Microfilaments (Week 5-2: Cytoskeletal System II)

Microfilaments

• Smallest of the cytoskeletal filament, with a diameter of 7 nm

• Contains G-actin monomers and have (+) / (-) polarity

Functions of Microfilaments

1. Muscle Contraction

2. Cell Migration, Amoeboid Movement, and Cytoplasmic Streaming 

3. Development & Maintenance of Cell Shape

4. Structural core of Microvilli 

Actin

• Building block of microfilaments

• Highly abundant protein that exists in two forms: monomeric (G-actin) and filamentous (F-actin)

G actin (Globular Actin)

• Single, globular shaped monomer that can bind ATP or ADP

• ATP-bound G-actin is more likely to polymerize into filaments

F actin (Filamentous Actin)

• Actual microfilaments made of G-actin monomers polymerized into long, helical strands

Actin filaments Polarity

• Plus end is fast-growing end

• Minus end is slow growing or depolymerizing end

Actin proteins can be categorized into

1. Muscle-specific actins (α-Actins)

• Found in skeletal, cardiac, and smooth muscle cells

• Play a major role in muscle contraction by interacting with myosin

2. Nonmuscle actins (β- and γ-Actins)

• Found in cytoskeletons of all cell types

• Involved in cell motility, and maintaining cell shape

Phases of G-actin polymerization

1. Lag Phase (Nucleation Phase)

• G-actin monomers slowly form small oligomers (seeds) to initiate polymerization

• This step is rate-limiting because monomers must first assemble to form a stable nucleus

2. Elongation Phase

• Once a nucleus forms, G-actin monomers rapidly add to both ends, primarily at the plus-end (+)

• ATP-actin incorporates into the growing filament, stabilizing it

3. Steady-State Phase (Treadmilling)

• Actin filaments reach an equilibrium where polymerization at the plus-end (+) matches depolymerization at the minus-end (-)

• ATP-actin at the plus-end converts to ADP-actin toward the minus-end, leading to filament turnover

Structure of F-actin filaments

1. Double Helix Structure

• F-actin consists of two linear strands of polymerized G-actin twisted into a right-handed helix

• Each strand is composed of head-to-tail G-actin monomers, giving actin filaments a polarized structure

2. Fixed Orientation of Actin Monomers

• All G-actin monomers in an actin filament face the same direction, giving F-actin intrinsic polarity

• Plus-end (+): Fast-growing, prefers ATP-actin

• Minus-end (-): Slow-growing, prefers ADP-actin, making it more likely to depolymerize

Demonstration of Microfilament Polarity: The Arrowhead Decoration Assay

• Myosin subfragment 1 (S1) is a fragment of myosin that binds to actin filaments

• When S1 monomers attach to actin, they form a distinctive arrowhead pattern along the filament

• This pattern allows researchers to identify actin filament polarity 

• The plus-end (+), where actin monomers add, is called the barbed end

• The minus-end (-), where actin monomers are more likely to dissociate, is called the pointed end

Barbed End

Where actin monomers add at the plus end

Pointed End

Where actin monomers depolymerize at the minus end

Polarity of Microfilaments

1. Rapid Addition or Loss of G-actin at the Plus-End (+) 

2.  Slower Addition & More Depolymerization at the Minus-End (-) 

3. ATP Hydrolysis in Actin Filaments

Rapid Addition or Loss of G-actin at the Plus-End (+) 

• G-actin monomers add to the plus-end

• The critical concentration (Cc) is lower at the plus-end, making polymerization easier

 Slower Addition & More Depolymerization at the Minus-End (-) 

• Depolymerization is more likely at the minus-end, where ADP-actin is exposed

• The critical concentration (Cc) is higher at the minus-end, meaning it requires more free G-actin to grow 

ATP Hydrolysis in Actin Filaments

• ATP-bound G-actin is added to the growing plus-end

• Once incorporated into the filament, ATP-actin is slowly hydrolyzed to ADP-actin

• Over time, the filament is primarily composed of ADP-actin, except for the growing plus-end, which retains ATP-actin (promotes stability and polymerization)

Stress Fibers - actin structure

• Organized bundles of actin filaments that allow cells to adhere tightly to the underlying substratum (extracellular surface)

• Just beneath the plasma membrane, a dense cross-linked actin network supports cell shape

• This gel-like network aids in membrane tension distribution and involved in signalling/membrane trafficking

Lamellipodia - actin structure

Broad, sheet-like protrusions formed by a dense, branched actin network

Filopodia - actin structure

Thin, finger-like projections formed by parallel bundles of actin filaments

Facilitators of Microfilament Assembly: Actin-Binding Proteins

Thymosin β4 – Inhibitor of Polymerization

• Binds to free G-actin and prevents it from polymerizing into F-actin

• Acts as an actin reservoir, ensuring that cells do not polymerize actin uncontrollably

Profilin – Promoter of Polymerization 

• Profilin competes with thymosin β4 for binding to G-actin

• Promotes actin polymerization by facilitating ATP exchange on G-actin

• Delivers ATP-actin to the plus-end (+) of growing microfilaments, increasing filament elongation

How is actin polymerization regulated?

