Cytoskeleton Notes: Actin, Microtubules, and Intermediate Filaments (Lecture Content)

Actin filaments (microfilaments)

• Abundance and basic properties
  • Actin is one of the most abundant cellular proteins; typical cellular concentration is $[Actin] \,\approx\ 20-70\ \mu\text{M}$, up to $1\ \text{mM}$ in muscle cells.
  • Actin exists as G-actin (globular, monomer) and polymerizes into F-actin (filament).
  • Filament polarity is defined by asymmetry of the G-actin monomer.
  • Filament bundling provides rigidity.
• Structure
  • G-actin (globular) = monomer; F-actin (filament) = polymer; structure is a two-stranded helix.
  • Filament diameter: $d \approx 5-9\ \text{nm}.$
  • Filaments form networks that support cell shape, motility, and contractility.
• Polymerization dynamics
  • Growth occurs primarily at the plus end (barbed end, often denoted +).
  • Step 1: An ATP-bound actin monomer binds to a growing filament.
  • Step 2: ATP bound to the incorporation molecule is slowly hydrolyzed after incorporation; ADP-actin remains bound within the filament.
  • The minus end (pointed end) is less favorable for addition and more prone to depolymerization.
• Functional roles
  • Actin networks drive locomotion, contractility, and cytokinesis in cells.
  • Polymerized actin provides structural support and enables force generation during cell movement.

Microtubules

• Structure and dimensions
  • Microtubules are hollow tubes composed of protofilaments formed from heterodimers of tubulin: $\alpha\text{-tubulin}$ and $\beta\text{-tubulin}$.
  • Diameter: $d \approx 25\ \text{nm}.$
  • Subunit: tubulin dimer; each dimer binds GTP/GDP.
  • The plus end is the primary site of rapid growth; the minus end is usually anchored at the microtubule organizing center (MTOC).
• Nucleation and dynamics
  • GTP binding and hydrolysis drive dynamics:
  • The tubulin dimer can bind GTP (on both subunits, but hydrolysis occurs after incorporation mainly in the ß-tubulin).
  • Incorporation of a GTP-tubulin dimer promotes GTPase activity of ß-tubulin, hydrolyzing GTP to GDP.
  • α-tubulin is always GTP-bound (structurally stable).
  • Dynamic instability at the plus end involves: catastrophe and rescue.
  • Catastrophe: GDP-tubulin dissociates from the plus end, causing shrinkage.
  • Rescue: GTP-tubulin associates to the plus end, promoting growth.
• Nucleation centers and MTOCs
  • Microtubules originate from microtubule organizing centers (MTOCs) in many cells; nucleation occurs at minus ends.
  • Centrosome is a major MTOC in many cells.
  • Gamma-tubulin nucleates microtubules at the minus end via the gamma-tubulin ring complex ((\gamma\text{-TuRC})).
  • Centrosome structure includes pericentriolar material and a pair of centrioles.
• Dynamics and regulation
  • Regulation of microtubule dynamics (assembly/disassembly) mainly occurs at the plus end.
  • Regulation ensures proper intracellular transport, mitosis, and overall cellular organization.
• Microtubule motors and directionality
  • Kinesins move toward the plus end (anterograde transport, toward the cell periphery).
  • Dyneins move toward the minus end (retrograde transport, toward MTOC).
  • Motor proteins convert chemical energy into mechanical work to transport cargo along microtubules.
• Motor protein comparisons (typical speeds)
  • Kinesin-1: ≈ $1.8\times 10^3\ \text{nm/s}$
  • Cytoplasmic dynein: ≈ $1.0\times 10^3\ \text{nm/s}$
  • Myosin V (actin motor, for context): ≈ $2.0\times 10^2\ \text{nm/s}$
• Examples and visual evidence
  • Movement of vesicles/endosomes in axons can be visualized with fluorescent imaging, illustrating directional transport along secretory and endocytic pathways.
• Key terms to remember
  • Plus end, minus end; GTP cap; catastrophe; rescue; MTOC; centrosome; (\gamma\text{-TuRC}); nucleation; protofilament.

Intermediate filaments

• General properties
  • Intermediate filaments are rope-like, fibrous networks composed of coiled-coil dimers.
  • Subunits form parallel dimers that assemble into anti-parallel tetramers, which then bundle into rope-like filaments.
  • Filaments are relatively non-polar, lacking the distinct plus/minus ends seen in actin and microtubules.
  • Diameter ≈ $10\ \text{nm}$.
  • They are structurally stable and can withstand high tensile forces.
• Organization and assembly
  • Basic organization: parallel dimers -> antiparallel tetramers -> rope-like filaments.
  • Subunits/protofilaments are symmetric (non-polar).
• Major classes/types
  • Nuclear lamins (A, B, C): form the nuclear lamina underneath the inner nuclear envelope.
  • Keratins (type I acidic and type II neutral/basic): epithelial cell IFs; contribute to tissue integrity and differentiation states.
  • Others: vimentin, desmin, GFAP, peripherin (mesenchymal, muscle, glial, neuronal contexts).
• Lamins and nuclear structure
  • Lamins support nuclear strength and shape; interact with transmembrane and chromatin-associated factors.
  • Nuclear lamina interactions with nuclear pores, Emerin, LBR, MAN1, LAPs, BAF, etc., help organize chromatin and nuclear envelope architecture.
• Keratins and cell adhesion
  • Keratins connect to desmosomes (cell–cell adhesion) and hemidesmosomes (cell–basal lamina adhesion), contributing to tissue mechanical strength.
  • Keratin expression is tissue-specific and can be diagnostic in cancer (keratin expression patterns used to identify tissue origin).
  • Epidermolysis bullosa simplex results from keratin mutations compromising epithelial cell integrity.
• Keratin expression and disease diagnostic relevance
  • Tissue-specific keratin expression profiles are used in cancer diagnostics (e.g., breast cancer metastasis in lymph nodes visualized with breast-specific keratin antibodies).
• Nuclear lamins and disease relevance
  • Mutations in nuclear lamins are linked to human diseases; Lamin A (LMNA) mutations cause Hutchinson–Gilford Progeria Syndrome (Progeria) with characteristic misshapen nuclei.
  • Example references: Progeria Foundation; Scaffidi et al., PLoS Biol. 2005.
• Summary of IF features
  • Structural role: mechanical resilience and nuclear support.
  • Non-polar, stable filaments form networks that resist tension.
  • Diverse family with tissue-specific expression; important in development, disease, and diagnostics.

