8.2 Cytoskeleton & Cell Junctions

The eukaryotic cytoskeleton is a dynamic intracellular network of protein filaments extending throughout the cytoplasm. It provides structural scaffolding to maintain cell shape, organizes organelle localization, generates contractile forces for motility and division, and facilitates intracellular transport. Multicellular organization further depends on specialized cell junctions that anchor adjacent cells to one another and to the extracellular matrix.

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Microfilaments: Actin Dynamics, Polarity & Motility

Microfilaments are the narrowest cytoskeletal fibers (~7 nm in diameter), composed of globular actin monomers (G-actin) that polymerize into two-stranded helical filaments (F-actin).

[Minus (-) End: Pointed]                                   [Plus (+) End: Barbed]
   ADP-Actin Dissociation <--- [F-Actin Filament] <--- ATP-Actin Monomer Addition
   (Rate = k_off)                                          (Rate = k_on)

Structural Polarity & Treadmilling

Actin filaments possess structural polarity, defined by asymmetrical monomer binding orientations:

• Plus (+) End (Barbed End): Fast-growing end where ATP-bound G-actin monomers assemble rapidly.
• Minus (-) End (Pointed End): Slow-growing end where disassembly predominates.

Upon incorporation into F-actin, G-actin hydrolyzes its bound ATP to ADP + $\text{P}i$. Inorganic phosphate is slowly released, leaving ADP-bound actin within the older core of the filament. Because ATP-bound actin has a lower critical concentration ($C_c^+$) for polymerization than ADP-bound actin ($C_c^-$): $C_c^+ < C_{\text{steady}} < C_c^-$ At steady-state monomer concentrations ($C{\text{steady}}$), the rate of ATP-actin addition at the (+) end precisely equals the rate of ADP-actin dissociation at the (-) end. Monomers continuously migrate through the filament from (+) to (-) end without altering total filament length—a phenomenon known as treadmilling.

Actin Regulatory Proteins & Cell Motility

Cellular actin architecture is controlled by specific actin-binding proteins:

• Profilin: Promotes ADP-to-ATP exchange on G-actin, accelerating assembly at the (+) end.
• Cofilin: Binds ADP-actin filaments and enhances severing and depolymerization at the (-) end.
• Arp2/3 Complex: Nucleates new actin branches at a $70^\circ$ angle relative to existing filaments, forming dense, gel-like actin networks responsible for lamellipodia (sheet-like membrane protrusions at the leading edge of migrating cells).
• Formins: Nucleate linear, unbranched actin filaments found in filopodia (finger-like sensory projections) and stress fibers.

Physiological Functions of Microfilaments

1. Cytokinesis: During M-phase, a contractile ring comprised of F-actin and Myosin II motor proteins forms beneath the equatorial plasma membrane. Myosin-driven sliding of actin filaments constricts the ring, creating the cleavage furrow that pinches the cell into two daughter cells.
2. Muscle Contraction: In skeletal and cardiac muscle, F-actin forms the thin filaments of the sarcomere, interacting with thick myosin filaments during cross-bridge cycling.
3. Microvilli Structure: Parallel bundles of F-actin cross-linked by villin and fimbrin support microvilli, increasing the absorptive surface area of intestinal epithelial cells.

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Microtubules: Tubulin Architecture, Dynamic Instability & Motor Proteins

Microtubules are rigid, hollow cylindrical tubes with an outer diameter of ~25 nm and an inner lumen of ~15 nm. They are composed of heterodimers of $\alpha$-tubulin and $\beta$-tubulin aligned end-to-end into linear protofilaments. Typically, 13 protofilaments associate laterally to form the cylindrical microtubule wall.

[ Centrosome / MTOC ]                                     [ Cell Periphery / (+) End ]
  (-) End anchored via gamma-TuRC                           GTP Cap ---> Assembly
  Stable (-) end                                            Loss of GTP Cap ---> Catastrophe
  <------------------ DYNEIN (Retrograde) -------------------
  ------------------- KINESIN (Anterograde) ----------------->

Structural Polarity & Dynamic Instability

Microtubules are polar structures:

• Plus (+) End: Exposes $\beta$-tubulin subunits; location of rapid assembly and disassembly.
• Minus (-) End: Exposes $\alpha$-tubulin subunits; typically anchored at the microtubule organizing center.

Both $\alpha$- and $\beta$-tubulin subunits bind GTP. However, while GTP bound to $\alpha$-tubulin is structurally permanent, GTP bound to $\beta$-tubulin is hydrolyzed to GDP shortly after incorporation into the polymer. Microtubules exhibit dynamic instability, alternating stochastically between phases of rapid growth (polymerization) and rapid shrinkage (depolymerization):

• GTP Cap & Growth: As long as the rate of GTP-tubulin addition at the (+) end exceeds the rate of GTP hydrolysis, the (+) end retains a protective GTP cap. GTP-tubulin dimers form straight protofilaments that pack tightly.
• Catastrophe: If GTP hydrolysis catches up to the (+) end and the GTP cap is lost, GDP-tubulin subunits exposed at the tip undergo a conformational transition, curving outward. The protofilaments peel apart rapidly, leading to explosive depolymerization termed catastrophe.
• Rescue: Re-acquisition of a GTP cap halts depolymerization, initiating growth (rescue).

