Week 10 Microfilaments

Cytoskeletal Systems

The three structural system in eukaryotic cells including:

• Microfilaments

• Microtubules

• Intermediate filaments

Responsible for cell shape, structure, and movement

What are microfilaments monomers?

Globular actin that polymerizes into actin filaments

What is the microtubule monomer

tubulin

Microfilament → function, dynamic, monomer, motor proteins 

Actin-based filaments that are dynamic, flexible, and critical for cell motility, muscle contraction, and maintaining cell shape

• Yes dynamic

• Monomer: alpha, beta, gamma actin

• Track for motor proteins: myosins 

Intermediate Filament → function, dynamic, monomer, motor proteins 

Cytoskeletal filament composed of various proteisn that provide tensil strength and structural stability

• Not bery dynamic

• Monomer: many different kinds of proteins 

• Track for motor proteins: no 

Microtubule →  function, dynamic, monomer, motor proteins 

Hallow tubes composed of alpha and beta tubulin dimers, involved in vesicle transport, mitosis, and cilia/flagella structure

• Highly dynamic

• Monomer: alpha and beta tubulin

• Track for motor proteins: Kinesins and dynsins

Apical vs basolateral surfaces

In polarized epithelial cells, the apical surface faces the lumen, while the basolateral surface interfaces with neighboring cells and extracellular matrix

Microvilli

Actin-based plasma membrane projections that increase the surface area for absorption

• Abundant in intestinal epithelial cells 

Cell Cortex

A dense network of actin filaments beneath the plasma membrane that supports shape and enables cell movement 

Adherens Belt

A continuous ring of actin filaments linked to cadherin junctions that maintain tissue integrity and cell-cell adhesion

Contractile Ring

A transient actin myosin ring that constricts during cytokinesis to divide cells

Filopodia

Long, unbranched actin protrusions used for sensing the environment

• Guided by Cdc42 GTPase signaling and formin activation

Lamellipodia

Broad, sheet like branched actin extensions that form a formal front

• Formed via Rac GTPase signaling and Arp2/3 branching

• Drives membrane protrusion during cell crawling

Stress Fibers

Contractile bundles of actin and myosin generated by Rho GTPase signaling and formin activation

• Provides traction during cell movement

G-actin

globular actin monomoer that binds ATP, polymerizes into F-actin filaments

F-actin

filamentous polymer of G-actin arranged helically

• exhibits structural and kinetic polarity with distinct (+) and (-) ends 

• (+) end → binds ATP bound actin at a faster rate

• (-) end → contains ADP bound actin with the ATP binding cleft facing this direction

• Structural asymmetry allows directional movement and treadmilling

ATP hydrolysis in Actin

Actin binds ATP during polymerization; ATP hydrolysis to ADP destabilizes older filament regions and promotes turnover

Nucelation

The rate-limiting first step of actin polymerization where a stable trimer “seed” forms

Elongation

Rapid addition of ATP-bound G-actin to the (+) end following nucleation

Critical concentration

The G-actin concentration where polymerization and depolymerization rates are balanced

Treadmilling

Dynamic process where actin monomers add to the (+) end and dissociate from the (-) end, maintaining filament length but driving movement and turnover

Cofilin

Binds ADP actin, increasing depolymerization rate at the (-) end and recycles actin monomers

Profilin

Promotes ADP → ATP exchange in G-actin, enhancing polymerization at the (+) end

Thymosin-B4

Sequesters ATP bound G actin, creating a reservoir of monomers ready for rapid filament assembly

CapZ

Caps the (+) end to halt filament elongation, leading to net depolymerization from the (-) end

Tropomudulin

Caps the (-) end, stabilizing filaments and preventing disassembly

Formin

Nucleates and elongates unbranced actin filaments by facilitating monomer addition at the (+) end 

Arp2/3 complex

Increases branched actin filament network by nucleating new filaments at a 70 degree angle from existing ones

Nucleation Promoting Factors

NPF proteins activate Arp2/3 to initiate actin branching

• E.g. WASp

• E.g. WAVE

Listeria ActA

bacterial surface protein that recruits Arp2/3 and Profilin to polymerize actin at one pole, propelling the bacterium through host cytoplasm

Endocytosis via actin

actin polymerization by Arp2/3 pushes invaginating vesicles inward, providing the force for receptor mediated uptake 

• This process can also be powered by myosin’s 

Myosin

ATP-dependent motor protein that interacts with actin filaments to generate movement via conformational changes 

Myosin Class I

Links actin to membrane bound proteins

• Aids in endocytosis and phagocytosis

Myosin Class II

Forms bipolar filaments responsible for muscle contraction and cellular tension generation

Myosin Class V

Transports vesicles and organelles along actin filaments within the cytoplasm

Cdc42

GTPase that activates formin to build parallel actin filaments in filopodia

Rac

GTPase that activates Arp2/3 to produce lamellipodia, enablign forward membrane protrusiton

Rho

GTPase that stimulates formin to form stress fibers and focal adhesions for traction

• Myosin dependent

Coordinated Cell Movement

Suquential activation of Cdc42, Rac, and Rho drives directed cell crawling 

• Filopodia senses

• Lamellipodia extend

• Stress fibers contract the rear 

Focal Adhesion

Integrin-based anchoring points where actin stress fibers attach to the ECM to generate traction 

How can actin polymerization be measured?

• Sedimentation

  • Centrifuge sample and polymerized filaments are in the pellet while monomer actin is in the supernant 

• Fluorescence Microscopy

  • Measure speed of actin growth

• Viscosity

  • Ball Fall Test - ball will not sink as far in a more viscous solution

  • Rotational viscometer - how hard is it to spin a router

  • More actin filaments = higher viscosity