Study Notes on Microfilaments and Cytoskeletal Dynamics

Chapter 17 - Cell Organization and Movement I: Microfilaments

Introduction

• Chapter derived from Molecular Cell Biology, Eighth Edition by W. H. Freeman and Company.

• Created by Thomas Deerinck and Mark Ellisman.

Components of the Cytoskeleton

Microfilaments

• Functions:

  • Determine cell shapes.

  • Involved in endocytosis and exocytosis.

  • Facilitate phagocytosis.

  • Enable ameboid movements.

Microtubules

• Functions:

  • Provide scaffolding for cellular structure.

  • Transport organelles within cells.

  • Essential for cell division.

  • Propel movement through structures like flagella.

Intermediate Filaments

• Functions:

  • Serve as cell coating.

  • Important for cell interactions and structural integrity.

Overview of Cytoskeletal Applications

Filament Systems in Animal Cells

• Physical and biochemical properties characterized.

• Differentiation between biological properties of filament types:

  • Microfilaments (Actin).

  • Microtubules (Tubulin).

  • Intermediate Filaments.

Cytoskeleton of Epithelial and Migrating Cells

• Key components:

  • Microfilaments

  • Microtubules

  • Intermediate Filaments

• Adhesion molecules include:

  • Anchoring junctions

  • Tight junctions

  • Gap junctions

• Extracellular matrix elements:

  • Basal membrane

  • Connective tissues

  • Structural proteins: collagen/elastin

  • Hydration agents: hyaluronic acid

  • Adhesive substances: glycoproteins

  • Fibroblasts.

Microfilament Structures

Actin-Based Structures

• Microfilaments can form various structures:

  • Distinct activities for different cell types.

  • Apical region in polarized epithelial cells has microvillar core bundles of actin filaments.

  • Fluorescent phalloidin binds specifically to F-actin, exposing microfilament arrangements in motile cells.

Dynamics of Actin Filaments

Regulation of Microfilament Stability

• The actin cytoskeleton is highly dynamic and its assembly/disassembly is tightly controlled.

  • Stability duration varies by structure.

  • Some structures are stable for hours; others dynamic with growth/shrinking within seconds.

  • Two main functions:

  1. Cell Movement: Actin organization changes generate forces impacting cell shape and intracellular movement (treadmilling).

  2. Fiber Contraction: Myosin II interacts with actin to provide contractile forces.

Actin-Binding Proteins

• Stabilization and disassembly of filaments affected by several actin-binding proteins.

Regulation of Cytoskeleton by Cell Signaling

• Cell-surface receptors convey external signals across the plasma membrane, activating cytosolic signaling pathways that manipulate cytoskeleton organization and function.

• Integration of signals from various receptor types leads to diverse cytoskeletal configurations and localized activities.

Structures of Monomeric G-Actin and F-Actin

Actin Characteristics

• Highly conserved, ancient protein.

• Identical at 80% amino acid sequence across species (e.g., amoebae vs. animals).

• Six human actin genes: expressed in various cell types, ~93% identical.

• Actin represents up to 10% of muscle protein, 1–5% in other cells; concentrations can reach 5 mM in microvilli.

Structure of G-actin and F-actin

• (a) G-actin:

  • Monomer size: $(5.5 imes 5.5 imes 3.5 ext{ nm})$

  • Central cleft divides monomer into lobes and subdomains (I-IV).

  • ATP/ADP binding occurs at the cleft's bottom.

• (b) F-actin:

  • Composed of two helically wound strands with a repeating unit of 28 subunits covering $(72 ext{ nm})$.

  • Polarity: ATP-binding cleft orientation determines ends ($ext{(−) end}$ and $(+) ext{ end}$).

  • (c) TEM Representation: Negatively stained actin filaments depicted as long, twisted strands.

Actin Polymerization and Stability

Phases of G-actin to F-actin Transition

• Three phases of in vitro polymerization: Nucleation, Elongation, Steady state.

  • Nucleation: Formation initiation of actin filaments,

  • Elongation: Rapid assembly of actin subunits at the $(+)$ end faster than $(−)$ end.