• If ATP-bound G-actin is abundant, actin filaments grow until monomers become limiting

• Thymosin β4 isolates G-actin, preventing excessive polymerization.

• Profilin frees G-actin from thymosin β4, promoting actin filament growth

Proteins that regulate actin polymerization

1. ADF/Cofilin 

• ADF (Actin Depolymerizing Factor) and Cofilin bind to ADP-actin in both G-actin and F-actin

• They increase turnover of ADP-actin at the minus-end (-) by severing filaments to create more filaments ends available for depolymerization

2. Formins

• Formins promote actin polymerization by associating with the plus-end (+) of microfilaments

• Unlike Arp2/3 (which nucleates branched filaments), formins facilitate linear actin filament growth

• Most formins act as Rho-GTPase effector proteins, meaning they respond to Rho signaling pathways to regulate cytoskeletal changes

A protein that cuts off actin filament

Gelsolin

• Breaks actin filaments into smaller fragments

• Caps the newly exposed plus-ends (+), preventing further polymerization

proteins that cap actin filament

1. CapZ 

• Binds to the plus-end (+) of actin filaments

• Stabilizes actin filaments through preventing further addition of G-actin monomers 

2. Tropomodulin

• Binds to the minus-end (-) of actin filaments, preventing subunit loss

A protein that crosslinks actin filaments

• Form a loose actin network of crosslinked filaments

  Filamin

  • Acts like a molecular "splice," linking actin filaments at intersecting points

A protein that bundles actin filaments

• α-Actinin

  • Bundles actin filaments into tightly organized arrays (focal adhesions) 

Microvilli

• Considered an example of microfilament assembly in the cell

• Microvilli are finger-like projections on the apical surface of intestinal mucosal cells that increase surface area for nutrient absorption.

• Their core structure consists of actin microfilaments oriented with plus-ends (+) toward the tip.

• Myosin I and calmodulin crosslink actin filaments to the plasma membrane, while fascin and villin stabilize the structure by crosslinking actin filaments within the bundle.

• At the base, the actin filament bundle extends into the terminal web, a dense network composed primarily of myosin II and spectrin, which links actin filaments to membrane proteins and possibly intermediate filaments, reinforcing the cytoskeleton
  

Actin associated proteins can connect microfilaments to plasma membrane

• Microfilaments are linked to the plasma membrane to facilitate cell movement, shape maintenance, and cytokinesis

• These connections allow the actin cytoskeleton to exert force on the membrane

• Examples of such proteins include ankyrin and spectrin

Proteins that promote actin branching and growth

Arp2/3 (Actin-Related Protein 2/3) Complex:

• Nucleates new actin filaments by binding to the sides of pre-existing filaments, forming branched actin networks

• The Arp2/3 complex is inactive until activated by nucleation-promoting factors (NPFs)

• WASP (Wiskott-Aldrich Syndrome Protein) and WAVE/Scar are key activators of Arp2/3

• Once activated, Arp2/3 binds to an actin filament and initiates branching, allowing the formation of dense, branched actin networks

Proteins that regulate the formation of long actin filaments

Formins: Nucleators of Long Actin Filaments

• Key proteins that regulate the formation of long, linear actin filaments instead of branched networks 

• Binds to the plus-end (+) of actin filaments and promotes continuous polymerization

• Moves along the growing filament to ensure elongation without branching

• Prevents capping proteins from terminating filament growth

Microtubules and microfilaments are similar in that both 

Have intrinsic directionality because one end of each structure is distinct from other end

A bacterial toxin sopE2 causes cells to form lamellipodia-like ruffles when injected artificially. The best interpretation of this result is that sopE2

Is an actin-binding protein 

Microfilaments and intermediate filaments are different in that only 

Microfilaments are composed of proteins bound to a ribonucleotide

Cellular Factors Regulating Microfilament Assembly

1. PIP2 phospholipid that regulates actin assembly

2. Rho-family GTPases

PIP2 - Phosphatidylinositol-4,5-bisphosphate

• A plasma membrane phospholipid that interacts with actin-binding proteins 

• Recruits actin-associated proteins to the membrane, regulating their activity

• This influences the formation, stability and breakdown of MF’s

Rho-Family GTPases

• Monomeric G proteins that regulate when and where actin-based structures form

• They act as molecular switches, toggling between an active GTP-bound state and an inactive GDP-bound state

Rho, Rac, & Cdc42 activations

• Rho activation results in formation of stress fibers, essential for cell adhesion 

• Rac activation results in lamellipodia extension, essential for cell migration 

• Cdc42 activation results in the formation of filopodia, essential for cell movement

Regulation of Rho-GTPases

Rho GTPases are regulated by three key classes of proteins:

1. Guanine-Nucleotide Exchange Factors (GEFs)

• Activate Rho GTPases by exchanging bound GDP for GTP

2. GTPase-Activating Proteins (GAPs)

• Inactivate Rho GTPases by promoting GTP hydrolysis to GDP

3. Guanine-Nucleotide Dissociation Inhibitors (GDIs)

• Isolate inactive Rho GTPases in the cytosol, preventing activation