BIOL 331 - Actin, Cell Motility, and Muscle Contractions (Lectures 15-17)

actin

the most abundant protein in most cells, filaments are composed of globular subunits (G-actin), have polarity

involved in cellular motile processes (eg. movement of vesicles, phagocytosis, cytokinesis), provides structural support (shape of cells), support for cellular projections

barbed end

positive (+) end

pointed end

negative (-) end

actin polarity

barbed and pointed ends, individual G-actin monomers have directionality and are added to the filament in a particular orientation, filament is a double-stranded helix with parallel strands, ends are named based on binding of a fragment of the myosin motor protein (S1)

ATP-actin

incorporated into the filament, actin hydrolyzes it to ADP

added to both ends but with faster addition at the barbed (+) end

critical concentrations

minimal concentration of available ATP-actin required to elongate

barbed and pointed ends have different values, barbed (+) end is much lower (0.1 uM), pointed end is (1.5 uM)

actin filament assembly and disassembly

1. preformed actin filament (seed) in the presence of ATP-actin

2. at high ATP-actin concentrations, it is added to both ends

3. concentration reaches the critical concentration of the pointed end, addition stops at the pointed end

4. loss of subunits occurs at the pointed end because ADP-actin dissociates more readily than ATP-actin but addition continues at the barbed end

5. relative position of subunits is continually moving (treadmilling)

steady state

when the rate of addition at one end is the same as the rate of loss at the other end (occurs at about 0.3 uM available ATP-actin)

conventional myosin type II

moves towards the + end (barbed end) of actin, involved in muscle contraction

myosin II motor head

binds the actin filament, binds and hydrolyzes ATP, conserved sequences

myosin II neck

or lever arm, moves during the power stroke

myosin II tail

intertwining of two heavy chains, allows the formation of filaments of myosin

unconventional myosin type V

moves processively along actin filaments towards the +/barbed end, moves in a hand-over-hand motion, long necks act as swinging arms, can take very large steps (36 nm - the length of one helical turn of an actin filament)

can associate with vesicles and organelles - eg. bound to a vesicle via adaptors (Rab27a)

some vesicles contain both microtubule motors and actin filament motors

• movement over long distances occurs mostly on microtubbules

• local movement in the outskirts of the cells - actin filaments

myosin type II filaments

tails allow the protein to form filaments, tails point towards the center and heads point towards the outside

bipolar - motor domains are oriented at opposite filament ends

= thick filaments

muscle fiber

a skeletal muscle cell, skeletal muscles usually anchored to bone, contains multiple nuclei and hundreds of myofibrils

myofibrils

composed of repeating contractile units called sarcomeres

sarcomeres

contractile unit with a characteristic banding pattern (Z line, M line, I bands, H zone, A band)

Z line

contains proteins important for sarcomere structure stability

M line

dark staining in the center of the sarcomere, contains anchoring proteins

I band

contains only thin filaments (actin), decreases in length with the muscle contracts

H zone

contains only thick filaments (myosin), decreases in length with the muscle contracts

A band

dark staining (overlap of thick and thin filaments), also includes the H zone

length does not change during muscle contraction

muscle contraction

thin filaments slide towards the centre of the sarcomere

I band and H zone decrease in length, A band stays the same

molecular basis - myosin II heads in a thick filament bind to six surrounding actin filaments, myosin II is non-processive (only in contact with actin for a fraction of the time, myosin heads are not synchronized)

actin-myosin contraction cycle

1. ATP binds to myosin head and myosin dissociates from actin

2. ATP hydrolysis, ADP and Pi remain bound to myosin

3. energized myosin binds actin

4. release of phosphate triggers conformational change - power stroke - actin moves towards the centre of the sarcomere

