Cytoskeleton – Actin Filaments, Dynamics, Motor Proteins & Pharmacology

The Cytoskeleton – General Concepts

• Complex, highly dynamic network of protein filaments that fills the cytoplasm of all eukaryotic cells.
  • Three filament systems work collectively to give a cell its strength, shape, internal organization and motility.
  • Provides the physical basis for cell–cell and cell–ECM interactions, determining cell polarity and tissue architecture.
• Core purposes
  • Maintain & modify cell shape (externally and internally).
  • Resist mechanical stress and deformation.
  • Anchor the cell to neighbours and to the extracellular matrix (ECM), thereby stabilising tissues.
  • Facilitate directed movement (migration, organelle transport, chromosome segregation, muscle contraction).
• Enables formation of specialised structures
  • Microvilli – apical protrusions that enlarge membrane surface area.
  • Desmosomes & Adherens Junctions – mechanical and signalling junctions between cells.
  • Maintenance of apical vs. basolateral membrane domains in epithelia.

Classification of Cytoskeletal Filaments

• Actin filaments (Microfilaments)
  • Diameter: $7 \text{ nm}$
  • Govern cell shape and locomotion
• Microtubules
  • Diameter: $24 \text{ nm}$
  • Establish positions of membrane-bound organelles, conduct intracellular transport, build the mitotic spindle.
• Intermediate filaments
  • Diameter: $8{-}12 \text{ nm}$
  • Provide mechanical strength and resilience.
• Microscopy
  • Each filament class can be visualised by immunofluorescence staining with specific antibodies or binding probes.

Shared Architectural Theme

• Actin & microtubules: built from compact, globular subunits (G-actin, α/β-tubulin) that assemble into helical polymers.
• Intermediate filaments: composed of elongated, fibrous subunits that intertwine into strong ropelike structures.
• Assembly relies on end-to-end and side-to-side contacts → yields polarised, self-organising polymers.

Actin Filaments – Structure & Properties

• Ubiquitous in eukaryotic cells; execute mechanical & motile duties.
• Filament (F-actin) = right-handed, two-stranded helix of G-actin monomers.
  • Filament ends are polar
  • (+) end grows faster
  • (–) end grows more slowly and depolymerises more readily.
  • Tough yet flexible, generally shorter than microtubules.
• Essential functions
  • Mechanical support of membrane protrusions (microvilli, lamellipodia, filopodia).
  • Cell crawling, phagocytosis, cytokinesis.
  • Basis for muscle contraction (with myosin).

Actin Dynamics – Polymerisation/Depolymerisation

• Three kinetic phases (in vitro & in vivo)
  1. Nucleation – formation of a stable actin trimer.
  2. Elongation – rapid monomer addition at both ends (faster at the $+$ end).
  3. Steady state / Treadmilling – net addition at $+$ end equals loss at $-$ end.
• Nucleotide state controls stability
  • Each free monomer binds ATP.
  • After assembly, ATP → ADP $+$ $P_i$ (hydrolysis)
  • $\text{ATP–actin}$: high affinity for neighbours → stabilises filament.
  • $\text{ADP–actin}$: weakened binding → favours dissociation.
• Key accessory proteins
  • Profilin – binds G-actin, blocks nucleation but feeds monomers to $+$ end.
  • Cofilin – binds ADP-actin within filament, accelerates disassembly at $-$ end.

Spatial Organisation by Actin-Binding Proteins (ABPs)

• Bundle-forming (rigid, parallel) cross-linkers
  • Example: Fascin – generates tight linear bundles (filopodia, stress fibres).
  • In microvilli: Villin & Fimbrin pack filaments closely → increase surface area & support contraction (with Myosin I + Calmodulin).
• Gel-forming (flexible) cross-linkers
  • Example: Filamin – intersects filaments nearly at right angles → forms loose 3-D meshworks, endows cytoplasm with gel-like consistency.

Actin-Driven Cell Motility

• Three universal steps
  1. Protrusion of leading edge via actin polymerisation (lamellipodia/filopodia).
  2. Attachment to substrate through integrins & ECM linkers.
  3. Traction – rear of cell contracts (actomyosin stress fibres) & moves body forward.
• Cell types employing this mechanism
  • Amoebae, neutrophils, fibroblasts, neuronal growth cones, etc.
• Visual demonstrations
  • Videos cited (amoeba crawling; neutrophil phagocytosing MRSA) illustrate rapid, actin-mediated shape changes.

Actin in Cytokinesis

• Post-mitosis, a contractile ring of actin + Myosin II forms beneath plasma membrane.
• Constriction pinches parent cell into two daughter cells.

Motor Proteins – The Myosin Family

• All actin-dependent motors are myosins; share a motor head domain with ATPase activity.
  • ATP hydrolysis → conformational change → movement along actin.
• Major classes highlighted
  • Myosin I: single head, short tail → vesicle & membrane transport; links actin to plasma membrane.
  • Myosin II: two heads + long coiled-coil tail; forms bipolar thick filaments → muscle contraction & stress fibre tension.
  • Myosin V: processive two-headed motor, long lever arms → long-range cargo transport (mRNAs, organelles, mitochondria) & peripheral tethering.

Actin & Myosin in Muscle Tissue

• Myofibril = chain of repeating sarcomeres (muscle’s functional unit).
• Each sarcomere contains
  • Thin filaments: actin + regulatory proteins (tropomyosin, troponins TnI/TnC/TnT).
  • Thick filaments: myosin II.
• Sliding Filament Mechanism
  • Cross-bridge cycle (ATP-driven):
  1. ATP binding → myosin detaches from actin.
  2. ATP hydrolysis → ‘cocked’ myosin head (ADP $+$ $P_i$) aligns with new actin site.
  3. Pi release → strong binding → power stroke (head pivots, thin filament slides).
  4. ADP release completes cycle; remains attached until new ATP binds.
• Ca$^{2+}$ regulation
  • At rest: tropomyosin/troponin block myosin-binding sites on actin.
  • Neural signal → depolarisation → $\text{Ca}^{2+}$ release from sarcoplasmic reticulum.
  • $\text{Ca}^{2+}$ binds TnC, shifting complex, exposing binding sites → contraction proceeds.

Rigor Mortis (Forensic/Industrial Relevance)

• Post-mortem: no O$_2$ → no ATP production.
  • Myosin cannot detach from actin → muscle rigidity.
  • SR deteriorates → excess cytosolic $\text{Ca}^{2+}$ reinforces contraction.
• Important in forensic timing of death & meat industry (meat tenderness, economics).

Drugs & Toxins Affecting Actin (and Microtubules)

• Experimental & clinical value: manipulate cytoskeletal dynamics.

Actin-Specific Compounds (Table 16-2)

• Phalloidin vs. α-Amanitin
  • Both from the deadly Amanita mushroom group.
  • Phalloidin targets actin (cytoskeletal poison; useful probe).
  • α-Amanitin is not a cytoskeletal drug; it inhibits RNA-polymerase II → blocks transcription (illustrates specificity of toxins).

Microtubule-Specific Compounds (for contrast)

Ethical, Practical & Clinical Connections

• Understanding cytoskeletal drugs informs cancer chemotherapy, antifungal strategies, and cell biology research.
• Cytoskeletal malfunctions underlie diseases (muscular dystrophies, cardiomyopathies, neurodegeneration).
• Knowledge of actin–myosin mechanics guides biomaterial design, tissue engineering, and robotic actuation models.