for reconstructing the image made by X-rays instead being recorded directly on the photographic film. The CT scanning technique is used for the diagnosis of the diseases of brain, spinal cord, chest, abdomen and also for the detection of benign and malignant tumours. Thus, it helps to determine the feasibility of operation treatment and also to assess the results of the treatment.

(ii) Prosthesis : Prosthesis is the implantation of an artificial substitute for any body part within the body. It enables the physical handicapped person to live a comfortable and productive life.

Internal prosthesis devices include intraocular lens, dentures. Prosthesis devices also include nose implant for cosmetic reshaping, electronic hearing aids in the ear, artificial arm or leg.

(iii) Pacemaker : It is electronic cardiac-support device that produces rhythmic electrical impulses that take over the regulation of the heartbeat in patients with certain types of -heart disease.

When electrical conduction system is interrupted, as is the case in a number of diseases including congestive heart failure and as an after effect of heart surgery, the condition is called heart block. An artificial pacemaker may be employed temporarily until normal conduction returns or permanently to overcome the block.

The first pacemaker were of a type called asynchronous, or fixed, and they generated regular discharges that overrode the natural pacemaker. The rate of asynchronous pacemakers may be set at the factory or may be altered by the physician, but once set they will continue to generate an electric pulse at regular intervals. Most are set at 70 to 75 beats per minute. .

More recent devices are synchronous, or demand pacemakers that trigger heart contractions only when the normal beat is interrupted. Most pacemakers of this type are designed to generate a pulse when the natural heart rate falls below 68 to 72 beats per minute. These instruments have a sensing electrode that detects the atrial impulse.

Question 10.
(a) Explain the role of a Genetic Counsellor. [4]
(b) Write the causative agent and the main symptoms of the following diseases : [4]
(i) Poliomyelitis
(ii) Typhoid
(iii) Tuberculosis
(iv) Cholera
(c) State two similarities between the chromosomes of man and apes. . [2]
Answer:
(a) The area of health care which provides advice by the expert geneticists on genetic problems is called genetic counselling. It is not a technology but uses biochemical, statistical and physiological techniques to determine chances of occurrence of the actual disease. It thus plays an important role in the welfare of healthy society.

Genetic counselling is advisable for such persons who :

  • have a birth defect due to genetic disorder.
  • plan to have children after the age of 35.
  • have had spontaneous abortions.
  • have a close relative with a genetic disorder/disability.
  • are parents of a child which has a birth defect or genetic disorder.
  • have ethnic disorders like sickle cell anaemia etc. Genetic counselling is helpful to those couples who think that there may be a risk of having a child with a congenital disease. The genetic counsellor then identifies the disorder and advises accordingly. This may allow couples to select the children free from sex-linked abnormalities in the children. Genetic counsellor thus helps in prenatal diagnosis.

Genetic counselling given by the experts is helpful to the prospective parents about the chance of their conceiving children with hereditary disorders. Due to growing knowledge of inheritance, now we have come to know that numerous disabilities have genetic origin. Some of these genetic disorders cannot be predicted easily but other can be. This has enabled us to predict the occurrence of certain genetic disorders such as haemophilia, cystic fibrosis, some kinds of muscular dystrophy etc. if we have proper information about the history of the disorder in the related families. Through genetic counselling, the history of a genetic disorder of the related families is researched and on the basis of this study, the parents are advised on the likelihood of that certain disorder arising in their children.

Disease Causative Agent Symptoms
(i) Poliomyelitis Antivirus or Poliovirus Infection of CNS, voluntary muscles fail to work and affected limb or limbs get paralysed, making the patient handicapped, stiffness of neck.
(ii) Typhoid Salmonella typhi Continued fever, slow pulse, abdominal tenderness, dry coated tongue, soap-like stool
(iii) Tuberculosis Mycobacterium tuberculosis Fever, general weakness, Loss of appetite, persistent coughing with yellowish blood-stained saliva /sputum, pain in chest, loss of weight
(iv) Cholera Vibrio cholerae The stool has rice, water appearance, vomiting, acute diarrhoea, in advance stages cholera results in dehydration and loss of minerals

(c) (i) The somatic number of chromosomes in the cells of human body is 46 and that of great apes is 48 chromosomes. Man is presumed to have descended from a 48 chromosome stock by a centric fusion, however, DNA content is same.
(ii) Comparison of human and ape chromosomes shows that the banding pattern of individual human chromosomes is very similar and in some cases identical to the banding pattern of apparently homologous chromosomes in great apes. The banding pattern of chromosome numbers 3 and 6 of man and chimpanzee shows remarkable similarity in the bands indicating a common origin.

The number and gross morphology of chromosomes in different human races is the same. This shows that morphological differences in the human races are very significant from evolutionary point of view.


Ordering of myosin II filaments driven by mechanical forces: experiments and theory

Myosin II filaments form ordered superstructures in both cross-striated muscle and non-muscle cells. In cross-striated muscle, myosin II (thick) filaments, actin (thin) filaments and elastic titin filaments comprise the stereotypical contractile units of muscles called sarcomeres. Linear chains of sarcomeres, called myofibrils, are aligned laterally in registry to form cross-striated muscle cells. The experimentally observed dependence of the registered organization of myofibrils on extracellular matrix elasticity has been proposed to arise from the interactions of sarcomeric contractile elements (considered as force dipoles) through the matrix. Non-muscle cells form small bipolar filaments built of less than 30 myosin II molecules. These filaments are associated in registry forming superstructures (‘stacks’) orthogonal to actin filament bundles. Formation of myosin II filament stacks requires the myosin II ATPase activity and function of the actin filament crosslinking, polymerizing and depolymerizing proteins. We propose that the myosin II filaments embedded into elastic, intervening actin network (IVN) function as force dipoles that interact attractively through the IVN. This is in analogy with the theoretical picture developed for myofibrils where the elastic medium is now the actin cytoskeleton itself. Myosin stack formation in non-muscle cells provides a novel mechanism for the self-organization of the actin cytoskeleton at the level of the entire cell.

This article is part of the theme issue ‘Self-organization in cell biology’.

1. Introduction

Myosins comprise a superfamily of molecular motors that interact with actin filaments and use the energy of ATP hydrolysis to move along these filaments thereby producing active mechanical forces [1,2]. The part of the myosin heavy chain molecule that interacts with actin filaments, known as the myosin head, is conserved among all members of the myosin superfamily, consisting of more than 30 classes [2]. Other parts of the myosin molecule, the neck and the tail, can differ significantly in different myosin classes. Compared to other myosin classes, the myosin II sub-family has the unique feature that its extended tails can interact with each other to form functional, bipolar filaments. Interaction of the bipolar myosin II filaments with arrays of oppositely oriented actin filaments pulls these arrays towards each other producing contractions typical for both muscle and non-muscle cells [3,4].

