Microfilaments, Muscle and Myosins Notes

Microfilaments, Muscle and Myosins

Roles of Actin Networks at the Cell Cortex

Actin networks play crucial roles at the cell cortex.

Mechanism of Actin Filament Nucleation and Disassembly

The regulation of actin filament nucleation and disassembly is essential for cellular processes.

Actin Binding Proteins and Their Function

Various actin-binding proteins modulate actin filament dynamics and function.

Myosin Motors: Structure and Mechanism of Action

Myosin motors are responsible for movement along actin filaments.

Myosin II and Skeletal Muscle Contraction

Myosin II plays a critical role in skeletal muscle contraction.

Myosin V Motor Movement and Function

Myosin V is involved in the transport of vesicles and organelles.

Overview of Cytoskeletal Filaments

Microfilaments:
- Diameter: 7-9 nm
- Subunit: Actin
Microtubules:
- Diameter: $\sim 25$ nm
- Subunit: $\alpha \beta$ -Tubulin dimer
Intermediate Filaments:
- Diameter: 10 nm
- Subunit: Various

Comparison of Microfilaments, Microtubules, and Intermediate Filaments

Feature	Microfilaments (Actin)	Microtubules ( $\alpha \beta$ -Tubulin)	Intermediate Filaments (IF)
Nucleotide Binding	Actin binds ATP	$\alpha \beta$ -tubulin binds GTP	IF subunits don't bind a nucleotide
Mechanical Properties	Rigid gels, networks, linear bundles	Rigid, not easily bent	Great tensile strength
Assembly Regulation	Regulated from many locations	Regulated from few locations	Assembled onto pre-existing filaments
Dynamics	Highly dynamic	Highly dynamic	Less dynamic
Polarity	Polarized	Polarized	Unpolarized
Tracks For	Myosins	Kinesins and dyneins	No motors
Functions	Contractile machinery, cell cortex network	Organelle organization and transport	Cell and tissue integrity

Localization of Microfilaments, Microtubules, and Intermediate Filaments

Microfilaments:
- Microvilli
- Cell junctions
- Leading edge
- Cell cortex
- Adherens belt
- Filopodia
- Lamellipodium
- Stress fibers
- Contractile ring
Microtubules:
- Basolateral domain
Intermediate filaments:
- Apical domain
- Extracellular matrix

Actin Structure and Assembly

Actin is a globular protein (G-actin) with a central cleft that binds ATP.
Actin filaments (F-actin) consist of two strands of subunits arranged in a helix.
A repeating unit of 28 subunits (14 per strand) spans 72 nm.
The filament has a clockwise helical twist with symmetry every 36 nm.
The ATP-binding cleft faces the minus end, giving the filament polarity.
G-actin monomers polymerize to form F-actin filaments.
The rate of ATP-G-actin addition is 10 times faster at the plus (+) end than the minus (-) end. Dissociation rates are similar at both ends.
ATP bound to actin is hydrolyzed to ADP-Pi, and Pi is slowly released, resulting in filaments containing ATP-actin, ADP-Pi-actin, and ADP-actin.
ATP-actin preferentially adds to the plus end, while ADP-actin disassembles at the minus end, causing treadmilling.
$Cc = k<em>{off} / k</em>{on}$ , where Cc is the critical concentration.

Compounds Affecting Actin Filament Stability

Cytochalasin D: Binds actin monomers and prevents polymerization.
Latrunculin A: Binds actin monomers and prevents polymerization.
Phalloidin: Binds and stabilizes actin filaments; can be fluorescently labeled for staining.

Regulation of Actin Polymerization

G-actin concentration is 1000 times greater than the critical concentration in cells, but most G-actin is bound to Thymosin- $\beta 4$ , preventing its incorporation into filaments.
Thymosin- $\beta 4$ inhibits actin filament assembly by sequestering actin monomers.
Profilin binds G-actin, promoting ATP exchange and incorporation into filaments. It is located at the plasma membrane bound to PIP2.