Microtubule Organizing Center (MTOC) & Centrosomes

In animal interphase cells, the primary MTOC is the centrosome, located near the nucleus. The centrosome contains a pair of barrel-shaped centrioles oriented perpendicular ($90^\circ$) to each other, surrounded by pericentriolar material (PCM). Each centriole consists of a cylindrical array of 9 triplets of microtubules ($9 \times 3 + 0$ structural arrangement). The PCM contains $\gamma$-tubulin ring complexes ($\gamma$-TuRCs), which serve as nucleation templates anchoring the (-) ends of microtubules while (+) ends project outward toward the cell periphery.

Molecular Motor Proteins: Kinesin vs. Dynein

Microtubules serve as intracellular tracks for ATP-dependent motor proteins that transport vesicles, organelles, and protein complexes:

• Kinesin: Most kinesins move in an anterograde direction toward the plus (+) end of microtubules (from the cell center to the cell periphery / axon terminal). Structure: Heavy chains with globular motor heads that hydrolyze ATP and walk along tubulin, attached to light chains that bind specific cellular cargo.
• Dynein: Cytoplasmic dynein moves in a retrograde direction toward the minus (-) end of microtubules (from the cell periphery back to the centrosome / soma). Transports endosomes, lysosomes, and retrogradely transported axonal signals.

MCAT Neurobiology Trap: Fast axonal transport in neurons relies on kinesin to transport newly synthesized neurotransmitter vesicles from the soma to the axon terminal (+ end), whereas dynein transports recycled endosomes and neurotrophic signals back to the soma (- end). Pathogens such as Rabies virus, Herpes simplex virus, Poliovirus, and Tetanus toxin exploit dynein for retrograde transport into the central nervous system.

Axonemal Structure of Cilia and Flagella

Motile cilia and flagella are specialized cell-surface projections built upon a central microtubule core called the axoneme:

• Axoneme Architecture: Possesses a $9 + 2$ arrangement, consisting of 9 outer doublet microtubules surrounding 2 central singlet microtubules.
• Dynein Arms & Nexin: Each outer doublet features inner and outer axonemal dynein arms that hydrolyze ATP to walk along the adjacent doublet. Flexible protein bridges composed of nexin link adjacent doublets, converting the sliding force generated by dynein into bending waves.
• Basal Body: The axoneme is anchored at its base by a basal body, which shares the identical $9 \times 3 + 0$ triplet array of centrioles.

Clinical Correlation: Primary Ciliary Dyskinesia (Kartagener Syndrome) An autosomal recessive disorder caused by mutations in genes encoding axonemal dynein arms. Results in immotile cilia and flagella, presenting clinically with chronic sinusitis, bronchiectasis, male infertility (immotile sperm), and situs inversus (transposition of visceral organs due to loss of embryonic nodal ciliary fluid flow).

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Intermediate Filaments: Non-Polar Structural Mechanical Integrity

Intermediate filaments have an intermediate diameter (~10 nm) between microfilaments (7 nm) and microtubules (25 nm). Unlike microfilaments and microtubules, intermediate filaments are extraordinarily stable, non-polar fibers specialized for absorbing mechanical stress and maintaining cellular structural integrity.

[Monomer] ---> [Parallel Dimer] ---> [Antiparallel Tetramer] ---> [Protofilament] ---> [Non-Polar Filament (10 nm)]
                                            |
                             Antiparallel orientation cancels polarity
                             (No + or - ends; No ATP/GTP required)

Assembly & Absence of Polarity

Intermediate filament proteins possess a central conserved $\alpha$-helical rod domain. Two monomers wrap around each other to form a parallel coiled-coil dimer. Two dimers associate in an staggered, antiparallel fashion to form a non-covalent tetramer. Because the tetramer subunits are oriented in opposite directions, intermediate filaments lack structural polarity (there are no + or - ends). They do not bind ATP or GTP, nor do they undergo treadmilling or dynamic instability.