This review is devoted to the organization of myosin II filaments in muscle and in particular non-muscle cells. We discuss experimental data that elucidate the organization of myosin II filaments into superstructures comprising of tens to hundreds of filaments. These superstructures or arrays not only generate cell contractility but also determine the global organization of actin filaments at the level of the entire cell. Actin and myosin II filaments, together with numerous accessory proteins, form characteristic actomyosin structures such as sarcomeres in cross-striated muscle cells and different kinds of actin bundles and networks in non-muscle cells. These various types of organization of the actomyosin cytoskeleton determine cell contractility, adhesion, locomotion and morphogenesis. We further propose and discuss a unifying theory that explains the formation of the myosin superstructures comprising numerous, laterally aligned myosin filaments. This theory considers myosin II filaments as force dipoles embedded in an elastic medium and predicts registered organization of these filaments by attractive forces between these dipoles.

2. Myosin II filaments and their arrangement in cross-striated muscle

Each myosin II molecule is a hexamer consisting of two heavy chains and two pairs of different light chains. Each heavy chain consists of an N-terminal head (motor) domain and a long α-helical rod/tail domain connected by a neck (lever arm) domain (figure 1a). A two-headed myosin II molecule is assembled by formation of a coiled coil between the α-helical tail domains of two heavy chains forming a myosin II rod. In a majority of myosin II heavy chain types, the short sequence at the C-terminus is non-helical and forms a so called C-terminal tailpiece [5–7]. Two different light chains associate with the neck regions of each heavy chain (figure 1a) [3,4]. These myosin II hexamers assemble into different types of superstructures in different cell types. The most complex and highly ordered myosin II organization is found in cross-striated (skeletal and cardiac) muscles [4,8] (figure 1b).

Figure 1. (a) A cartoon depicting the generic organization of myosin II molecules. Myosin II is a hexameric protein composed of two heavy chains and two pairs of light chains: the essential light chains (ELC) and the regulatory light chains (RLC). Each myosin II heavy chain is composed of an N-terminal motor domain containing ATP- and actin-binding sites, a neck domain (lever arm) that binds ELC and RLC, α-helical rod domain and C-terminal tail. The two myosin II heavy chains dimerize via interactions between the rod domains, together forming an α-helical coiled coil. (b) Modes of formation of myosin II dimers. Two myosin molecules can interact in either antiparallel (left) or parallel (right) fashion. The interaction is mediated by the rods and tails of individual myosin molecules and depends on the charge distribution along the rods. The shifts between individual myosin II molecules in the dimers can have only particular discrete values. (c) Three general types of myosin II filaments. Left: Myosin II filaments of striated muscles consist of several hundreds of myosin II molecules and accessory proteins (see text). The myosin heads are symmetrically located on the sides of bipolar filaments leaving a bare zone in the middle. The length of human striated muscle filaments is about 1600 nm. Middle: Myosin II filaments in non-muscle cells have a uniform length of 300 nm and consist of approximately 30 myosin hexameric molecules (for A and B heavy chain isoforms) or approximately 15 molecules (for C isoform). Right: Smooth muscle myosin II molecules usually form long side-polar (face-polar) filaments lacking the bare zone. (Online version in colour.)

Muscle tissue is highly contractile and produces a wide range of active forces in order to move the skeleton of all bilaterian animals. The basic contractile unit of cross-striated body muscles is known as the sarcomere [4,8,9]. Each stereotyped sarcomere is bordered by two Z-discs, which anchor the barbed (plus) ends of two arrays of highly organized polar actin (thin) filaments, facing in opposite directions (figure 2). This organization ensures that the linear actin (thin) filaments face with their pointed (minus) ends towards the centre of the sarcomere, where the bipolar myosin (thick) filaments are located. Each Z-disc contains many accessory proteins: most importantly, actin filament crosslinker α-actinin, α-actinin binding ZASP family proteins (ZASP stands for ‘Z-band alternatively spliced PDZ motif-containing protein’), actin filament capping cap-Z protein [8,14], as well as formins [15–17]. These proteins crosslink, cap, or promote polymerization of actin filaments. The Z-disc also anchors the N-terminus of the largest human protein named titin, which extends with its C-terminus all the way to the M-line in the middle of the sarcomere (see below) and thus stably links the actin with the myosin filaments (figure 2) [18]. In its fully extended state, titin is more than 1.5 µm long, which fuelled the idea of it being a molecular ruler that determines the stereotyped length of sarcomeres [19]. The sarcomeres in relaxed human skeletal muscles are between 3.0 and 3.4 µm long, depending on the muscle sub-type [20,21]. In mammalian cardiomyocytes, the sarcomeres are shorter (1.6–2.3 µm in length) [10,22,23]. In mature muscles, titin molecules span half of the sarcomere, such that 6 titin molecules connect each myosin filament with Z-discs at each side [24].

Figure 2. Striated muscle morphology. (a) Top: A cartoon depicting skeletal muscle fibres attached to tendons at both ends. Note the straightness of the muscle fibres indicating tension in the muscle-tendon system. Middle: Zoom-in showing the linear organization of striated myofibrils. Note the lateral alignment of the myofibrils resulting in muscle fibre cross-striation. Bottom: Further zoom-in depicting the organization of an individual sarcomere in striated muscle. Note the stereotyped length of sarcomeres (3.0–3.4 µm), which is generally orders of magnitude smaller than the total muscle fibre length. In cardiomyocytes, the sarcomere length is slightly smaller than in skeletal muscles (1.6–2.3 µm) [10]. This scheme was modified from Lemke & Schnorrer [11]. (b,c) Immuno-stainings of myofibrils in a cultured rat cardiomyocyte (b, MHC in red, myomesin in yellow) and in a Drosophila leg muscle (c, actin in red, Z-disc associated protein kettin [12] in green) displaying highly registered myofibrils resulting in cross-striated muscle patterns. (d) Not all Drosophila muscle types are cross-striated. Although the individual myofibrils of the Drosophila flight muscles (actin in red, kettin in green) are highly regular, they are not registered with their neighbouring myofibrils. See Spletter & Schnorrer [13] for a brief review of different muscle types. Scale bars correspond to 10 µm. Part b is courtesy of Dr. Yfat Yahalom-Ronen (Weizmann Institute of Science), c and d were acquired by Christiane Barz (Max Planck Institute of Biochemistry).

Thick filaments of striated muscles are very large, elongated molecular complexes of about 1600 nm in length and 30 nm in diameter (in vertebrates), containing about 300 muscle myosin II isoform hexamers [25] together with a number of accessory proteins [10]. The muscle myosin II hexamers are organized within a bipolar filament, with the heads present at both ends and a bare zone in the middle part of the filament (figure 1c). The exact structure of a thick filament is somewhat different for different species, but the general principles proposed more than 40 years ago [26] are still valid for all types of striated muscle thick filaments [4]. Recent cryo-electron tomography studies revealed the details of thick filament organization in insect flight muscle at almost atomic resolution [27]. The filaments are formed by interactions of the rod domains of the individual myosin heavy chain molecules. The central bare zone is built of anti-parallel myosin rods, being the base for a perfectly bipolar filament. The laterally protruding myosin motor heads are organized in a helical lattice [4,28]. It was established that only particular shifts between myosin rods interacting in antiparallel or parallel fashion are allowed (figure 1b) [26], which can be explained by an uneven distribution of positive and negative charges along the length of the rod [29,30]. These permitted configurations are thought to determine the organization of the entire filament [4,26].