  • Steady state achieved as assembly matches disassembly.

Actin Treadmilling

• Actin filament assembly is biased:

  • Faster assembly at the $(+)$ end compared to disassembly at the $(−)$ end.

  • Explanation of steady state dynamics: unstable filament regions maintain net assembly rates, resulting in treadmilling.

Experimental Evidence on Actin Dynamics

Myosin Decorated Filament Growth Rates

• Demonstrations that actin filaments grow at different rates:

  • Fast at $(+)$ end vs slow at $(−)$ end reflected by ratios of assembly/disassembly rates.

Regulation of Filaments by Actin-Binding Proteins

Key Functionality of Actin-Binding Proteins

• Various actin-binding proteins regulate polymerization rates and G-actin availability:

  • Profilin:

  • Binds ADP-G-actin, catalyzes ATP exchange, influencing filament assembly at different ends.

  • Cofilin:

  • Enhances depolymerization via fragmentation of ADP-actin regions.

  • Thymosin-β4:

  • Acts as a reservoir for ATP-G-actin, regulating the availability and polymerization.

Capping Proteins

Role of Capping Proteins in Actin Filaments

• Capping proteins influence assembly/disassembly dynamics:

  • CapZ limits assembly at the $(+)$ end.

  • Tropomodulin stabilizes the $(−)$ end by blocking depolymerization.

Interaction of Actin with Other Proteins

Bundles and Networks

• Actin filaments link with proteins to create:

  • Bundles: Crosslinked tightly for muscle contraction and force generation.

  • Networks: Loosely crosslinked for structural uniformity and support.

Actin Cross-Linking Proteins

Cross-Linking Proteins and Their Functions

• Diverse proteins contributing to actin structures include:

  • Fimbrin: tight bundles like microvilli.

  • Alpha-actinin: looser bundles.

  • Spectrin: flexible networks for structure underneath plasma membranes.

  • Filamin: spring-like regions for creating gels and networks.

  • Dystrophin: links actin to membrane proteins; mutations can cause diseases such as muscular dystrophies.

Nucleation Promoting Factors (NPFs)

NPF Family Members

• Mammalian WASP superfamily includes:

  • WASP: specific to hematopoietic cells.

  • N-WASP: present in neural tissue.

  • WAVE1, WAVE2, WAVE3: different tissue expressions.

• NPFs activate nucleators such as Arp2/3.

Clinical Significance of NPF Dysregulation

Diseases Associated with NPFs

• Mutations/dysregulation in WASP/WAVE linked to:

  • Wiskott-Aldrich Syndrome: immunodeficiency, eczema, thrombocytopenia from WASP mutations.

  • Cancer: aberrant activity in promoting invasive behaviors through NPFs.

Actin Nucleation Factors

Categories of Actin Nucleation Factors

• Three classes involved in actin polymerization:

  • Arp2/3 complex with NPFs.

  • Formins.

  • Tandem-monomer-binding nucleators.

Nucleation Mechanism by the Arp2/3 Complex

Steps of Arp2/3 Activation and Function

• Activation process leads to branched filament formation:

  1. NPFs engage on actin monomers.

  2. NPF-actin complexes activate Arp2/3.

  3. Arp2/3 binds edges of existing filaments prompting new growth.

  4. Fresh G-actins polymerize at the new $(+)$ end forming branches.

Functional Roles of Actin Structures

Arp2/3 Functionality

• Mediation of cellular locomotion and vesicle transport.

• Drugs modulating actin dynamics for research and visualization.

Listeria and Actin Polymerization

Mechanism of Listeria Movement

• Listeria monocytogenes hijacks actin polymerization for motility, using ActA to activate Arp2/3, pushing itself via actin comet tails.

Endocytosis and Arp2/3 Dependency

Clathrin-Mediated Endocytosis

• NPFs activate the Arp2/3 for rapid actin assembly, facilitating the transport of internalized vesicles.

Cytoskeletal Element Drugs

Common Drugs Affecting Cytoskeletal Dynamics

• Depolymerizing Drugs for Actin: Cytochalasin, Latrunculin, and Swinholide.