Cofilin and Actin Filament Disassembly

Cofilin (Actin de-stabilizing factor or ADF) binds to ADP-actin on the sides of the filament, inducing fragmentation.
Cofilin replenishes the pool of free ADP-actin, which is then recharged by profilin.
Dissociation of ADP-actin filaments occurs in two steps: cofilin chops off 18-20mers, and Aip1 (actin-interacting protein 1) breaks these into monomers.

Actin-Binding Proteins: The Toolbox

Monomer Binding Proteins:
- Profilin, Thymosin $\beta 4$
Filament Severing Proteins:
- Cofilin
Filament Capping Proteins:
- Plus end: CapZ
- Minus end: Tropomodulin
Filament Nucleators:
- Straight: Formin
- Branched: Arp2/Arp3 complex
Bundling Proteins:
- Fimbrin, $\alpha$ -Actinin
Filament Side Binders:
- Tropomyosin
Molecular Motors:
- Myosins (14 classes)

Spatial Regulation of Actin Filament Formation

Formin is a multi-domain protein regulating straight actin filament formation.
It contains a Rho GTPase binding domain (RBD), a profilin-ATP-actin binding domain (FH1), and a filament nucleating domain (FH2).
When unbound to Rho, the RBD inhibits the FH2 domain.
Upon Rho GTPase activation, formin is recruited to the plasma membrane, releasing FH1 and FH2 domains to trigger straight actin filament formation.

Arp2/Arp3 Complex and Branched Actin Filaments

The Arp2/Arp3 complex induces branching of actin filaments at a fixed angle of 70 degrees.
It is located near the cell membrane and activated by proteins like WASp and WAVE.

WASp and Regulation of Branching

WASp is a multi-domain protein with an RBD, an actin-binding domain, and an Arp2/Arp3 complex binding domain.
When not bound to Rho, the RBD prevents binding to the Arp2/Arp3 complex.
Upon Cdc42 GTPase activation (e.g., in response to a chemotactic signal), WASp is recruited to the plasma membrane.
This releases the Arp2/Arp3 binding domain to trigger branched actin filament formation.

Packing of Actin Filaments

Actin filaments can be packed into tight or loose bundles.
$\alpha$ -actinin forms dimers that bundle either parallel or anti-parallel filaments loosely, allowing Myosin II access for chemotaxis and cytokinesis.
Fimbrin bundles filaments of the same polarity tightly, excluding Myosin II, as seen in microvilli.

Myosin Motors and Their Roles

Myosin motors are involved in:
- Organelle organization
- Chromosome segregation
- Protein and RNA transport
- Cell division
- Cell motility and chemotaxis
- Maintaining cell integrity
- Food mastication
- Digestion
- Blood circulation
- Communication
- Reproduction
- Body movement

Myosin Superfamily Members

Examples:
- Myosin I: Defects can cause deafness.
- Myosin II: Defects can cause seizures.
- Myosin V: Defects can cause deafness/blindness.

Myosin Tail and Function

The myosin tail determines its binding target and function.
Myosin I binds membranes and is involved in endocytosis.
Myosin II forms dimers, associating in a bidirectionally symmetrical configuration (Myosin II bouquet).
Myosin V dimerizes and binds vesicles via adaptor molecules for transport to the plus ends of actin filaments.

Myosin II Filament Structure

Myosin II filaments are formed from bi-symmetrical bouquets of Myosin II dimers.
Each dimer is connected by a coiled-coil tail region.
Chymotrypsin cleavage removes most of the tail, leaving the heads together.
Papain further digests the tail (S2 region), leaving monomeric S1 heads with motor activity and light chain binding sites.

Actin Filament Polarity

Actin filament polarity can be visualized using purified monomeric myosin S1 heads in electron micrographs.
This gives filaments an arrowhead appearance, with the arrow pointing towards the minus end.
Tropomyosin binding changes on muscle actin, inhibiting myosin binding when muscle is inactive; it moves to allow myosin binding when muscle is active.

Myosin Motility

Myosin motility can be studied by binding myosin to a coverslip and observing fluorescent actin filaments moving over it.
The mechanism of Myosin II serves as a model for other myosins.
Myosins act independently with two heads so that the action of one head can be considered alone.