In mature vertebrate sarcomeres, a prominent myosin accessory protein is myosin binding protein C (MyBP-C or C-protein) [31]. The C-terminal domains of MyBP-C run along the myosin filament surface, while the N-terminus extends toward neighbouring actin filaments [32] and is proposed to modulate the actomyosin interactions during muscle contractions [33]. In insects and other invertebrates, the thick filaments have a core made of the protein paramyosin, which is homologous to the rod part of the myosin II molecule [27]. Different versions of paramyosin are present in functionally different insect muscle types [34,35]. Another feature of myofibrils in the specialized stretch-activated insect indirect flight muscles is that individual fibrils do not align with the neighbours (the organization type is called ‘fibrillar’ muscle, figure 2c,d) [35]. However, the molecular mechanism or particular proteins responsible for the lack of alignment of the individual myofibrils in insect flight muscles remains to be identified.

At a larger scale, all myosin filaments in each sarcomere are organized into an ordered array aligned at a structure known as the M-line that connects the centre of the bare zones of the individual filaments (figure 2a) [8,9,36]. The M-line consists of several protein components among which the myomesin family elastic proteins, including M-protein, play a major role [36,37]. Another large sarcomeric protein that is found at the Z-disc and the M-line of mature vertebrate muscles is obscurin. It has significant similarities to the titin protein family [38]. However, in insects, obscurin is largely present at the M-line, where it is required for the symmetrical assembly and alignment of the thick filaments during flight muscle development [39].

At the extreme end are the very long and well-aligned thick filaments found in the catch muscle of certain molluscs (e.g. in anterior byssus retractor muscle (ABRM) muscle of Mytilus), in which the thick filaments are up to 25 µm long and stacked up to 75 µm in diameter [40]. This particular organization is believed to be caused by particularly high paramyosin concentrations present in the core of the thick filament [41]. These very long thick filaments in mollusc catch muscle contain a long region of acto-myosin overlap and thus enable a very high force production, three to four times greater than other muscles [42].

Actin filaments in the sarcomere are also associated with several important accessory proteins. One of them is nebulin, another giant protein, which in vertebrate muscle spans almost the entire length of each actin filament. Nebulin regulates actin dynamics and possibly determines thin filament length [43]. In addition, each actin filament is associated with two strands of tropomyosin molecules assembled head-to-tail along two helices of the actin filament [4,44]. The regulatory protein troponin is periodically located along actin/tropomyosin filament and regulates the position of the tropomyosin strands on actin filament in a Ca ++ -dependent manner. This ensures that an interaction of the myosin II motor heads with the actin filament, and thus productive force generation, is possible only when the Ca ++ concentration in the cytoplasm exceeds a threshold value [44]. This type of contractility regulation is specific for striated muscles but not for smooth muscles or non-muscle cells which contain several isoforms of tropomyosin but do not contain troponin.

Most insect body muscles and probably all vertebrate skeletal muscles as well as the heart are cross-striated fibres. This means that in addition to the precise organization within each sarcomere and the periodic organization of the sarcomeres into chains called myofibrils, even the myofibrils are eventually organized in registry, such that Z-bands of the neighbouring myofibrils are minimally shifted relative to each other (figure 2). In mature cardiomyocytes, the Z-bands of the neighbouring myofibrils are connected by intermediate filaments consisting of the protein desmin [8,45]. In addition, the Z-bands of the peripheral myofibrils are linked to the transmembrane integrin molecules and glycoproteins of the dystroglycan complex that together form structures called ‘costameres’, the regions where the cross-striated muscle fibres adhere laterally to the extracellular matrix (ECM) [45,46]. At least as important is the solid anchorage of the two terminal Z-disc of each myofibril at the long muscle ends to the ECM, a process that is also integrin mediated [47–49]. Among the integrin linker proteins that mediate the association of integrin with myofibrils at costameres or terminal Z-discs are talin, kindlin, vinculin, filamin and other molecules found also in cell matrix adhesions of other types [46,50,51].

How the ordered organization of myosin molecules and the entire structure of sarcomeres and myofibrils emerge during muscle differentiation is still poorly understood. The sarcomeres do not exist in isolation. They are connected at their symmetrical Z-discs forming myofibrils (figure 2). Each myofibril spans the entire muscle cell, mechanically connecting the two muscle-tendon attachment sites in the case of human skeletal or insect body muscles. In cardiac muscle, the myofibrils of the cardiomyocytes are connected with intercalated discs (fascia adherens), the junctions between the neighbouring cardiomyocytes [52]. In Drosophila, it was shown that mechanical tension is essential for myofibrillogenesis in vivo [49]. During muscle development myotubes connect both ends to tendons and build up mechanical tension. In turn, tension triggers the simultaneous self-organization of actin, myosin and titin complexes into immature myofibrils, which span from one muscle-tendon attachment site to the other [11,49]. These immature myofibrils then become contractile and their spontaneous twitchings are required for the lateral alignment of neighbouring myofibrils into the highly registered lateral organization of Z-discs and M-lines in cross-striated muscle [53]. These conclusions from developing insect muscles are also supported by data gained in the developing zebrafish body muscles, which showed that developmental contractions are required to form regular cross-striated sarcomeres [54].

Apart from actin and muscle myosin filaments, titin is essential to assemble sarcomeres and myofibrils [55–58]. As titin stably connects the Z-discs with the thick filaments and contains an endogenous mechanical spring domain, it is the major source of passive muscle elasticity in mature muscle fibres [43,59]. The extension of titin's spring occurs at the low pN range and is fully reversible [60,61]. Hence, it is very likely that forces across the titin molecule, which will eventually connect thin and thick filaments, play an important role in the myofibril assembly process [11].

Thus, it is becoming increasingly clear that forces generated by the myosin filaments play an important role in the myofibril self-organization process. A theory explaining one aspect of this process, the registered organization of myofibrils, will be discussed below.

3. Registry of myofibrils in striated muscle in culture and in vivo

The organization of myofibrils into registry occurs by lateral ordering of neighbouring myofibrils. This order is manifested by the alignment of the location of their Z-discs and M-lines (figure 2) [53,62].

Experimental observations of cross-striation in embryonic muscle cells cultured on synthetic, deformable substrates [63–66] suggest that the registry of neighbouring myofibrils is a mechanically regulated process. The extent of striation is maximal on optimally rigid substrates, which are neither too soft nor too rigid [63,64]. The observation of strong striations indicates the local registry of myofibrils. By systematically quantifying the registry while varying the rigidity of the underlying gel substrate, it was demonstrated that both striation and the contractile strains produced by beating cardiac muscle cells show a maximal value in a certain range of substrate stiffness [64,65]. Interestingly, the intermediate stiffness found for maximal striation and beating strength of about 5 kPa corresponds to the native stiffness of embryonic heart tissue. The requirement of proper mechanical forces during muscle development is consistent with aforementioned results that the twitchings of immature myofibrils are required for their lateral alignment [53].

