Cytoskeleton: Structure and Functions — Comprehensive Notes

Cytoskeleton Filament System: Overview

• Filament systems in cells consist of three main types of filaments:

  • Microtubule (tubulin)

  • Microfilament (actin)

  • Intermediate filament (e.g., vimentin)

• Functions of the cytoskeleton:

  • Provide structural support

  • Enable motility

  • Regulation of cellular processes

• Cytoskeletal proteins can be stained by Coomassie Blue.

Structural Roles and Basic Organization

• Structural support:

  • Mechanical support to maintain cell shape

  • Anchorage site for organelles

• Dynamics:

  • Filaments can disassemble in one location and reassemble elsewhere to change cell shape

Cytoskeletal Filaments: General Principles

• Filaments are built from smaller protein subunits (soluble) into large filamentous structures

• Assembly is a dynamic process involving nucleation, elongation, and disassembly

• Subunit exchange occurs: disassembly of subunits at one site allows rapid diffusion and reassociation at another site

• Protofilaments and stability:

  • Multiple protofilaments provide strength and adaptability

  • Stability increases with more protofilaments and cross-contacts

  • Example: a single protofilament is thermally unstable; multiple protofilaments (e.g., 5 or more; microtubules typically have ~13) are thermally stable

Actin-Based Microfilaments: Actin (Microfilaments)

Actin Monomer: G-Actin

• Molecular weight: $MW = 43\,\mathrm{kDa}$

• Length: $\approx 375\,\mathrm{a.a.}$

• Bound nucleotide: $1\ \text{bound ATP/ADP}$

• Subdomains: $4$

• Structure: globular actin monomer (G-actin)

Actin Filament: F-Actin

• One twist: $37\,\mathrm{nm}$

• Thickness: $\approx 7\,\mathrm{nm}$

• Length in vitro: >$10\ \mu\mathrm{m}$; in vivo: $1-2\ \mu\mathrm{m}$

• Structure: double helix; semi-flexible polymer chain

Actin Filament Polarity

• Filaments possess directional polarity:

  • Barbed or plus end

  • Pointed or minus end

• Bound myosin heads create arrowhead appearance in EM images at the plus end

Actin Polymerization: G-Actin to F-Actin

• Monomers polymerize to form long filaments (F-actin)

• Addition preferentially at the barbed (+) end: $k<em>{on}^{+}\approx 5-10\times k</em>{on}^{-}$

• Polymerization requires ATP hydrolysis: $\mathrm{ATP: →: ADP + P_i}$

Phases of Polymerization

• Lag phase (nucleation): formation of a nucleus

• Elongation: rapid addition of monomers at the + end

• Equilibrium: addition at + end equals removal from the - end

• Described as: lag phase, elongation, equilibrium

Assembly Dynamics of Actin

• During rapid assembly: actin-ATP cap stabilizes the growing end

• After incorporation, ATP is hydrolyzed to ADP

• Plus end (fast-growing end) vs minus end (slow-growing end) dynamics

• Site of preferential depolymerization is the − end in steady-state conditions

Treadmilling

• State of equilibrium where monomers add at the + end and dissociate from the − end at the same rate

• Filament length remains approximately constant

• Conceptual expression: rate{+} = rate{-}

Capping Proteins

• Bind at ends of actin filaments to regulate growth and disassembly

• Roles:

  • Stabilize filaments by capping

  • Inhibit polymerization at ends

• Examples:

  • Tropomodulins cap the minus end, preventing dissociation

  • CapZ caps the plus end, inhibiting polymerization

Cross-Linking Proteins and Bundling

• Actin filaments are organized into bundles or networks by cross-linking proteins

• Most cross-linkers are dimeric or have two actin-binding domains

• Examples:

  • α-Actinin, villin, fimbrin promote parallel bundles

  • Filamins form V-shaped cross-links creating loose networks

• Specific bundle architectures:

  • Parallel bundles (e.g., stressed by motors such as myosin II) vs loose networks

Cross-Linking: Specific Proteins and Architectures

• α-Actinin, fimbrin, and villin form parallel cables

  • Bundles can be loose or tight; tight packing can exclude certain motor proteins like myosin II

• Filamins promote loose networks with hinge regions (flexible)

• Spectrin links actin networks to the plasma membrane, contributing to membrane stability

Mechanical Properties of Cross-Linked Networks

• Static connections resist slow changes; rapid deformations are resisted by network organization

• Adaptation occurs through reorganization of cross-links

Actin-Containing Cell Structures

Microvilli

• Each microvillus contains several dozen actin filaments with their + ends outward