• Stabilizing Drugs: Phalloidin, Jasplakinolide.

Cytoskeleton-Associated Disorders

Summary of Key Disorders

Stress Fibers and Their Functions

Actin-Myosin Interactions

• Actin-Myosin complexes involved in:

  • Migration, phagocytosis, signal transduction, cell division, muscle contraction.

• ATP-dependent processes integral for contractile forces.

• Stress fibers are crucial in many cultured animal cells.

• Highlighted example includes human umbilical vein endothelial cells showing interconnected stress fibers.

Chapter 17 - Cell Organization and Movement I: Microfilaments

Components of the Cytoskeleton

Microfilaments

• Functions:

  • Determine cell shapes.

  • Involved in endocytosis and exocytosis.

  • Facilitate phagocytosis.

  • Enable ameboid movements.

Microtubules

• Functions:

  • Provide scaffolding for cellular structure.

  • Transport organelles within cells.

  • Essential for cell division.

  • Propel movement through structures like flagella.

Intermediate Filaments

• Functions:

  • Serve as cell coating.

  • Important for cell interactions and structural integrity.

Overview of Cytoskeletal Applications

Filament Systems in Animal Cells

• Differentiation between biological properties of filament types:

  • Microfilaments (Actin).

  • Microtubules (Tubulin).

  • Intermediate Filaments.

Cytoskeleton of Epithelial and Migrating Cells

• Key components:

  • Microfilaments

  • Microtubules

  • Intermediate Filaments

• Adhesion molecules include:

  • Anchoring junctions

  • Tight junctions

  • Gap junctions

• Extracellular matrix elements:

  • Basal membrane

  • Connective tissues

  • Structural proteins: collagen/elastin

  • Hydration agents: hyaluronic acid

  • Adhesive substances: glycoproteins

  • Fibroblasts.

Microfilament Structures

Actin-Based Structures

• Microfilaments can form various structures, such as microvillar core bundles of actin filaments in the apical region of polarized epithelial cells.

• Fluorescent phalloidin binds specifically to F-actin, exposing microfilament arrangements in motile cells.

Dynamics of Actin Filaments

Regulation of Microfilament Stability

• The actin cytoskeleton is highly dynamic; its assembly/disassembly is tightly controlled.

• Stability duration varies by structure, from hours to seconds.

• Two main functions:

  1. Cell Movement: Actin organization changes generate forces impacting cell shape and intracellular movement (treadmilling).

  2. Fiber Contraction: Myosin II interacts with actin to provide contractile forces.

Actin-Binding Proteins

• Stabilization and disassembly of filaments are affected by several actin-binding proteins.

Regulation of Cytoskeleton by Cell Signaling

• Cell-surface receptors convey external signals, activating cytosolic signaling pathways that manipulate cytoskeleton organization and function.

Structures of Monomeric G-Actin and F-Actin

Actin Characteristics

• Highly conserved, ancient protein, ~80% identical amino acid sequence across species.

• Represents up to 10% of muscle protein, 1–5% in other cells.

Structure of G-actin and F-actin

• (a) G-actin:

  • Monomer size: $(5.5 imes 5.5 imes 3.5 ext{ nm})$

  • Central cleft divides monomer, where ATP/ADP binding occurs.

• (b) F-actin:

  • Composed of two helically wound strands with a repeating unit of 28 subunits covering $(72 ext{ nm})$.

  • Polarity: ATP-binding cleft orientation determines ends ($ext{(−) end}$ and $(+) ext{ end}$).

• (c) TEM Representation: Negatively stained actin filaments depicted as long, twisted strands.

Actin Polymerization and Stability

Phases of G-actin to F-actin Transition

• Three phases of in vitro polymerization:

  • Nucleation: Initiation of actin filament formation.

  • Elongation: Rapid assembly of actin subunits, predominantly at the $(+)$ end.

  • Steady state: Achieved as assembly matches disassembly.

Actin Treadmilling

• Actin filament assembly is biased: faster at the $(+)$ end than disassembly at the $(−)$ end.