This dependence of muscle structure and function on the stiffness of the environment and the forces produced by the muscle can be understood in terms of elastic interactions between fibrils by deformations of the underlying substrate induced by actomyosin contractile forces. The theoretical treatment of the properties and interactions of active force dipoles [67] provides a coarse-grained model for a cell's cytoskeletal contractility. Such ideas have been applied to quantitatively measure the mechanical response of single cells [63,68,69] or cytoskeletal elements in various situations [70]. Examples include the orientation of fibroblasts under an external stretch [71] or the substrate rigidity-dependent mutual orientation of stress fibres of mesenchymal stem cells [72]. In the case of muscle, each myofibril is modelled as a periodic array of contractile, equal and opposite forces of each of the myosin II heads these are termed force dipoles (figure 3a,b) by analogy with electrical dipoles that are equal and opposite charges separated by a finite distance. (We stress, however, that the force dipoles are only analogous to electrical dipoles and not identical with them even the mathematical properties of the two are different since charge is a scalar (direction-independent) quantity, while force is a vector (direction-dependent) quantity below we denote vectors by boldface and their components by an index.) The force dipoles deform the elastic substrate via the coupling of the contractile acto-myosin to the crosslinkers (Z-discs) these forces are then exerted on the substrate at the costamere adhesion sites of the Z-discs [62].

Figure 3. Illustration of striated muscle myofibrils interacting through deformations of soft substrate. (a) View of whole muscle cell cultured on an elastic substrate. Three myofibrils that span the cell are shown, with dark squares indicating the position of the myosin filament. Z-discs are not shown. Each end of a myofibril is anchored to a grey rectangle symbolizing a muscle-tendon attachment site in the case of human skeletal or insect body muscles, or an intercalated disc connecting neighbouring cardiomyocytes in the case of heart muscle. (b) Each myofibril, a series of sarcomeres, is mechanically coupled to the elastic substrate at the Z-discs through the transmembrane integrin-containing adhesive complexes known as costameres. (c) Schematic of deformation pattern transduced in the substrate by a single fibril. The light and dark regions represent regions where the substrate is expanded or contracted. The deformations decay with transverse distance away from the myofibril. (d) A pair of neighbouring fibrils represented as arrays of force dipoles. They are out of registry by a distance Δ and experience a substrate-mediated elastic force that tends to drive them towards mutual registry [62].

We now sketch the basic elements of the force dipole theory of deformation-induced interactions among distinct and well-separated acto-myosin units using a model in which the intervening elastic medium (or substrate) is represented as a linear elastic medium (i.e. where the stress is proportional to the strain). The displacement at a point r located on the surface (z = 0) of a semi-infinite linearly elastic medium (or substrate) caused by a force acting in the direction j = x,y at another location (chosen to be the origin) on the surface of this semi-infinite medium is given by Landau et al. [73],

Considering about 100 myosin filament heads in a sarcomere [25], each producing a contractile force of about 1 pN [75], a reasonable estimate for the contractile force per sarcomere is about 100 pN [62]. In this case, each force dipole is the pair of forces acting on the elastic substrate through the costameres at the Z-discs as shown in figure 3b. The spatial extent of such a dipole therefore corresponds to the size of a sarcomere. The transduction of these sarcomeric forces into the medium or substrate by adhesions coupling the two, induces a spatially, periodically patterned strain field within the substrate with alternating regions of compression and expansion shown respectively in figure 3c as dark and light regions. Given the strains induced by one myofibril, a sarcomere in the neighbouring myofibril located in an expanded region of the medium can then actively compress this region, restoring the medium closer to its undeformed state and thereby lowering the overall deformation energy of the elastic medium. This medium-based bias in the positioning of a neighbouring contractile unit (figure 3d), templates a registered configuration of neighbouring fibrils.

By including the molecular noise inherent in cells, such a theoretical approach can map the substrate rigidity dependence of registry onto that of the measured beating strains generated by cardiomyocytes [76]. The good agreement of theory with experiments relates the correlated beating of heart cells to the structural registry of the myofibrils, which in turn is regulated by their elastic environment. Finally, we comment on the possibility of registry driven by mechanical interactions in muscle tissue in vivo. Both the cell culture experiments [63] described in this section and in vivo studies of insect muscle [49] show that mechanical factors such as stiffness and tension are important in the registry or cross-striation of myofibrils. The proposed theoretical model is based on elastic deformations that arise from actively contractile myofibrils that are under tension. Further, immature muscle fibrils (premyofibrils) are found to be less ordered in both cases. This is consistent with a gradual progression towards order suggested by a force-driven mechanism.

4. Is there higher-order organization of myosin II filaments in smooth muscle cells?

Vertebrate smooth muscle cells, as evident from their nomenclature, have no apparent striations and therefore do not exhibit the highly ordered sarcomeric organization typical for striated muscle [4,77]. The homologues of Z-discs in smooth muscle are the dense bodies, which in analogy to Z-discs keep together the barbed ends of the actin filaments and are enriched in α-actinin [78]. As in the Z-discs of vertebrate striated muscle, the dense bodies are linked by desmin intermediate filaments, which are very abundant in smooth muscle [78,79]. Structures similar to dense bodies, known as dense plaques, are associated with the plasma membrane and mediate the adhesion of smooth muscle cells to the ECM. These structures are homologous to focal adhesions in non-muscle cells and contain characteristic proteins such as vinculin and integrin [80,81]. There is no homologue of the M-band in smooth muscle cells and the smooth muscle myosin filaments do not form ordered arrays, but rather interdigitate with the actin filaments out of registry. The organization and dynamics of the filaments formed by smooth muscle myosin molecules containing a smooth muscle-specific heavy chain isoform is generally different from that in striated muscle. In the majority of vertebrate smooth muscles, myosin filaments are not bipolar but contain myosin heads all along their length (so-called face-polar or side-polar filaments figure 1c) [82,83]. Such filaments or ribbons can be polymerized from purified smooth muscle myosin in vitro [83]. It is even claimed that the length of myosin II filaments in smooth muscle cells is not constant but varies within a broad range [84] even though there is no complete agreement on this subject [85,86].

Smooth muscle cells do not express troponin and, therefore, the Ca ++ -dependent regulation of the actin and myosin filament interactions is significantly different from that of striated muscle. Both ATPase activity of the smooth muscle myosin II and the ability to assemble into filaments are regulated by phosphorylation of the regulatory light chains (RLCs) [87–91]. A similar type of myosin II regulation occurs in non-muscle cells [3]. In cardiac striated muscle, RLC phosphorylation is constitutive and seems to be needed for the optimal contractility function [92].

Even though the degree of order in the organization of smooth muscle actin and myosin filaments is apparently less than in striated muscle, the degree of actomyosin order in the smooth muscles could be underestimated. In fact, some structures in smooth muscle could exhibit rather regular organization. For example, vinculin-containing dense plaques are quite regularly arranged in cultured smooth muscle cells [80]. Further regularity in the organization of the actomyosin cytoskeleton in smooth muscle cells will hopefully be revealed in future studies using super-resolution light microscopy.