• Filaments are tightly packed and crosslinked by actin-bundling proteins

• Inner membrane surface connected by lateral cross-links to maintain structure

Filopodia (microspikes)

• Bundles of parallel actin filaments

• Plus ends oriented toward the filopodial tip

• Cross-linked by fascin to form tight, stiff bundles

• Closely spaced filaments provide stiffness for exploration of the extracellular environment

Lamellipodia

• Contain extensively branched networks of actin filaments

• Plus (barbed) ends orient toward the plasma membrane

• Forward extension occurs by growth of actin filaments near the membrane

Microtubules: Tubulin-Based Polymers

General Properties

• Polymers of tubulin

• Dynamically unstable: undergo rapid cycles of assembly and disassembly

• Primary functions:

  • Vesicle and organelle transport (along microtubule tracks)

  • Cilia and flagella structure

  • Mitotic spindle formation during cell division

Structure and Nucleation

• Microtubules are stiff, hollow cylinders composed of α- and β-tubulin heterodimers

• Diameter: $d \approx 25\,\mathrm{nm}$; thickness ~25 nm

• Protofilaments: typically $n_{pf} = 13$ per microtubule

• Polarity:

  • + end: rapid polymerization

  • − end: slower polymerization

• Centrosome nucleation center:

  • γ-tubulin in the centrosome is necessary for nucleation of polymers

  • α- and β-tubulin heterodimers polymerize at the + end outward into the cytoplasm; the − end is anchored in the centrosome

Tubulin Monomer Details

• Monomer molecular weight: $MW \approx 50\,\mathrm{kDa}$

• Tubulin exists as a heterodimer: $\alpha\text{-tubulin}$ and $\beta\text{-tubulin}$

• Nucleotide state per dimer: $1\ bound\ GTP \text{or} \text GDP$ depending on incorporation state

• End designation: the + end and − end refer to growth polarity

Microtubule Polymerization and GTP Hydrolysis

• Process: tubulin dimers add to growing ends to form protofilaments, which later close to form a hollow tube

• GTP-tubulin cap concept:

  • GTP-bound tubulin added to the + end stabilizes growth

  • After incorporation, GTP is hydrolyzed to GDP within the lattice

• Hydrolysis dynamics govern stability: GDP-tubulin is less stable than GTP-tubulin

• GDP/GTP state drives dynamic instability and catastrophe/rescue events

Formation Pathway (Simplified)

• Dimer → oligomer → protofilament → sheet of protofilaments → closed microtubule

• Extensions involve sequential steps of nucleation and elongation until a stable microtubule is formed

GTP Cap and Stability (Illustrative Idea)

• GTP cap presence keeps rapid polymerization favorable at the + end; loss of cap leads to catastrophe

• GDP-tubulin region behind the cap is less stable and prone to depolymerization

Dynamic Instability of Microtubules

• Microtubules exhibit dynamic instability: rapid cycles of growth and shrinkage

• Phases:

  • Growth with a GTP cap at the + end

  • Catastrophe: rapid shrinkage when the GTP cap is lost

  • Rescue: re-establishment of a growth-cap structure allowing renewed growth

• Conceptual illustration (not exact kinetics):

  • Growth phase: GTP cap stabilizes plus end

  • Catastrophe: cap is lost; rapid depolymerization ensues

  • Rescue: regain cap and resume growth

• Structural scale events involve transitions between GTP- and GDP-tubulin-rich regions along the microtubule

Dynamic Instability and Cell Division

• Dynamic instability supports chromosome movement during mitosis

• In interphase: microtubules explore the cytoplasm; in mitosis: population dynamics facilitate accurate chromosome alignment and separation

• Conceptual arrangement:

  • Interphase nucleus and centrosome organize microtubule networks for cellular transport

  • Mitotic cell configuration reorganizes into spindle apparatus to separate chromosomes

Intermediate Filaments: Structure and Versatility

Overview

• Formed by a large, heterogeneous group of proteins

• Major classes include:

  • Keratin (epithelial cells)

  • Neurofilaments (neurons)

  • Vimentin-containing filaments (fibroblasts, glial cells, muscle cells)

  • Nuclear lamina (all nucleated cells)

• Properties:

  • No direct energy requirement for filament assembly

  • Filaments are not polarized

  • Provide mechanical resilience and structural integrity

Formation and Architecture

• Assembly begins with an N-terminal head domain, which is variable in size and structure

• Coiled-coil dimer formation

• Tetramer assembly: two dimers align to form a tetramer

• Protofilament formation: tetramers assemble into protofilaments

• Filament formation: multiple protofilaments twist together to form a ropelike filament

