Module 2: Cytoskeletal System II

Microfilaments

• smallest of the cytoskeletal filament (7nm wide)

• has two intertwined chains of F-actin

• consist of ATP (nucleotide substrate)

• contains G-actin monomers

• has defined polarity (±)

Functions of microfilaments

1. Muscle Contraction

   • actin filaments interact with myosin motor proteins

   • this interaction allows muscles to contract

   • found in skeletal, cardiac and smooth muscle cells

2. Cell Migration, Amoeboid Movement, and Cytoplasmic Streaming

   • actin polymerize and depolymerize at the leading edge to push it forward

   • cells crawl using flowing extensions of their cytoplasm

   • helps move organelles and materials within the cell

3. Maintenance of Cell Shape

   • actin filaments form a dense network beneath the plasma membrane

   • helps maintain shape and resist mechanical stress

4. Structural Core of Microvilli

   • they form the core of microvilli

     • tiny finger-like projections that increase surface area to aid in absorption

Actin

• building block of microfilaments

• highly abundant protein

• exists in two forms:

  • G-actin (monomeric)

  • F-actin (filamentous)

Polarity of Actin

• actin filaments have two ends:

  • plus end: grows faster

  • minus end: grows slowly or may shrink

• this polarity allows for directed growth and movement

G-Actin (Globular Actin)

• each G-actin can bind ATP or ADP

• ATP-bound G-actin is more likely to join the growing filament

F-Actin (Filamentous Actin)

• when G-actin monomers polymerize, they form F-actin

  • F-actin: long twisted strands that make up microfilaments

2 types of actin

1. Alpha Actin

   • found in muscle cells (smooth, cardiac, skeletal)

   • interact with myosin to power muscle contraction

2. Beta & Y-Actin

   • found in non-muscle cells

   • maintain cell shape and movement

3 phases of actin polymerization

1. Lag Phase (Nucleation Phase)

   • G-actin monomers form small oligomers

     • seeds that initiate polymerization

   • rate-determining step (slowest) because it takes longer to form a stable nucleus

2. Elongation Phase

   • once a nucleus forms, G-actin quickly adds to both ends of the filament (mostly the plus end)

   • ATP-actin is added, which stabilizes the filament and allows it to grow quickly

     • ATP-actin is the active form

3. Steady-State Phase (Treadmilling)

   • equilibrium is reached when:

     • polymerization at plus end = depolymerization at minus end

   • filament stays the same length but actin monomers flow through it

   • over time, ATP-actin is converted to ADP-actin as it moves towards the minus end, leading to filament turnover

Actin Polymerization

• where actin filaments (F-actin) are built by joining G-actin monomers into long, stable chains

• this occurs in 3 phases

• the process is reversible - meaning filaments can grow or shrink depending on conditions

Structure of F-Actin

1. Double Helix

   • F-actin is made of two strands of G-actin twisted around each other into a right-handed helix

2. Polarity

   • all G-actin monomers are arranged in the same direction

   • creating F-actin intrinsic polarity

     • plus end: prefers ATP actin, faster-growing

     • minus end: prefers ADP actin, slow-growing (can depolymerize)

Arrowhead Decoration Assay

• demonstrates how polarity is shown in microfilaments

• scientists use a piece of the myosin protein called S1 fragment

• S1 binds to actin filaments in a specific orientation

• when S1 binds along the filament, it forms a pattern that looks like arrowheads

  • minus end: pointed end

  • plus end: barbed end (open side)

• this allows researchers to see the end differences, proving actin filaments are polarized

Polarity of Microfilaments

• Plus End (barbed end)

  • ATP bound G-actin monomers are added quickly

  • easier for actin to polymerize due to lower Cc

  • filaments grow at this end

• Minus End (pointed end)

  • ADP-actin is more common

  • makes it less stable

  • higher Cc, requires more free G-actin to grow

  • depolymerization is more likely at this end

• ATP Hydrolysis in Actin Filaments

  • when ATP-actin adds to plus end, it is stable

  • after being added, ATP is slowly hydrolyzed to ADP

  • plus end remains rich in ATP-actin → keeps growing

  • minus end is composed of ADP-actin → falls apart

4 actin based structures in cells

• actin filaments can be reorganized into different structures, depending on the cell’s needs

1. Stress Fibers

   • thick bundles of actin filaments

   • allows cells to anchor tightly to the extracellular surface

2. Cortical Actin Network

   • found just beneath the plasma membrane

   • forms a dense, cross-linked gel of actin filaments

3. Lamellipodia

   • broad, sheet-like extensions

   • formed by a branched network of actin

   • found at the leading edge of crawling cells

   • allows them to move along a surface

4. Filopodia

   • thin, finger-like projections

   • formed by parallel bundles of actin

   • found at the leading edge of crawling cells

   • allows them to move along a surface

What facilitates microfilament assembly?