Myosin II Mechanism

In the absence of actin, Pi dissociation from the myosin head is slow.
Actin binding increases the rate of Pi dissociation 300-fold, to approximately 10 ATP molecules per myosin per second.
Pi dissociation induces the “power stroke.”
Conformational changes in the head (motor) domain are amplified by a “converter” region, causing the lever-like neck to rotate.
This is further amplified by the lever arm (neck domain), moving the actin filament by a few nanometers.

Sliding Filament Theory

Myofibrils are composed of sarcomeres, the basic units of muscle action.
Myofibrils contains muscle fibers, and muscle cells are multinucleated.
Each myosin thick filament is surrounded by 6 thin microfilaments (actin filaments).
Plus ends of actin filaments are capped by CapZ in the Z disk, while minus ends are capped by tropomodulin.
Nebulin determines the length of the actin filaments.

Muscle Cell Structure

Muscle cells are syncytial and multinucleated, up to ~50 cm long, formed by myoblast fusion.
Myofibrils contain repeating sarcomeres, the base unit of muscle action.
Electron micrographs show alternating light and dark bands with Z lines in the middle of light bands.
Each sarcomere is the region between Z lines, with one dark band and half a light band on either side.
Dark bands are composed of myosin II filaments, and light bands of actin filaments.
The darkest region contains overlapping myosin and actin filaments in a regular pattern.
During muscle contraction, the light band disappears as the overlap between myosin and actin increases.

Nerve Impulses and Muscle Contraction

Nerve impulses trigger muscle contraction by depolarizing the sarcolemma (plasma membrane).
Transverse tubules (T-tubules) are invaginations of the plasma membrane adjacent to the sarcoplasmic reticulum (SR) around each myofibril.
Action potentials open voltage-gated $Ca^{++}$ channels, releasing a burst of $Ca^{++}$ into the cytosol.
This $Ca^{++}$ binds to channels in the SR, triggering massive release of $Ca^{++}$ into the cytosol, causing myosin to bind to actin.

Calcium and Regulatory Proteins

Elevated $Ca^{++}$ concentration causes conformational changes in tropomyosin (TM) and the troponin (TN) complex.
In the absence of $Ca^{++}$ , tropomyosin covers the myosin-binding site on actin.
In the presence of $Ca^{++}$ , the myosin-binding site is exposed.
The troponin complex consists of TN-T, TN-I, and TN-C. TN-C is the $Ca^{++}$ -binding subunit.
$Ca^{++}$ binding to TN-C induces a conformational change, altering tropomyosin.

Functions of Non-Muscle Myosin II

Non-muscle Myosin II can bind loosely-packed contractile bundles of actin filaments

Measuring Myosin Motor Properties

The movement of myosin motors depends on step size (neck linker length) and duty ratio (ATPase activity).
Processive motors need a high duty ratio and large step size.
Laser-based optical tweezers measure step size, processivity, and force generation.
Myosin is bound to a bead, and an actin filament held by lasers is lowered towards the bead.
ATP addition stimulates the ATPase cycle, and myosin moves the actin filament.
Distance and force are calculated via computer.

Myosin II and V Characteristics

Myosin II takes 5-10 nm steps with a 10% duty ratio, so one or more Myosin II heads are always bound.
Myosin V takes successive 36 nm steps and has a 70% duty ratio.

Myosin V Mechanism

Myosin V moves by a hand-over-hand mechanism with 72 nm steps because the distance traveled between the two heads are 72nm instead of the single motor of 36nm step if it moved by a inchworm manner.
Fluorescent probes attached to one head confirm 72 nm steps.
Myosin V walks down one side of the actin filament.

Vesicle Transport

Vesicles can bind Kinesin and Dynein for long-range transport on microtubules and Myosin V for short-range transport on actin filaments to the plasma membrane.
Vesicles move from microtubules to actin filaments near the cell cortex and eventually undergo exocytosis.
The Arp2/3 complex is observed at the intersection of branched actin filaments.