5. Ordered arrays of myosin II filaments in non-muscle cells

The first indication that non-muscle cells can also assemble myosin filaments was obtained long ago using immuno-electron microscopy [93]. Higher quality images of such filaments were later provided by the technique of platinum replicas of permeabilized cells, from which the actin filaments were removed by incubation with the actin depolymerizing protein gelsolin [94,95]. Transmission electron microscopy of such replicas revealed that in cultured fibroblasts, numerous bipolar non-muscle myosin filaments of uniform length (approx. 300 nm) were present. These often appeared as groups of parallel filaments termed ribbons or stacks [94]. Later, such stacks were also observed in other cell types using similar techniques [95,96]. Biochemically, non-muscle isoforms of vertebrates are represented by three types of heavy chain, MHC-A (Myh9), -B (Myh10) and -C (Myh14), which interact with the same essential and regulatory light chains [3,97]. Each of the non-muscle myosin II types can assemble into filaments in vitro [98]. It was documented that myosin IIA and IIB molecules can co-assemble into a single filament [99–101], even though these isoforms usually segregate from each other resulting in anterior and posterior accumulation of myosin IIA and IIB, respectively, in polarized cells [101–103]. Interestingly, all isoforms of non-muscle myosin II can co-assemble with myosin 18A, which by itself can form antiparallel dimers, but not large filaments, and lacks the ATPase activity [104]. Thus, myosin 18A may regulate the assembly of other non-muscle myosin II filaments in vivo or mediate their interactions with some associated proteins [104].

Experiments with selective knockdown of myosin IIA or IIB revealed that myosin IIA tends to contribute more to overall cell contractility, while myosin IIB tends to be more involved in the regulation of cell front-rear polarity, guidance of cell migration, and matrix remodelling [3,105–109]. Partition between myosin IIA and IIB functions may also depend on cell type.

Regular fluorescence microscopy has insufficient resolution for the visualization of the 300 nm small myosin II filaments in non-muscle cells. However, even in the early immunofluorescence studies of myosin II localization, the obvious periodic distribution of myosin II entities (striations) was observed [110,111]. The recent introduction of super-resolution imaging, especially structured illumination microscopy (SIM) [112,113], has permitted the better visualization of myosin II filaments and super-structures formed by such filaments, as well as their dynamics in living cells [114–117].

The myosin filaments and filament stacks in the cells are mainly associated with bundles of actin filaments. The main myosin II-containing structures are the organized, parallel actin bundles or stress fibres typical of polarized fibroblast-like cells (figure 4). Related types of structures comprising myosin filament stacks are the so-called arcs or circumferential actin bundles that emerge at the periphery of spreading cells [114]. A recent study of the distribution of myosin IIA filaments revealed that in both stress fibres and arcs, myosin filaments are oriented parallel to the actin filaments and co-localize with the regions enriched by pointed (minus) ends of actin filaments associated with the protein tropomodulin (figure 4) [117]. This means that myosin filaments connect arrays of actin filaments with opposite polarity. Consistent with previous studies [93,94,111], along the length of the stress fibres or arcs, the myosin filaments are distributed in a periodic fashion, alternating with regions enriched in the actin filament crosslinking proteins α-actinin-1 and -4 [117]. The α-actinin enriched regions are also enriched with the barbed (plus) ends of actin filaments, which can incorporate monomeric (G) actin [117]. The sites with the most prominent G-actin incorporation are focal adhesions, molecular complexes connecting stress fibre ends with transmembrane integrin molecules [118].

Figure 4. Myosin IIA filaments and filament stacks in REF52 rat embryo fibroblasts. (a) Complementary distribution of myosin II filaments and α-actinin-enriched domains in REF52 cells co-expressing α-actinin-1-mCherry (blue) and RLC-GFP (yellow). The RLC images of the boxed regions shown at high magnification in the inset on the right display a putative myosin filament and a myosin filament stack, as indicated in the schematic. The images were taken using a Nikon 3D-SIM microscope. Scale bar, 5 µm. (b) Schematic depicting the organization of actin and myosin II filaments in the quasi-sarcomeric units of stress fibres or transverse arcs. A single ‘sarcomere’ is shown. Barbed ends of actin filaments (visualized by G-actin incorporation) are located in the α-actinin-enriched zones, while pointed ends (decorated by tropmodulin 3) overlap with myosin filament stacks.

Periodic organization of myosin II and α-actinin in stress fibres and circumferential arcs is somewhat similar to the organization of periodic sarcomeres in myofibrils (see above). However, there are several important differences between the structure of myofibrils compared with that of non-muscle stress fibres or arcs. First of all, the myosin II filaments of non-muscle cells are about fivefold shorter than thick filaments in striated muscles (300 nm versus 1600 nm). Further, the widths of the α-actinin-rich zones in stress fibres and centrally located arcs, unlike those in Z-discs, are not uniform, but show a broad length distribution (in a range between 300 and 1000 nm). In peripheral arcs, such zones are even wider than in central arcs and in stress fibres [117]. Moreover, while sarcomeric contraction causes a shortening of the distance between Z-discs, the contraction of stress fibres or arcs seems to involve a decrease in the lengths of the α-actinin-rich zones themselves [117].

Homologues of the M-line protein obscurin which hold together myosin filaments in myofibrils [39] were recently found in several types of non-muscle tissues and organs including brain, skin, kidney, liver, spleen, and lung [119] as well as in cultured epithelial cells [120]. However, direct evidence that obscurin or obscurin-like proteins mediate the link between myosin II filaments in non-muscle cells is missing. Moreover, even though there are data in the literature indicating the presence of titin-related molecules in non-muscle cells [121], and some authors attribute the high stress fibre elasticity to the presence of such molecules [122,123], the existence of the giant elastic titin-like filaments connecting the actin and myosin II filaments in non-muscle cells has not been confirmed. The elastic response of the stress fibres may rather be related to the function of the mechanoresponsive α-actinin-binding protein zyxin [124,125].

The deep structural differences between myosin filament organization in myofibrils as compared to the stress fibres and arcs of non-muscle cells are in line with the very different rates of filament turnover. In experiments with fluorescence recovery after photo-bleaching (FRAP), the recovery of the fluorescence of labelled non-muscle myosin light or heavy chains was observed in less than a minute suggesting rapid turnover of the myosin filaments [117], while the characteristic FRAP time for myosin filaments in muscle cells exceeded an hour [126].

Interestingly, besides stress fibres and circumferential arcs, registered stacks of myosin II filaments were also found in other domains of the actin cytoskeleton. SIM microscopy revealed such structures in the contractile ring of dividing human cells during cytokinesis [115]. Periodic distribution of myosin II clusters in the contractile ring was also noticed in other studies [127]. The architecture of the actomyosin contractile ring is not fully understood even though several models have been suggested [128–130]. The periodic distribution of myosin stacks in the contractile ring highlight an interesting similarity with circumferential actin bundles in interphase cells. Another type of actomyosin ring containing periodically distributed myosin II filament stacks is represented by adhesion belts of epithelial cells in the highly ordered epithelial layer of the organ of Corti [131]. Here, unlike other types of myosin filament structures in non-muscle cells, the myosin filaments are formed by myosin IIC isoform molecules. A peculiar feature of these adhesion belts is that they are organized in registry so that arrays of myosin filaments in one cell are located exactly opposite to the symmetrical array in a neighbouring adherent cell [131]. An ordered organization of myosin IIA filament clusters was also found in the cadherin-mediated junctions between human intestinal Caco-2 epithelial cells [132].