Actin-Binding Proteins

• regulates microfilament assembly

• they control how much G-actin is available and how fast actin filaments grow or shrink

How is actin polymerization regulated?

1. If ATP G-actin is abundant

   • actin filament will grow quickly at the plus end

   • growth continues until G-actin runs low

2. Thymosin β4 controls excess growth

   • binds to G-actin

   • keeps it in an inactive storage form

   • prevents uncontrolled filament growth

3. Profilin promotes filament growth

   • releases G-actin from thymosin β4

   • helps G-actin swap ADP for ATP

   • delivers ATP-actin to growing plus end of microfilaments

4 actin-binding proteins that regulate actin polymerization

1. Thymosin β4: Inhibits Actin Growth

   • binds to free G-actin

   • prevents G-actin from assembling into F-actin

   • act as storage system to hold back excess G-actin from overpolymerization

2. Profilin: Promotes Actin Growth

   • competes with thymosin for binding to G-actin

   • facilitates ATP exchange (swap ADP for ATP) on G-actin for polymerization

   • delivers ATP-actin to the plus end, speeding up growth

3. ADF/Cofilin: Break Down Filaments

   • actin depolymerizing proteins

   • binds to ADP-actin, both as G-actin and F-actin

   • sever filaments to create more ends available for depolymerization

     • increasing filament turnover at the minus end

4. Formins: Promotes Actin Growth

   • promote actin polymerization

   • binds to plus end of microfilament

   • facilitate linear actin filament growth (unlike Arp2/3 that nucleates branched filaments)

   • activated by Rho-GTPase signalling

   • responds to signals that regulate cell shape, movement and cell division

5 actin-binding proteins that regulate microfilament length

1. Gelsolin

   • sever actin filaments

2. CapZ

   • cap actin filaments

3. Tropomodulin

   • cap actin filaments

4. Filamin

   • cross-link actin filaments

5. α-Actinin

   • bundle actin filaments

Protein that sever actin filaments

Gelsolin

• cuts actin filaments into smaller segments

• caps the plus end after cutting to prevent further growth

Proteins that cap actin filaments

• both proteins block the ends of filaments, so no more monomers can be added or removed

  1. CapZ

  • binds to plus end

  • stops G-actin from being added, which stabilizes filament

  2. Tropomodulin

  • binds to minus end

  • prevents subunit loss

Protein that cross-link actin filaments

Filamin

• works like molecular splice

• links intersecting filaments, forming net-like structure

Protein that bundle actin filaments

α-Actinin

• organize actin filaments into tight and parallel bundles

• found in focal adhesions

What is an example of microfilament assembly?

Microvilli

• tiny, finger-like projections on apical (top) surface of intestinal muscosal cells

• increases surface area for nutrient absorption

• contains a core of tightly bundled actin filaments

• these filaments are polarized, with their plus ends pointing toward the tip of the microvillus

Key proteins involved:

1. Cross-linking to the plasma membrane

   • Myosin I & Calmodulin

2. Cross-linking within the actin bundle

   • Fascin & Villin

Terminal Web:

• occurs at the base of microvillus

• where actin bundles extend into a dense network

  • this network links actin filaments, reinforcing cytoskeleton

• primary components: myosin II & spectrin

Actin-associated proteins that connect to plasma membrane

• Ankyrin & Spectrin

  • critical for cell movement

  • maintaining shape

  • cytokinesis

Proteins that promote actin branching

• Arp2/3 Complex

  • creates branched actin structures (in lamellipodia)

  • binds to the side of existing actin filament

  • starts a new filament at an angle

  • remains inactive until turned on by NPFs

    (nucleation-promoting factors)

