Unit 3: Cytoskeleton and Nucleus

I. Orientation — Dynamic cellular architecture

The cytoskeleton is a remodelled network of protein polymers that organizes cell shape, internal transport, movement, and division, while the nucleus stores and selectively exchanges genetic information. Its central principle is regulated organization: filament assembly, disassembly, motor activity, and nuclear transport are controlled according to cellular location and physiological state.

• Three filament systems: Microtubules are hollow tubulin polymers; intermediate filaments are rope-like, tough polymers; actin filaments are thin, polar polymers.
• Dynamic versus stable behavior: Microtubules and actin commonly undergo rapid assembly and disassembly, whereas intermediate filaments are generally more mechanically stable.
• Polarity: Microtubules and actin have structurally distinct plus and minus ends, allowing directional motor movement; most intermediate filaments lack functional polarity.
• Nuclear compartmentalization: The nuclear envelope separates nucleoplasm from cytoplasm but permits selective exchange through nuclear pore complexes.
• Energy and regulation: GTP powers tubulin polymerization dynamics, ATP supports actin cycling and motor activity, and Ran-GTP controls nuclear transport directionality.
• Mechanical integration: Cytoskeletal filaments connect to membranes, organelles, cell junctions, and the nuclear lamina to distribute forces across the cell.

II. Microtubules — Polar tracks and structural organizers

Microtubules are cylindrical polymers built from α/β-tubulin heterodimers. Their hollow structure and dynamic polarity make them suitable both for long-range intracellular transport and for rapid reorganization during mitosis.

A. Structure and functions of Microtubules

Microtubule structure determines its mechanical strength, directional transport, and ability to reorganize.

• Molecular organization: A microtubule usually contains 13 protofilaments arranged into a hollow tube approximately 25 nm in diameter; each protofilament consists of head-to-tail α/β-tubulin dimers.
• Polarity: The β-tubulin-exposed plus end generally grows faster, while the α-tubulin-exposed minus end is often anchored at a microtubule-organizing center (MTOC).
• Dynamic instability: GTP-bound tubulin adds preferentially at the plus end. GTP hydrolysis creates a GDP-tubulin lattice that is less stable, producing catastrophe or rescue.
• Cell organization: Centrosomes organize radial microtubule arrays in many animal cells; cilia and flagella contain stable axonemal microtubules.
• Intracellular transport: Kinesins usually move toward plus ends and dynein toward minus ends, carrying vesicles, mitochondria, and protein complexes.
• Cell division: Mitotic spindle microtubules attach to kinetochores, overlap with antiparallel microtubules, or contact the cell cortex to position and separate chromosomes.
• Pharmacological evidence: Taxol stabilizes microtubules, whereas colchicine and nocodazole inhibit polymerization; both types of treatment can block mitosis.

III. Intermediate filaments — Tensile-strength networks

Intermediate filaments are approximately 10 nm in diameter and are assembled from elongated fibrous proteins. Their major role is to resist stretching and preserve cellular and tissue integrity.

A. Structure and functions of Intermediate filaments

Intermediate-filament architecture explains their high tensile strength and comparatively low polarity.

• Subunit assembly: Coiled-coil dimers form antiparallel tetramers; tetramers pack laterally and longitudinally into rope-like filaments.
• Nonpolar structure: Antiparallel assembly places opposite ends together, so intermediate filaments lack distinct plus and minus ends and do not support conventional directional motor transport.
• Major families: Keratins occur in epithelial cells; vimentin occurs in mesenchymal cells; desmin occurs in muscle; neurofilament proteins occur in neurons; lamins occur at the inner nuclear envelope.
• Mechanical function: Keratin networks connect to desmosomes and hemidesmosomes, distributing tensile stress across epithelial sheets.
• Cellular resilience: Vimentin supports fibroblast shape and organelle positioning; desmin links contractile structures in muscle.
• Nuclear protection: Lamins provide a mechanically supportive meshwork beneath the inner nuclear membrane and help organize chromatin.
• Disease connection: Mutations in keratins can cause fragile skin, while lamin mutations are associated with laminopathies such as muscular dystrophy and progeroid syndromes.

IV. Actin Filaments — Force generation and cell shape

Actin filaments, or microfilaments, are approximately 7 nm in diameter and form branched or bundled networks. Their rapid remodeling and interaction with myosin allow cells to change shape and generate force.

A. Structure and functions of Actin Filaments

Actin filaments are polar polymers whose organization is controlled by nucleators, capping proteins, severing proteins, and actin-binding proteins.

• Polymer structure: Globular G-actin monomers polymerize into filamentous F-actin, producing a two-stranded helical filament with distinct plus, or barbed, and minus, or pointed, ends.
• ATP-dependent dynamics: ATP-actin adds more rapidly at the plus end; after incorporation, ATP is hydrolyzed to ADP, weakening older filament regions.
• Treadmilling: When addition at the plus end balances loss at the minus end, subunits appear to flow through the filament without net length change.
• Cell cortex: A cortical actin network beneath the plasma membrane supports membrane shape and participates in endocytosis and exocytosis.
• Cell movement: Arp2/3-mediated branching pushes lamellipodia forward; formins produce long, unbranched filaments in filopodia and contractile structures.
• Force production: Myosin II slides antiparallel actin filaments in a sarcomere or contractile ring, using ATP hydrolysis to generate contraction.
• Cell division: A cytokinetic actomyosin ring constricts the cell membrane during animal-cell cytokinesis.
• Tissue attachment: Actin connects to cadherin-based adherens junctions and integrin-associated focal adhesions, coupling cell shape to extracellular matrix forces.