• Steady state dynamics involve unstable filament regions maintaining net assembly rates, resulting in treadmilling.

Experimental Evidence on Actin Dynamics

Myosin Decorated Filament Growth Rates

• Actin filaments grow at different rates: fast at $(+)$ end vs. slow at $(−)$ end.

Regulation of Filaments by Actin-Binding Proteins

Key Functionality of Actin-Binding Proteins

• Regulate polymerization rates and G-actin availability:

  • Profilin: Binds ADP-G-actin, catalyzes ATP exchange, influencing filament assembly.

  • Cofilin: Enhances depolymerization through fragmentation of ADP-actin regions.

  • Thymosin-β4: Acts as a reservoir for ATP-G-actin, regulating its availability for polymerization.

Capping Proteins

Role of Capping Proteins in Actin Filaments

• Influence assembly/disassembly dynamics:

  • CapZ limits assembly at the $(+)$ end.

  • Tropomodulin stabilizes the $(−)$ end by blocking depolymerization.

Interaction of Actin with Other Proteins

Bundles and Networks

• Actin filaments link with proteins to create:

  • Bundles: Crosslinked tightly for muscle contraction and force generation.

  • Networks: Loosely crosslinked for structural uniformity and support.

Actin Cross-Linking Proteins

Cross-Linking Proteins and Their Functions

• Diverse proteins contributing to actin structures include:

  • Fimbrin: forms tight bundles (e.g., microvilli).

  • Alpha-actinin: forms looser bundles.

  • Spectrin: creates flexible networks beneath plasma membranes.

  • Filamin: creates gels and networks with spring-like regions.

  • Dystrophin: links actin to membrane proteins; mutations can cause muscular dystrophies.

Nucleation Promoting Factors (NPFs)

NPF Family Members

• Mammalian WASP superfamily (e.g., WASP, N-WASP, WAVE1/2/3) activates nucleators such as Arp2/3.

Clinical Significance of NPF Dysregulation

Diseases Associated with NPFs

• Mutations/dysregulation in WASP/WAVE linked to:

  • Wiskott-Aldrich Syndrome: immunodeficiency, eczema, thrombocytopenia from WASP mutations.

  • Cancer: aberrant activity in promoting invasive behaviors.

Actin Nucleation Factors

Categories of Actin Nucleation Factors

• Three classes involved in actin polymerization:

  • Arp2/3 complex with NPFs.

  • Formins.

  • Tandem-monomer-binding nucleators.

Nucleation Mechanism by the Arp2/3 Complex

Steps of Arp2/3 Activation and Function

• Activation leads to branched filament formation:

  1. NPFs engage on actin monomers.

  2. NPF-actin complexes activate Arp2/3.

  3. Arp2/3 binds edges of existing filaments, prompting new growth.

  4. Fresh G-actins polymerize at the new $(+)$ end, forming branches.

Functional Roles of Actin Structures

Arp2/3 Functionality

• Mediates cellular locomotion and vesicle transport.

Listeria and Actin Polymerization

Mechanism of Listeria Movement

• Listeria monocytogenes hijacks actin polymerization, using ActA to activate Arp2/3 and propel itself via actin comet tails.

Endocytosis and Arp2/3 Dependency

Clathrin-Mediated Endocytosis

• NPFs activate Arp2/3 for rapid actin assembly, facilitating the transport of internalized vesicles.

Cytoskeletal Element Drugs

Common Drugs Affecting Cytoskeletal Dynamics

• Depolymerizing Drugs for Actin: Cytochalasin, Latrunculin, Swinholide.

• Stabilizing Drugs: Phalloidin, Jasplakinolide.

Cytoskeleton-Associated Disorders

Stress Fibers and Their Functions

Actin-Myosin Interactions

• Actin-Myosin complexes are involved in migration, phagocytosis, signal transduction, cell division, and muscle contraction.

• These are ATP-dependent processes integral for contractile forces.

• Stress fibers are crucial in many cultured animal cells, connecting to form interconnected networks.