    • proteins that activate ARP2/3, to start branching

    • examples: WASP & WAVE / Scar

Proteins that promote long, unbranched actin filament

• Formins

  • found in filopodia and stress fibers

  • help build long and linear actin filaments

  • binds to plus end and stays attached as filament grows

  • prevents capping proteins from stopping filament growth

2 cellular factors that regulate microfilament assembly

1. Phospholipids (PIP2)

   • regulate actin assembly

   • found in the plasma membrane

   • binds to actin-binding proteins

   • recruit and activate actin-associated proteins to the membrane

2. Rho-Family GTPases

   • monomeric G-proteins that act as on/off switches

     • active when bound to GTP

     • inactive when bound to GDP

regulates when and where actin-based structures form

• 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 formation of filopodia

  • essential for cell movement

3 classes of proteins that regulate Rho-GTPases

1. GEFs

   • guanine-nucleotide exchange factors

   • turns on Rho-GTPase by exchanging GDP for GTP

2. GAPs

   • GTPase-activating proteins

   • turns off Rho-GTPase by hydrolyzing GTP to GDP

3. GDIs

   • guanine-nucleotide dissociation inhibitors

   • isolate inactive Rho-GTPase in the cytosol

   • prevents activation

Microtubules and microfilaments are similar in that both 

1. Form hollow tubes in the cytosol

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

3. Are composed of monomers bound to guanosine ribonucleotides

4. Are essential for maintaining the cell shape of animal cells

5. Are composed of monomers bound to adenosine ribonucleotides

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 

1. Is similar to the Rho-like GTPase protein Cdc-42

2. Is a MAP

3. Binds to intermediate filaments 

4. Is a type of catastrophin  

5. Is an actin-binding protein

Is an actin-binding protein

Microfilaments and intermediate filaments are different in that only 

1. Microfilaments are dynamic

2. Intermediate filaments are dynamic

3. Microfilaments are composed of proteins bound to a ribonucleotide

4. Intermediate filaments are important for maintaining the shape of animal cells

5. Microfilaments are stronger 

Microfilaments are composed of proteins bound to a ribonucleotide

• microfilaments have actin which binds to ATP or ADP (which are ribonucleotides)

• But, intermediate filaments doesnt bind GTP or ATP

Intermediate Filaments

• consist of 8 protofilaments joined end to end w/ staggered overlaps

• key component in the cytoskeleton of animal cells

• absent in plant cells

• 8-12 nm wide

• made of 6 classes of protein monomers

• lacks polarity

• lacks GTP or ATP

Functions of intermediate filaments

• provide structural support

• maintenance of animal cell shape

• formation of nuclear lamina and scaffolding

• keeping myofibrils intact

• strengthening nerve cell axons

Keratin

• most abundant intermediate filament in animal cells

• found in epithelial tissues (like skin)

• provide toughness and protection

4 key characteristics of intermediate filaments

1. Most stable

   • IFs are tough and resist mechanical stress

   • least dynamic (do not grow/shrink quickly)

   • provide long term structural support

2. Least soluble

   • insoluble in most conditions

   • ideal for giving cells durable strength

3. Tissue-specific

   • different cells produce different types of IFs

     • unlike actin (microfilaments) and tubulin (microtubules) that are universal

   • classified into 6 major types based on amino acid composition

4. Supports the entire cytoskeleton

   • IFs serve as a scaffold that connects actin filaments and microtubules

   • maintains overall cell shape and stability

2 functional properties of intermediate filaments

1. Tension-Bearing Role

   • IFs are built to resist pulling and stretching forces

   • provide structural integrity to cells and tissues

2. Chemical Stability

   • IFs are more chemically stable than MTs or MFs

   • do not break down easily in harsh chemical environments

   • ideal for long-term structural support in cells

Structure of intermediate filaments

• core subunit is a dimer

  • made of two IF proteins twisted together in a coiled-coil shape

• IFs are made from fibrous dimers, not gobular

  • structurally different from actin and tubulin

  each IF has:

  • homologous central rodlike domain of 310-318 amino acids in length

  • N-terminal (head): variable between IF types

  • C-terminal (tail): also variable

• these domains determine specific properties and functions of various intermediate filaments

4 steps of intermediate filament assembly

1. Dimer Formation

   • 2 IF polypeptides twist together in the same direction (parallel)

   • forms alpha-coiled coil dimer (building block of IFs)

2. Tetramer Formation

   • two dimers align side by side, but in opposite direction

   • this forms a tetramer

   • since they are antiparallel, IFs have no polarity

     • unlike MT and MF that have distinct polarity

3. Protofilament Assembly

   • tetramers link end-to-end to form protofilaments

4. Final Filament Formation

   • 8 protofilaments overlap laterally to form mature IF

   • this thick bundling of protofilaments gives IFs their mechanical strength

How is the cytoskeleton a mechanically integrated structure?

• Microtubules

  • resists bending when a cell is compressed

• Microfilaments

  • serve as contractile elements that generate tension

• Intermediate filaments

  • considered elastic and can withstand tensile forces

• Spectraplakins

  • linker proteins that connect intermediate filaments, microfilaments, and microtubules

• Plectin

  • type of spectraplakin

  • crosslinks intermediate filaments (IFs) to microfilaments (MFs) and microtubules (MTs)