V. Nuclear Envelope — Boundary and exchange interface

The nuclear envelope is a double membrane system that encloses the genome while remaining continuous with the endoplasmic reticulum. Its pores, lamina, and membrane proteins coordinate nuclear shape, transport, and genome organization.

A. Nuclear Envelope- structure of nuclear pore complex

The nuclear envelope has two membranes interrupted by large nuclear pore complexes (NPCs), which form regulated gateways between nucleoplasm and cytoplasm.

• Double-membrane structure: The outer nuclear membrane is continuous with rough endoplasmic reticulum and may bear ribosomes; the inner nuclear membrane contains proteins that bind lamins and chromatin.
• Perinuclear space: The gap between the two membranes is continuous with the ER lumen and is typically about 30–50 nm wide.
• NPC architecture: Each NPC has eightfold rotational symmetry and contains cytoplasmic filaments, a central transport channel, a nuclear basket, and scaffold proteins.
• Nucleoporins: NPCs are built from nucleoporins, or Nups. Some contain phenylalanine-glycine (FG) repeats that create a selective permeability barrier.
• Passive and facilitated movement: Small solutes can diffuse through the NPC, while larger proteins and ribonucleoprotein complexes require transport receptors.
• Continuity and selectivity: Although the envelope is continuous with the ER, NPCs selectively regulate traffic rather than allowing unrestricted membrane or protein passage.

VI. Nuclear Lamina — Mechanical scaffold beneath the inner membrane

The nuclear lamina is a meshwork of lamin intermediate filaments and associated proteins lining the inner nuclear membrane. It supports nuclear mechanics and contributes to chromatin regulation.

A. Nuclear lamina

The nuclear lamina provides both structural reinforcement and a platform for organizing nuclear contents.

• Main components: Lamins A, C, B1, and B2 polymerize beneath the inner nuclear membrane; lamin B is permanently membrane-associated through a lipid modification, whereas lamin A undergoes precursor processing.
• Membrane attachment: Proteins such as emerin and lamin B receptor connect the lamina to the inner nuclear membrane and to chromatin-associated complexes.
• Nuclear shape: The lamina resists deformation caused by cytoskeletal forces; defects can produce misshapen or mechanically fragile nuclei.
• Chromatin organization: Lamina-associated domains are often enriched in transcriptionally inactive, heterochromatic DNA near the nuclear periphery.
• Cell-cycle remodeling: During mitosis, phosphorylation causes lamin disassembly; dephosphorylation after mitosis permits reassembly around daughter nuclei.
• Functional consequence: Lamina defects can impair mechanotransduction, DNA organization, and gene expression, explaining tissue-specific laminopathies.

VII. Protein import and export through Nuclear pore complex — Selective nucleocytoplasmic transport

Nuclear transport uses soluble receptors that recognize localization signals on cargo and interact transiently with FG-repeat nucleoporins. Directionality is generated by the Ran GTPase gradient.

A. Protein import and export through Nuclear pore complex

Import and export are receptor-mediated processes that distinguish cargo by molecular signals rather than simply by size.

• Import signal: A nuclear localization signal (NLS) is commonly rich in lysine and arginine; the classical NLS is recognized by importin-α, which binds importin-β.
• Import sequence:
  1. Importin-cargo complexes bind FG-Nups and pass through the NPC.
  2. In the nucleus, Ran-GTP binds importin-β.
  3. Cargo is released, and importins return to the cytoplasm.
• Export signal: A nuclear export signal (NES) is often leucine-rich and is recognized by exportin, especially CRM1/exportin 1.
• Export sequence:
  1. Exportin binds cargo and Ran-GTP in the nucleus.
  2. The complex crosses the NPC.
  3. Cytoplasmic Ran-GTP hydrolysis causes cargo release.
• RNA-related export: mRNA, tRNA, and ribosomal subunits use specialized export factors and are often exported only after correct processing and assembly.
• Transport size: A folded protein larger than approximately 40 kDa generally requires facilitated transport, although the effective limit depends on shape and NPC conditions.

VIII. Regulation of Nuclear protein import and export — Conditional access to the genome

Nuclear transport is regulated so that signaling proteins, transcription factors, and cell-cycle regulators enter or leave the nucleus only under appropriate conditions.

A. Regulation of Nuclear protein import and export

Regulation changes the accessibility, receptor affinity, or activity of transport cargo and thereby links cytoplasmic signals to gene expression.

• Phosphorylation: Protein kinases can expose or mask an NLS or NES. For example, phosphorylation of a transcription factor may promote import, while another phosphorylation event can retain it in the cytoplasm.
• Regulated masking: Cytoplasmic inhibitor proteins can bind an NLS. Release of the inhibitor unmasks the signal and permits import.
• Ran gradient: Ran-GTP is concentrated in the nucleus because the chromatin-associated guanine-nucleotide exchange factor RCC1 converts Ran-GDP to Ran-GTP there; Ran-GAP in the cytoplasm stimulates GTP hydrolysis.
• Directionality:
  1. Nuclear Ran-GTP promotes import-cargo release.
  2. Cytoplasmic Ran-GTP hydrolysis promotes export-cargo release.
     This spatial cycle prevents random bidirectional mixing.
• Transport receptor control: Importin and exportin availability, receptor phosphorylation, and competition among cargos alter transport rates.
• Cell-cycle control: Nuclear-envelope breakdown during open mitosis temporarily removes the NPC barrier; after mitosis, envelope reassembly restores regulated transport.
• Stress responses: Heat shock, oxidative stress, or DNA damage can change NPC permeability, modify transport factors, or redistribute transcription regulators.
• Export inhibition example: Leptomycin B inhibits CRM1, causing CRM1-dependent NES cargo to accumulate in the nucleus; this demonstrates that export depends on a specific receptor rather than passive diffusion alone.