How do these highly organized myosin II structures form and what could be their functions in non-muscle cells? To answer the first question, the processes of formation and assembly of myosin filaments should be studied. Myosin II in non-muscle cells is regulated mainly by phosphorylation of RLCs. In particular, myosin II molecules assemble into filaments only if the RLCs are phosphorylated [3,133]. This phosphorylation is mediated by several enzymes, notably myosin light chain kinase (MLCK) and Rho kinase (ROCK). ROCK, in addition, phosphorylates and inactivates the myosin light chain phosphatase (MLCP), which normally antagonizes with RLC phosphorylation [3]. Thus, ROCK and its upstream activator, small G-protein Rho, act as master regulators of myosin II filament formation in non-muscle cells. Inhibition of ROCK by a specific drug Y27632 results in the total disassembly of myosin II filaments in the central part of the cell [117]. There could, however, be spatial differences in the regulation of RLC phosphorylation. At the cell periphery, formation of the new myosin filaments is less sensitive to Y27632 treatment [116], probably because in this region it depends more on MLCK than on ROCK [116,134].

The assembly of myosin II molecules into filaments depends also on several other regulatory events including phosphorylation of the myosin II heavy chain [135–137] and action of a myosin chaperone UNC-45a [138]. Signalling networks regulating these processes are not completely known. In general, the conditions under which assembly of myosin II molecules into filaments can occur are satisfied at the cell periphery. This probably includes the activity of small G-proteins of the Rho family and their downstream protein kinase targets, which are required for proper phosphorylation of the subunits of myosin molecules. The assembly of new myosin filaments was found at focal adhesions, where the active G-protein Rac1 together with protein kinase C (PKC) stimulate the phosphorylation of myosin heavy chain at serine 1916 [137]. The assembly of new myosin II filaments was also observed in other peripheral regions of the cell such as at the lamellipodium–lamellum interface [115–117,139]. It might be that formation of new myosin II filaments in vivo is facilitated by their interactions with newly formed actin bundles which appear as a result of debranching of the Arp2/3 network in lamellipodia. This process may lead to formation of the transverse arcs at the rear border of lamellipodia [140–142].

As soon as a myosin filament is formed, it appears to be associated with a thin bundle of actin filaments [116,117] and begins to move towards the cell centre. In the course of such motion, the filament is incorporated into pre-existing superstructures (stacks) formed by other filaments. This process appears to underlie the formation and maintenance of the observed myosin stacks. It was recently shown that in addition to this process (known as concatenation), myosin filaments can undergo splitting or partitioning, such that a single filament is transformed into two separated ‘daughter’ filaments [115,116]. Such partitioning of myosin filaments was shown to depend on actin filament polymerization as well as the activity of MLCK [116]. The exact mechanism of the myosin filament partitioning is yet to be established conclusively. Obviously, understanding this phenomenon would shed light on the mechanism of myosin filament self-organization in non-muscle cells.

While expansion of filaments based on their partitioning could be one of the mechanisms by which myosin filaments form stacks, Hu et al. demonstrated several events when the movement of existing myosin filaments into registry with another and that of two existing myosin stacks into registry with each other occurred [117]. The formation of myosin filament stacks is sometimes preceded by a movement of myosin filaments for a distance of about 1 µm. Such long-distance movement is consistent with an emergent force between the filaments acting over a long range. In vitro experiments with purified myosin IIB have demonstrated formation of complexes consisting of a small number of neighbouring filaments [98] but not the large micron-sized stacks where registry occurs over more than 10 filaments observed in cells [117].

To systematically investigate the molecular requirements for myosin filament stack formation, Hu et al. disrupted the myosin filaments by cell treatment with ROCK inhibitor Y27632 and observed the recovery of the filaments and the filament stacks under various conditions. It was shown that not only the mechanochemical activity of myosin, but also the dynamics and organization of the actin filaments, are important for stack formation. Inhibitors of formin-driven actin polymerization or depolymerization did not interfere with the recovery of myosin II filaments but blocked their assembly into stacks. The actin-associated proteins involved in the process of stack formation include the formin Fmnl3, known to be a potent activator of actin filament elongation [143]. Similarly, formin mDia1 was shown to be required for the ordered organization of myosin IIA filaments associated with cell–cell junctions [132]. Cofilin1, a known actin filament severing and depolymerizing factor [144], was also shown to be involved in myosin IIA filament stack formation [117]. Finally, the formation of these stacks depended on the actin cross linking protein α-actinin-4 and to a lesser degree on α-actinin-1 [117].

To explain the process of actin-dependent myosin filament stack formation in non-muscle cells and, in particular, the emergence of a force that acts between myosin filaments with a range much larger than a typical molecular scale, we adapted the theory developed for the explanation of registered organization of sarcomeres in striated muscle.

6. Interactions between myosin II filaments through the intervening actin network: a theoretical model

In this section, we summarize a theory that shows how myosin filaments can interact via their mutual deformations of the intervening elastic medium. As discussed above for cells cultured on soft deformable substrates, parallel organization of stress fibres [72], as well as registered organization of myofibrils in cardiomyocytes [76], can be explained by the effective interaction of cellular contractile elements through the elastic substrates. We now adapt these ideas to the cytoskeleton of cells cultured on rigid substrates where interactions between myosin filaments via deformations of the relatively disorganized, intervening cytoskeletal network that surrounds and connects them [117,145,146] may register the myosin filaments into stacks.

The theory is motivated by the following observations [117]: (i) Registered stacks develop dynamically and can involve micron scale displacements of the myosin II filaments over a timescale of minutes. (ii) Myosin filaments move in association with actin fibres and the registry requires actin dynamics the stacks disassemble when actin polymerization is inhibited. (iii) Registry is lost when contractility is blocked by contractility inhibitors such as blebbistatin. The sensitivity to actomyosin dynamics cannot be attributed to only molecular-level, local turnover of actin and myosin filaments, since these kinetics that take place over tens of seconds, as determined by FRAP experiments, are much faster than the time-scales of minutes required for registry to be established [117].

These considerations lead us to focus theoretically on the role of force transmission due to actomyosin contractility instead of direct, molecular associations of myosin filaments. Even in the absence of a soft deformable substrate, elastic interactions between neighbouring actomyosin elements on organized stress fibres may be mediated by the actomyosin-induced deformations of the intervening, disordered network of actin and possibly other cytoskeletal components (henceforth termed intervening network or ‘IVN’). The existence of such a network can be inferred from previous light and electron microscopy studies. In particular, an actin intervening network can be seen in the recent STORM images [145] as well as in earlier electron microscopy studies [146]. We hypothesize that myosin filaments are interacting with each other through such a network (figure 5a–c). An actomyosin unit on one stress fibre deforms the IVN. This deformation interacts mechanically with a contractile actomyosin unit on a neighbouring stress fibre and results in forces (transmitted by the IVN) that cause the myosin filaments on the two neighbouring stress fibres to be ‘pushed’ into registry with each other to form the stacks across stress fibres that are not in direct molecular contact with each other [117].