• Typical organization shows an 8-tetramer repeating pattern forming a ~10 nm filament

• Key structural concept:

  • Central rod domain is an a-helix of ~310–350 amino acids

  • Helical, rope-like organization gives mechanical resilience

Mechanical Properties Across Filament Types

• Microtubules: easily deformed and can rupture under stress

• Actin filaments: relatively rigid but can rupture under sufficient force

• Intermediate filaments: easily deformed but resist rupture, helping maintain cell integrity

Motor Proteins: Driving Movement and Transport

General Concept

• Motor proteins bind to specific filament types and “walk” or slide along cytoskeletal tracks

• They convert chemical energy from ATP hydrolysis into mechanical work

• Primary roles:

  • Transport of organelles and vesicles

  • Organization and remodeling of cytoskeleton

Actin-Based Motors

• Myosins: major family of motors that move along actin filaments

• Myosin types include conventional Myosin II and non-conventional myosins (Myosin I–XVIII)

• Typical molecular weight: varies; examples include some myosins around tens to hundreds of kDa

Microtubule-Based Motors

• Kinesins:

  • Cytoskeletal kinesins; in neurons and other cells for cargo transport along axons

  • Conventional kinesins and isoforms

  • Typical molecular weight: ~$110\,\mathrm{kDa}$

  • Move toward the + end of microtubules

• Dyneins:

  • Flagellar and cytoplasmic dyneins

  • Molecular weight: ~$500\,\mathrm{kDa}$

  • Move toward the − end of microtubules

Nucleic Acid-Based Motors (Mentioned in Context)

• Some nucleic acid enzymes (DNA and RNA polymerases) conceptually act as motor-like motors in moving along templates, illustrating broader motor principles, but not classical cytoskeletal motors

Cellular Integration and Cross-Talk

• Put together: Microtubules, intermediate filaments and actin filaments are physically linked by adaptor proteins such as plectin, creating an integrated cytoskeletal network that coordinates movement, stability, and signaling across the cell

• This integrated network allows coordinated responses to mechanical and chemical cues, spatial organization of organelles, and efficient intracellular transport

Summary of Key Structural and Functional Themes

• Three filament systems with distinct properties:

  • Actin microfilaments: thin, flexible; form cortices, filopodia, lamellipodia, microvilli; motor interactions with myosin drive contraction and movement

  • Microtubules: hollow tubes; provide tracks for long-range transport; organize chromosomes during cell division; dynamic instability enables rapid remodeling

  • Intermediate filaments: rope-like, tensile strength; non-polar; provide mechanical resilience

• Motor proteins convert ATP energy into directed movement along filaments, enabling vesicle transport, organelle positioning, and force generation for movement and shape changes

• Filament dynamics (assembly/disassembly, treadmilling, catastrophe/rescue) govern cell shape changes, polarity, and division

• Structural proteins (cross-linking proteins, spectrin, filamins, α-actinin, fimbrin, villin, plectin) organize filaments into networks and bundles, modulating rigidity and flexibility

Notation and Key References from the Transcript (LaTeX-formatted summary)

• Actin monomer details: $MW = 43\,\mathrm{kDa},\; 375\,\mathrm{aa},\; 1\;\text{bound ATP/ADP},\; 4\ \text{subdomains}$

• Actin filament: $ext{twist} = 37\ \mathrm{nm},\; \text{thickness} \approx 7\ \mathrm{nm}$

• Actin polymerization: $k<em>{on}^{+} \approx 5-10 \times k</em>{on}^{-}$; ATP hydrolysis: $\mathrm{ATP} \rightarrow \mathrm{ADP} + P_i$

• Actin ends: barbed end (+) and pointed end (−)

• Microtubule dimensions and composition: $d \approx 25\ \mathrm{nm},\; n_{pf} = 13$; tubulin monomers: $MW \approx 50\,\mathrm{kDa},\; \alpha/\beta\text{-tubulin},\; 1\ \text{GTP or GDP bound}$

• Microtubule polarity and dynamics: + end rapid polymerization, − end slow polymerization; centrosomal nucleation via $\gamma\text{-tubulin}$; GTP cap concept and dynamic instability

• Intermediate filaments: ropelike filament structure; 8 tetramers twisted into a ~10 nm filament; diameter ~10 nm; no energy required for assembly; non-polar

• Motor proteins: Myosin (actin-based), Kinesin (plus-end directed on microtubules), Dynein (minus-end directed on microtubules); typical MWs: kinesin ~$110\,\mathrm{kDa}$, dynein ~$500\,\mathrm{kDa}$

• Structural integration: plectin links microtubules, intermediate filaments, and actin filaments to coordinate network

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