Tissue-Specific Classes of Intermediate Filaments

Intermediate filaments exhibit strict cell-type specificity, making them invaluable markers in tumor histopathology:

1. Keratins: Expressed in epithelial cells. Keratins form tonofilaments attached to desmosomes and hemidesmosomes, conferring resistance to mechanical friction. Mutations in keratin genes cause Epidermolysis Bullosa Simplex, characterized by severe skin blistering following mild mechanical trauma.
2. Vimentin: Expressed in mesenchymal cells (fibroblasts, endothelial cells, smooth muscle, leukocytes). Used clinically as a histological marker for mesenchymal tumors (sarcomas).
3. Desmin: Expressed in muscle cells (skeletal, cardiac, smooth). Links sarcomere Z-discs to the sarcolemma, maintaining myofibrillar alignment.
4. Neurofilaments (NF-L, NF-M, NF-H): Expressed in neurons, providing structural support within axons and determining axonal caliber (diameter), which directly dictates nerve conduction velocity.
5. Nuclear Lamins (Lamin A, B, C): Form the nuclear lamina beneath the INM. Phosphorylation of lamins by Cyclin B / CDK1 during prophase causes nuclear envelope breakdown.

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Cell Junctions: Tissue Architecture, Barrier Integrity & Intercellular Communication

In epithelial sheets and multicellular tissues, specialized plasma membrane junctional complexes maintain spatial organization, prevent paracellular leakage, and coordinate metabolic and electrical signaling.

[Apical Domain]
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  Tight Junctions (Zonula Occludens) --------> Paracellular Barrier (Claudins/Occludins)
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  Adherens Junctions (Zonula Adherens) ------> Actin Belt Coupling (E-Cadherin)
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  Desmosomes (Macula Adherens) --------------> Intermediate Filament Spot Welds (Desmoglein)
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  Gap Junctions (Nexus) ---------------------> Direct Passage < 1 kDa (Connexons)
      |
[Basolateral Domain]
  Hemidesmosomes ----------------------------> Basement Membrane Anchor (Integrins)

Tight Junctions (Zonula Occludens)

• Molecular Composition: Transmembrane proteins Claudins and Occludins interacting intercellularly, linked intracellularly to peripheral membrane proteins (ZO-1, ZO-2).
• Function: Form a continuous belt-like seal around the apical perimeter of epithelial cells, sealing the paracellular space. They establish a selective paracellular diffusion barrier restricting passive movement of water, ions, and solutes between cells. Furthermore, tight junctions act as a "fence" preventing lateral diffusion of lipids and membrane proteins between apical and basolateral plasma membrane domains, maintaining cell polarity.
• Key Locations: Blood-Brain Barrier (BBB) (non-fenestrated brain capillary endothelial cells), intestinal epithelium, renal distal tubules and collecting ducts.

Desmosomes (Macula Adherens) & Hemidesmosomes

• Desmosomes: Button-like "spot welds" that rivet adjacent cells together, resisting severe mechanical shearing stress. Transmembrane calcium-dependent adhesion proteins (Desmoglein and Desmocollin, members of the cadherin superfamily) bind across the intercellular space. Intracellularly, these cadherins attach to dense cytoplasmic plaques (containing Desmoplakin and Plakoglobin) that anchor keratin intermediate filaments.
  • Clinical Trap: Pemphigus Vulgaris: An autoimmune disease caused by IgG autoantibodies targeting Desmoglein-3. Disruption of desmosomes leads to loss of cell-to-cell adhesion (acantholysis), resulting in painful, flaccid intraepidermal skin blisters and mucosal erosions (positive Nikolsky sign).
• Hemidesmosomes: Anchor the basal surface of epithelial cells to the underlying extracellular matrix basement membrane. Transmembrane Integrins (specifically $\alpha_6\beta_4$ integrin) link basement membrane laminin to intracellular keratin intermediate filaments.
  • Clinical Trap: Bullous Pemphigoid: Autoantibodies against hemidesmosomal proteins (BP180/BP230), causing subepidermal tense blisters with a negative Nikolsky sign.
• Adherens Junctions (Zonula Adherens): Positioned just below tight junctions. Transmembrane E-cadherins bind adjacent cells and link intracellularly via $\alpha$- and $\beta$-catenins to the actin microfilament network, forming a continuous contractile belt.

Gap Junctions (Nexus)

• Molecular Composition: Composed of hexameric complexes of transmembrane proteins called Connexins. Six connexin subunits assemble to form a hollow transmembrane channel called a Connexon. When connexons on adjacent plasma membranes align end-to-end, they form a continuous intercellular aqueous pore (~1.5–2.0 nm diameter).
• Function: Provide direct cytoplasmic continuity between neighboring cells, allowing free paracellular passage of water, small inorganic ions ($\text{Na}^+, \text{K}^+, \text{Ca}^{2+}$), second messengers ($\text{cAMP}, \text{IP}_3, \text{cGMP}$), and small metabolites (molecules $< 1\text{ kDa}$ / $1000\text{ Da}$). Large macromolecules (proteins, nucleic acids, polysaccharides) are excluded.
• Key Locations: Cardiac muscle intercalated discs (allowing rapid passage of $\text{Ca}^{2+}$ and $\text{Na}^+$ ions to propagate electrical action potentials across the cardiac syncytium, ensuring coordinated contraction), vascular smooth muscle, and electrical synapses in the CNS.

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Cytoskeletal Element & Cell Junction Summary Matrix