Figure 5. (a) Cartoon showing myosin filaments associated with neighbouring stress fibres with an intervening network of disordered actin filaments (IVN) between the neighbouring fibres. A similar picture applies to transverse arcs. As the sketch shows, the same actin filament in the IVN may bind two neighbouring stress fibres and thus physically connect and mechanically couple them. (b) Schematic geometry of interaction of two parallel force dipoles (P1 and P2) separated by a transverse distance d. Each dipole corresponds to the pair of contractile forces applied by the bipolar myosin filament at the points of adhesive contact with the IVN. The two dipoles representing the pair of myosin filaments are initially shifted with respect to each other (out of registry) by a distance Δ along the x-direction. The dipole P1 deforms the elastic medium (IVN) which thus exerts a force on the second dipole P2. The component of this force in the x-direction, Fx, tends to move P2 into registry with P1. (c) The same geometry as in (b) except now we consider a line of dipoles uniformly separated by a distance a as a model for an entire fibre. The deformation of the elastic medium by the line of dipoles causes a spatially periodic force on a single dipole, P2, a transverse distance d apart and shifted from registry by Δ as in (b). (d) Plot showing elastic medium-mediated force (for a Poisson's ratio ν = 0.5, incompressible medium) on an actomyosin unit (force dipole) by another dipole as shown in (b). We plot the force, Fx on a dipole transmitted through the elastic medium (the intervening IVN), as a function of the distance of registry mismatch Δ/d. A positive displacement of P1 by a distance Δ away from registry, results in a force that acts on P1 in the negative x-direction, demonstrating a tendency towards registry (Δ = 0) of the two dipoles. (e) Plot showing elastic medium-mediated force Fx (for a ν = 0.5) on an actomyosin unit (force dipole P2) by a line of dipoles (as a model of a stress fibre) as shown in (c). This shows that the elastic medium-mediated forces promote registry of P2 with the nearest dipole on the line of dipoles. (f) Plot of the transverse force Fy between two force dipoles oriented in and offset along the x-direction by a distance Δ, as shown (b), as a function of their transverse separation d. The negative values of Fy for a range of transverse separations implies that the two dipoles with parallel organization are acted upon by attractive elastic forces mediated by the medium. (g) Plot of the transverse force Fy on a force dipole by a line of dipoles (as a model of a stress fibre) as shown in (c). This shows that the elastic medium-mediated forces promote attraction of a single actomyosin unit towards an already formed actin bundle. All forces are rescaled here by suitable scales for force dipole, separation distance and elastic modulus, and can be taken to be in arbitrary units.

ATP hydrolysis-induced activity causes actin-bound myosin II motors to produce pairwise contractile forces on actin filaments of reverse polarity [75]. Such a ‘pinching’ force pattern can be modelled as a force dipole [67,70] of magnitude P which comprises a pair of equal and oppositely directed myosin forces (figure 5b,c), each of magnitude F, separated by a distance a, so that P = F a. Two such contractile actomyosin units located on neighbouring bundles can interact mechanically through their mutual deformations of the intervening elastic medium with which they are in contact. The force of one such unit results in an elastic stress and displacement of the IVN near a neighbouring actomyosin unit located on an adjacent actin bundle, which then can respond by translating or orienting in response to the deformation of the IVN caused by the first unit. The actin filament IVN between neighbouring actin bundles (figure 5) is a crosslinked gel and can be modelled as a continuous, deformable elastic medium at sufficiently short timescales of seconds [147] appropriate to the local force transmission in our case. The continuum description is valid for a dense IVN (i.e. as long as the pore size of the disordered network is small compared to the size of a force dipole), here an actomyosin unit comprising the myosin filament and associated actin filaments.

We now consider the elastic deformation of the IVN in the presence of two such force dipoles (figure 5b), P1 at the origin of coordinates and P2 at r= (Δ, d, 0), representing two neighbouring myosin filaments on two adjacent actin bundles (oriented in the x-direction) that are separated by a transverse distance (in the y-direction), d. The corresponding force in the x-direction exerted on the dipole P2 by the elastic IVN due to its deformation by forces exerted by the first dipole, P1, is proportional to P2 (and inversely proportional to the rigidity of the IVN, E) as well as to the derivative of the local stress field (which is calculated from the displacement in equation (3.1)) induced by P1 [74],

The force between a pair of dipoles given by equation (6.1) is plotted in figure 5d as a function of the distance Δ by which they are misregistered in their mutual positions in the x-direction. This shows that if Δ > 0, that is P2 is shifted away from P1 in one direction, then the force from the medium on P2 (see equation (6.1)), due to the deformations induced by P1, acts in the opposite direction, i.e. it pushes P2 in the negative x-direction, towards Δ = 0. This shows that the deformation-induced mechanical interactions between dipoles in this simple geometry tend to register them. The interaction of a dipole with a neighbouring periodic series of dipoles arranged in parallel (representing myosin filaments along a stress fibre or a transverse arc) results in a periodic force profile which moves it into registry with the neighbouring array (figure 5e). (The preferred positions for myosin filaments are those with zero force from the medium and are seen to correspond to spatial registry, Δ = 0.) The details behind this theory have been derived in connection with the registry of sarcomeres [62,70]. The deformation-induced force in the longitudinal direction (along the stress fibre) may explain the sliding of two different stacks of myosin filaments into mutual registry as observed by Hu et al. [117].

We can also calculate the force on P2 in the direction transverse to the parallel bundles (stress fibres), i.e. y-direction, due to the elastic deformation of the IVN by P1. The resulting expression for the transverse force is

The physical origin of these forces is the tendency of the passive, intervening elastic medium to minimize its deformations in response to the active, ATP-dependent contractile forces generated by the actomyosin dipoles. Placing a contractile dipole in a region where the medium is already expanded by the other dipoles reduces the overall medium deformation. The contractile forces exerted by the two dipoles deform the elastic IVN and are everywhere locally balanced by the elastic restoring forces of the IVN that resist mechanical deformations. However, this local equilibrium does not necessarily imply global mechanical equilibrium (minimal elastic energy) of the system of dipoles plus medium. In addition, the contacts of the actomyosin force dipoles with the IVN are subject to dynamically changing, stochastic forces due to the thermal and molecular fluctuations within the actin IVN as well as deterministic forces that arise from the tendency of the elastic medium to resist deformation. The stochastic forces allow for local rearrangements of the dipoles while the deterministic forces eventually—at times significantly longer than those related to molecular turnover—result in translations of the dipoles in a manner that reduces the global deformation of the medium (here, the IVN). This occurs when the dipoles in neighbouring bundles are in registry, as we showed above. The recently demonstrated load-dependent changes in binding lifetimes of myosin to actin [148] may in principle affect the extent of myosin stacking.

A reason why the myosin stacking is lost when actin polymerization/depolymerization is suppressed might be related to the necessity for translocation of myosin filaments along the actin bundles/networks. In order for these actomyosin elements to move into registry and thus reach mechanical equilibrium with their neighbouring bundles, actin filaments in a bundle must depolymerize at one end of the myosin filament and polymerize at the other unless that happens, the myosin filaments are sterically hindered from moving and registry cannot occur. Since actin polymerization/depolymerization is also stochastic, this suggests that the time scales required for net motion in the direction required for stacking may be quite long, which is consistent with the observed stacking times of about tens of minutes [117] for myosins that must move about 1 µm into registry. Specifically, actin depolymerization proceeds at about 1 nm s −1 [75], so that for this to occur all along the acto-myosin unit of length about 1 µm will require thousands of seconds (tens of minutes).