5. ADP is released - actin and myosin remain attached until a new ATP binds

neuromuscular junction

point of contact between motor neuron and muscle fiber, site of transmission of the nerve impulse, stimulated by acetylcholine

muscle fibers within a motor unit are stimulated simultaneously by a single motor neuron

excitation-contraction coupling

transverse tubules propagate impulse to interior of cell, sarcoplasmic reticulum stores Ca2+ in lumen

arrival of action potential at the SR opens Ca2+ channels, release Ca2+ into the cytoplasm

transverse tubules

T tubules, membrane folds that propagate an impulse to the interior of a muscle cell

sarcoplasmic reticulum

special smooth ER in muscle cells, stores Ca2+ in lumen (pumped in from the cytosol)

opens Ca2+ channels in response to action potential, releases Ca2+ into cytoplasm

tropomyosin

rod shaped proteins in thin filaments, position is controlled by troponin

troponin

globular proteins in thin filaments, controls the position of tropomyosin

absence of Ca2+

thin filaments - tropomyosin blocks myosin-binding sites on actin

presence of Ca2+

thin filaments - Ca2+ binds troponin which moves tropomyosin, exposing the myosin-binding site on actin

cell cortex

actin network on the inner face of the plasma membrane, capable of dynamic remodelling

enable cells to crawl/move, enable phagocytosis, cellular constriction during cell division

actin-binding proteins

regulate the assembly, disassembly, and rearragements of actin networks, more than 100 different proteins (8 main categories)

8 categories of actin-binding proteins

1. filament nucleating

2. monomer-sequestering

3. end-blocking (capping)

4. monomer polymerizing

5. depolymerizing

6. cross-linking & bundling

7. filament-severing

8. membrane-binding

filament nucleating actin proteins

slowest step in the formation of an actin filament, proteins can enhance the rate at which actin filaments are formed, eg. Arp 2/3 complex, formins

Arp2/3 complex

filament nucleating actin-binding proteins, binds to the side of an existing filament (creates branches), remains at the pointed end (-) of the new branch, similar structure to actin monomers

formins

filament nucleating actin-binding proteins, generate unbranched filaments (de novo), stay associated with the barbed (+) end, promote rapid elongation of filaments

monomer-sequestering actin proteins

bind to actin-ATP monomers to prevent them from being added to the elongating filament, able to modulate the available monomer pool in certain regions at certain times, eg. thymosins

end-blocking (capping) actin proteins

regulate the length of actin filaments, bind at either end (+ or -)

monomer polymerizing actin proteins

binds to actin monomers to promote growth of actin filaments, promotes replacement of ADP with ATP on the actin monomers, eg. profilin

depolymerizing actin proteins

bind to actin-ADP at the pointed (-) end to promote depolymerization so cell can relocate monomers quickly, eg. cofilin

cross-linking and bundling actin proteins

multiple actin-binding sites, allowing them to alter the 3d organization of the actin filament network, eg. filamin (cross-linking), villin and fimbrin (bundling)

filament severing actin proteins

break an existing filament in two, eg. gelsolin and cofilin

membrane binding actin proteins

actin filaments linked to the plasma membrane, enabling the plasma membrane to protrude outward (cell locomotion) or inwards (phagocytosis), eg. spectrin in RBCs

profilin

a monomer polymerizing actin protein

cofilin

a depolymerizing actin protein

cell motility

1. movement is initiated by a protrusion of the cell in the direction of movement (lamellipodium)

2. a portion of the protrusion anchors to the surface below

3. the bulk of the cell is pulled towards the front, over the adhesive contacts

4. adhesive contacts break, causing retraction of the trailing edge (tail)

lamellipodium

the leading edge of a moving cell that extends over the surface, broad and flat, a dynamic actin network involved in formation

lamellipodium formation

1. stimulus is received at cell surface

2. Arp2/3 complex at the site of stimulation gets activated

3. Arp2/3 binds the side of an existing filament

4. ATP-actin monomers bind to the Arp2/3 complex, forming a new actin branch - polymerization promoted by profilin

5. additional Arp2/3 complexes can bind to the sides of the new filaments, forming additional branches, older filaments are capped at their barbed ends (+)

6. newer filaments continue to grow at the barbed end (fast growing end), pushing the membrane of the lamellipodium outward - older capped filaments undergo disassembly promoted by cofilin

traction forces

when the cell grips the surface (at adhesion points called focal adhesions)

focal adhesions

structures in the cell membrane where integrin proteins connect to actin

integrin proteins

transmembrane proteins that mediate the interaction between actin and extracellular components

contraction forces

pull the bulk of the cell forward, myosin found near the rear of the lamellipodium