Key concepts and relationships across the cytoskeleton

• Three major cytoskeleton systems
  • Actin filaments (microfilaments): 5-9 nm diameter; dynamic; supports locomotion and cytokinesis.
  • Microtubules: 25 nm diameter; dynamic instability; tracks for long-range transport; form MTOCs and spindle apparatus.
  • Intermediate filaments: 10 nm diameter; highly stable; provide mechanical strength and nuclear integrity; non-polar.
• MTOCs and centrosomes
  • MTOCs organize microtubules; most cells use centrosomes with a pair of centrioles and pericentriolar material.
  • Gamma-tubulin ring complexes ((\gamma\text{-TuRC})) nucleate microtubules at the minus ends, anchored at the centrosome.
• Polarity and directionality in transport
  • Actin networks drive short-range, cortical movements and contraction.
  • Microtubules provide long-range transport; kinesins move toward plus ends; dyneins move toward minus ends.
• Dynamics and energy use
  • Polymerization and depolymerization are driven by nucleotide states (ATP for actin; GTP for tubulin).
  • The conversion of nucleotide-bound states to GDP or ADP-bound states regulates stability and turnover.
• Disease relevance and practical implications
  • Lamin and keratin mutations underlie specific human diseases, highlighting the roles of these networks in tissue integrity and aging.
  • Keratin expression profiles assist in cancer diagnostics, highlighting the clinical relevance of IFs.

Exam-style review questions (with answers)

• Q: Which motor proteins are responsible for plus-end transport along microtubules in eukaryotic cells?

  • A: kinesins. (Answer: B)

• Q: What role does GTP hydrolysis play in the dynamic behavior of microtubules?

  • A: GTP hydrolysis induces microtubule depolymerization (catastrophe) when the GTP cap is lost. (Answer: B)

• Q: What is the primary function of the MTOC in eukaryotic cells?

  • A: Microtubule nucleation and organization (centrosomal guidance of microtubule arrays). (Answer: C)

Notable numbers and symbols to remember

• Actin filament diameter: $d\approx 5-9\ \text{nm}$
• Actin concentration: $[Actin] \approx 20-70\ \mu\text{M}$ (up to $1\ \text{mM}$ in muscle)
• Microtubule diameter: $d\approx 25\ \text{nm}$
• Tubulin dimer width: $\approx 8-9\ \text{nm}$
• Growth speeds (typical motors):
  • Kinesin-1: $v \approx 1.8\times 10^3\ \text{nm/s}$
  • Dynein: $v \approx 1.0\times 10^3\ \text{nm/s}$
  • Myosin V (context on actin): $v \approx 2.0\times 10^2\ \text{nm/s}$

Connections to broader principles

• Nucleotide state controls polymer stability across cytoskeleton components (ATP in actin; GTP in tubulin).
• Structural polarity (actin and microtubules) enables directional transport and force generation.
• Mechanical integrity of tissues depends on intermediate filaments and their network organization (lamins in the nucleus; keratins in epithelia).
• Crosstalk between cytoskeletal systems underlies processes such as cell division, vesicular trafficking, and cell migration.

References to slide content (conceptual anchors)

• Three cytoskeletal systems: actin filaments, microtubules, and intermediate filaments.
• Actin polymerization steps and polarity; polymer size and structure.
• Microtubule dynamics: GTP/GDP states, catastrophe/rescue, MTOCs and centrosomes, γ-TuRC nucleation.
• Motor proteins: kinesins (plus-end directed) and dyneins (minus-end directed); speeds and functional roles.
• Intermediate filaments: lamins and keratins; structural roles, assembly, non-polarity, tissue specificity.
• Disease links: Laminopathies (Progeria), Epidermolysis bullosa simplex, cancer diagnostics via keratin profiling.

Quick glossary

• G-actin: globular actin monomer
• F-actin: filamentous actin
• plus end: growing end of actin/microtubule
• minus end: typically the slower-growing/ minus-end
• MTOC: microtubule organizing center
• γ-TuRC: gamma-tubulin ring complex (nucleates microtubules)
• protofilament: linear chain of tubulin dimers forming part of a microtubule
• lamins: nuclear lamins A, B, C
• desmosomes/hemidesmosomes: cell–cell and cell–matrix adhesion structures linked to intermediate filaments
• progeria: accelerated aging syndrome associated with Lamin A mutations