In contrast to the deformation-induced forces described above, processes such as the expansion of myosin filaments by splitting or the preferential assembly of multiple filaments in close proximity to each other [115,116] might indeed explain the initial formation of filament stacks. However, as already discussed above, it is not at all clear that such an initial configuration can be maintained despite a variety of disordering forces in the cytoskeleton—random molecular-scale and motor-driven noise as well as fluctuations of the actin through polymerization/depolymerization processes. The interactions between myosin filaments through IVN provides a plausible mechanism for the observed long-range movements of myosin filaments to assemble stacks far from their point of initial formation [117] it is also robust to the presence of noise, which does, however, control the probability that the myosin position relative to those in a neighbouring stress fiber is controlled by the force. Local expansion events [115,116] could be acting in concert with a long-ranged mechanism such as those we describe here. Further experiments will be necessary to unravel the effects of these different mechanisms. Genetic perturbations that vary the degree of crosslinking and density of the intervening network could be a test of the effect of mechanical interactions. Another possible evidence would be the dependence of myosin filament stacking on substrate stiffness in cells cultured on compliant substrates.

7. Concluding remarks

Here we reviewed some aspects about the emergence of highly ordered organization of myosin II filaments in striated muscle and non-muscle cells. Cross-striated muscles show the highest-level of order—crystal-like organization of the myosin, actin and titin filaments. Recent studies showed that in non-muscle cells, myosin II filaments also form relatively well-ordered structures, such as myosin filament stacks associated with actin bundles in interphase cells, the contractile ring in dividing cells, or adhesion belts in cells that form cadherin mediated cell–cell junctions. The mechanisms involved in the establishment and maintenance of such organization are diverse and include interactions of myosin II molecules with each other, with the actin filaments, and with a variety of accessory proteins that regulate actin and myosin filament assembly and crosslinking. In this review, we focused on a basic process that may be a common denominator of these self-organized structures: long-range mechanical forces between actively contractile units (force dipoles) embedded in an elastic medium.

Previous studies demonstrated that the formation of striations corresponding to registered sarcomeric structures in newly formed myotubes [63] and embryonic cardiomyocytes [64] is better established on substrates of an optimal, intermediate rigidity rather than on those that are too soft or too rigid. This is corroborated by in vivo experiments demonstrating that mechanical forces are required throughout the muscle fibre in order to assemble the regular myofibrils during early muscle development [49]. The observation of an optimal substrate stiffness was successfully explained by a theory that assumed sarcomeric contractile elements (force dipoles) effectively interact with each other via the substrate to which they are connected via the costamere adhesions.

Non-muscle cells also form ordered actomyosin bundle arrays in a matrix rigidity-dependent manner [72,149,150]. However, well-ordered myosin stacks are formed even in cells attached to very stiff substrates (glass coverslips), which cannot mediate elastic interactions between contractile elements. Therefore, here we have extended the theory considering individual non-muscle myosin II filaments (instead of sarcomeres) as force dipoles and the disordered, intracellular intervening actin network (instead of the substrate) as the force-transmitting, elastic medium. This theory qualitatively explains the formation of registered organization of the myosin II filaments in non-muscle cells.

Contemporary models of myofibrillogenesis suggest that myofibrils are formed from precursors, the periodic actomyosin structures that comprise non-muscle myosin II filament arrays [151,152]. Formation of such precursor structures may proceed, similarly to formation of myosin II stacks in non-muscle cells, via interactions of myosin filaments with the intervening actin network. Thus, self-organization of myosin II filaments driven by effective, attractive forces between them (due to their mutual deformations of the intervening actin network) may be a general mechanism that drives the organization of actomyosin systems.

This mechanism can operate in concert with other mechanisms based on direct or indirect molecular interactions between myosin filaments. While the accessory molecules responsible for the formation of thick filaments and their arrays in cross-striated muscle are known, our knowledge of molecules with similar functions in non-muscle cells is limited. Factors regulating assembly and disassembly of non-muscle myosin filaments and, in particular, the duplication or partitioning of such filaments [115,116] obviously require further studies.

Finally, the processes of formation of myosin stacks could underlie the global self-organization of the entire actomyosin cytoskeleton in cells. While the formation of the actin filament arrays such as stress fibres or transverse arcs obviously depends on actin polymerization and crosslinking, it is well known that inhibition of the myosin filament assembly and/or activity results in disintegration of these arrays. We hypothesize that mechanical forces between myosin filaments play an important role in the organization and maintenance of these structures. An interesting question is how the ordered organization of actomyosin bundles affects the overall cell contractility. In cardiac muscle, more registered myofibrils tend to beat in concert [76] generating larger beating forces, but further study is required on the relationship between stacking of myosin filaments and contractility in non-muscle cells. The functions of ordered organization of myosin II filaments in the contractile ring [115] and adhesion belt [131,132] are also not clear and provide an interesting avenue for future research. All in all, the discovery of a global ordered organization of myosin II filaments in non-muscle cells opens a new page that challenges our experimental and theoretical understanding of the non-muscle actomyosin cytoskeleton.

Data Accessibility

This article has no additional data.

Competing interests

We declare we have no competing interests.

Funding

F.S. acknowledges support from the CNRS, the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant 310939, the excellence initiative Aix-Marseille University AMIDEX, the ANR-ACHN and the LabEX-INFORM. S.S. acknowledges funding from the Israel Science Foundation, the US-Israel Binational Science Foundation, and the Villalon and Perlman family foundations. S.S. and K.D. gratefully acknowledge previous collaborations with B. Friedrich, D. Discher and S. Majkut. K.D. acknowledges support from the University of Chicago Materials Research Science and Engineering Center funded by the National Science Foundation under award number DMR-1420709. A.D.B. acknowledges support from the National Research Foundation, Prime Minister’s Office, Singapore and the Ministry of Education under the Research Centres of Excellence programme (ref no. R-714-006-006-271). A.D.B. is also participating in EU Horizon 2020 InCeM project under the Marie Sklodowska-Curie grant no. 642866, and Maimonides program on Mechano-transduction and Integrative Biology supported by Ministry of Science, Technology & Space, Israel, and the Ministry of Foreign Affairs and the Ministry of Higher Education and Research of France.


↵ ¶ To whom correspondence should be addressed at: Department of Biology, San Diego State University, 5500 Campanile Drive, Life Sciences 371, San Diego, CA 92182-4614. E-mail: sbernstsunstroke.sdsu.edu .

↵ † Present address: Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10-CRC, Room 5-3288, 10 Center Drive, Bethesda, MD 20892.

↵ § Present address: Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: IFM, indirect flight muscle pCa, -log10 calcium concentration pm, paramyosin transgene pm S10A , pm with Ser-10 replaced by Ala pm S18A , pm with Ser-18 replaced by Ala pm S-A3 , pm with Ser-10, -13, and -18 replaced by Ala pm S-A4 , pm with Ser-9, -10, -13, and -18 replaced by Ala prm 1 , paramyosin functional null mutant.