Actin/Myosin, Muscle and Cell Motility

Structure and Function of myosin

• MT is responsible for movement of biomolecules and other cellular components over long distances (uses kinesin and dynein motors)

• Once MT ends, the cargo switches over to microfilaments for local movements

  • Actin microfilament-rich cell periphery which uses myosin motors

• Myosin motor proteins functions in cellular events

  • Muscle contraction

  • Cell movement

  • Phagocytosis

  • Vesicles transport

About Myosin Family

• Motor proteins that move on actin

• Large gamily of actin-based motors with different roles

• 17 different classes of myosins

• 40 different myosin

• Its grouped into unconventional (type 1, 3-17)

• Conventional is type 2 myosin

Myosin 1

• Motion of membrane along MFs and endocytosis

• 10-14nm step size

Myosin 2

• Slides MFs in muscles; contractile events like cytokinesis, cell migrati

• 2 globular domain of heavy chain which acts has actin binding domain

• Has regulatory light chain

• Essential light chain

• 2 heavy chain tails coiled around each other

• 5-10nm step size

Myosin 5

• Vesicle positioning and trafficking/organelle transport

• It is an efficient motor that moves "hand over hand" along actin MF

• Has 2 globular domains ofthe heavy chain for actin binding domain

• Regulatory and essential light chain

• Cargo binding site

• 36nm step size

y

Myosin 6

• Endocytosis; moves toward minus ends of MFs

• 2 globular domains

Myosin 7

Base of stereocilia in inner ear

Myosin 10

Tips of filopodia

Myosin 15

Tips of stereocilia in inner ear

Myosin 5 vs Kinesin

Similarities

• Similar amount of force and high efficient

• Moves towards + end of cytoskeletal component

• Uses ATP hydrolysis to change shape and exert force

• 1 head remains associated making them processive

• Similar shapes

 

Differences

• Myosin move on MF

• Kinesin move on Actin

• Myosin takes bigger steps

  • Kinesin takes 8nm steps

  • Myosin 5 takes 36nm steps due to MF have a helical shape

Myosin 2, Skeletal Musle

 first motor protein identified and best understoode

• 2 heavy chains (globular head, neck-region and a rod-like tail and 4 light chains (essential and regulatory)

  • S1 segment has an ATP binding site in the head

• Important for muscle contraction, cytokinesis, cell migration and focal adhesions

• It is able to form bipolar filaments with heads pointed away from the centre

  • Non of the unconventional myosin can form filaments

 

Levels of organization in muscles

• Made of muscle bundles surrounded by a sheath

  • Muscle bundles are made of a collection of muscle fibers

    • Fibers made of myofibrils

Muscle Fiber/Muscle cells

• Long thin multinucleated cells that’s specialized for contractile function

• Each cell has many myofibrils that’s 1-2 micrometers in diameter and extend the entire length of the cell

Myofibrils

Myofibrils are subdivided along its length into repeated units of sarcomeres

Sarcromers

• made of bundles of myosin 2 bipolar filaments and actin microfilaments

• The fundamental contractile unit

• Spends from 1 Z line to another Z line (Z line is the end of the actin filament)

• Positive end is at the Z line, end of actin

• Minus end is in the centre of the sarcromere

Thick Filaments

• Staggered array of myosin 2

• Myosin heads allow them to form cross-bridges between thick and thin filaments for muscle contraction

Thin Filaments

• Actin filaments with 2 other associated proteins

  • Tropomyosin

    • A long rod-like molecules which fits in grooves along the sides of F-actin helix

  • Troponin

    • Made of 3 polypeptides

      • TnT: tropomyosin binding

      • TnC: Calcium binding

      • TnI: actin binding

Other Proteins Associates in Sarcomeres

• Tropomodulin

  • Caps the minus ends of filaments to maintain stability

• Cap Z

  • Caps the positive end and attaches MF to the Z line

• Nebulin

  • Stabilize and binds the thin filament to Z line

• Alpha-actinin

  • Cross-link Z line to MF, keeps thin filament in parallel arrays

• Myomesin

  • Bundles of myosin molecules

  • 3rd most abundant protein

• Titin

  • Attaches thick filaments to the Z line and keeps thick filament in correct position relative to thin during contraction

  • Biggest protein

Sliding Filament Model

• Contraction is due to thin filaments sliding past the thick filaments

• Myosin walks towards the plus end of the actin MF which is towards the Z line

• Walking towards the Z line pulls acitn filaments towards the centre of the sarcomere

 

COntractile Cycle

1. ATP binds to myosin and it dissociates from actin

2. ATP hydrolyzes and puts myosin in energized state

3. Energized myosin binds to actin

4. Phosphate is released and changes the conformation of myosin and pulls actin toward the centre

   1. When phosphate is released it initiates the power stroke

5. ADP is released but myosin still remains bound to actin

6. ATP binds to myosin again to dissociate

 

Power stroke results in 10nM of movement

Purpose of Calcium in Muscle contraction

Neuronal signaling results in a rapid rise in Intracellular calcium in muscle cells

• Nerve impulse will cause an opening of a voltage gated calcium channel in the T-tubule

• This simulate the opening of calcium released in sarcoplasmic reticulum which leads to a rise in calcium in the cytoplasm by facilitated passive diffusion

  • Calcium is higher in SER and EC

• A sudden rise in cytosolic calcium will initiate a muscle contraction (Calcium spark)

• Contraction stops when calcium is actively transported (P2-type ATPases) back in SR

 

• Calcium will bind to troponin at the TnC which causes a change in conformation of troponin which will pull tropomyosin off the myosin binding site

Actin and myosin 2 form contractile bundles in non muscle cells

• Adherens belt

• Stress fibers

• Contractile ring

How do cells move

Actin-based motility in nonmuscle cells: cell crawling

When a signal is received a cell crawl towards it

1. Extension of a protrusion at the leading edge (lamellipodia and filopodia)

2. Attachment of the protrusion to the substrate

3. Generation of tension (contractile activity pulls the cell forward)

4. The tail or trailing edge releases its attachments and retracts

lamellipodium

A thin sheet of cytoplas

• Loose networks of actin that form a dendritic which is a tree-like networking pushing on the membrane

• A complex of actin-related proteins Arp2/3 complex nucleates new branches on the side of the filaments

• Arp2/3 branching is activated by a family of proteins that includes (WASP) and WAVE

 

filopodium

A thin pointed protrusion

Stress Fibers

Cells adhere tightly to the underlying substratum by organelle bundle

Chemotaxis

• Directional movement in response to a graded chemical stimulus

Directional migration occurs through the formation of protrusion predominantly one side of a cell

Diffusible molecules can act as cue for directional migration

Chemoattractants

Cell move towards a higher concentration of diffusible molecules

Chemorepellents

Cell moves toward a lower concentration of diffusible molecules