BC 465 Lecture 3

Cytoskeleton Overview

• Cells must organize internal components and adapt their organization in response to developmental cues, external signals, or intrinsic programs (e.g., migration, gastrulation, mitosis).
• Three filament systems (diameter, historical naming, major roles)
  • Actin / Microfilaments – $\sim 7\,\text{nm}$; first discovered, originally called "microfilaments" only.
  • Microtubules – $\sim 25\,\text{nm}$; largest; 13 protofilaments form a hollow tube.
  • Intermediate filaments – $\sim 10\,\text{nm}$; “intermediate” in size; diverse family.
• Beyond a mere “skeleton,” filaments drive force-generating events: division, motility, vesicle/organelle transport, signaling, transcriptional regulation.

General Properties Shared by Cytoskeletal Polymers

• Built from soluble subunits that can exist as monomers (or heterodimers for tubulin).
• Assemble into multi-protofilament structures (2 for actin, 13 for microtubules, variable for IFs) → lateral + longitudinal bonds impart high tensile strength (breaking a single bond no longer severs the whole filament).
• Dynamic Instability / Treadmilling – ability to polymerize & depolymerize rapidly underlies adaptability and force generation.
• In vitro, pure protein + minimal buffer + nucleotide (ATP for actin; GTP for tubulin) → spontaneous self-assembly (useful for kinetic assays).

Actin: Structure & Nomenclature

• Monomer (G-actin) ≈ $42\,\text{kDa}$, binds 1 ATP.
• Polymer (F-actin) = two twisted protofilaments; helical pitch ≈ $75\,\text{nm}$.
• Intrinsic polarity → structurally distinct ends:
  • Plus / barbed end – fast growth.
  • Minus / pointed end – slow growth.
• ATPase activity
  • ATP hydrolysis is slow in free monomer.
  • Rapidly accelerated once subunit incorporates into filament.

Polymerization Kinetics & Critical Concentration

• Typical in-tube polymerization curve (percent F-actin vs. time)
  1. Lag / Nucleation phase – rate-limiting, stochastic formation of stable nuclei; eliminated experimentally by adding short “seeds.”
  2. Elongation phase – rapid, linear increase in filament mass.
  3. Steady state – net assembly = net disassembly.
• Critical concentration (Cc) – free monomer concentration at which $\text{rate}</em>{\text{on}} = \text{rate}_{\text{off}}$.
  • If [G] > Cc → net assembly; if $[G] < C</em>c$ → net loss.
  • Ends have distinct $C_c$ values:
  • Plus end $C_c^+ \approx 0.1\,\mu\text{M}$.
  • Minus end $C_c^- \approx 0.8\,\mu\text{M}$.
  • Cytoplasmic G-actin concentration typically falls between these numbers ⇒ intrinsic treadmilling.

Molecular Basis of Treadmilling

• Three key facts
  1. ATP-actin adds preferentially; ADP-actin dissociates more readily.
  2. Hydrolysis lags behind addition at the plus end → “ATP cap.”
  3. Minus end becomes ADP-rich → higher off-rate.
• Net outcome: subunits add at + end, flow through filament, dissociate at − end (visualized as “conveyor-belt”).
• Force generation: treadmilling at the lamellipod pushes the plasma membrane forward during crawling.

Experimental Visualization – Photoactivatable GFP (PA-GFP)

• Mutated GFP remains dark until a $405\,\text{nm}$ pulse restores fluorescence.
• Strategy: express Actin-PA-GFP → activate a diffraction-limited spot → track the fluorescent cohort.
• Observation: activated stripe moves from leading edge toward cell interior, confirming plus-to-minus treadmilling flux.

Actin-Binding Proteins (ABPs)

Monomer Sequestration & Activation

• Thymosin – tiny (4-5 kDa); binds G-actin and sequesters it. Up to 40 % of cellular actin held inactive → reservoir for rapid use.
• Profilin – multifunctional:
  • Competes with thymosin; releases G-actin.
  • Catalyzes ADP→ATP exchange on G-actin (recharging).
  • Binds poly-proline “whiskers” of formins, delivering ATP-actin directly to the growing barbed end.

Nucleators

• Arp2/3 complex
  • Contains Arp2 + Arp3 (actin structural mimics) + 5 accessory subunits.
  • Activating factor (WASP/WAVE family) induces conformational change bringing Arp2 & Arp3 together → template for a new filament at $\sim 70^{\circ}$ branch off existing mother filament.
  • Generates dendritic networks in lamellipodia; also exploited by pathogens (see below).
• Formins
  • Dimeric; FH2 domains dimerize to hold two actin monomers → seed for an unbranched filament.
  • Remain processively attached to barbed end; FH1 “whiskers” recruit profilin-ATP-actin → rapid elongation.
  • Produce long, parallel bundles (filopodia, stress fibers, cytokinetic ring).

Severing & Depolymerization

• Cofilin / Actin Depolymerizing Factor (ADF)
  • Binds ADP-F-actin 1:1, introduces additional twist → thermally driven severing.
  • Preferentially targets minus-end, ADP-rich regions → accelerates filament turnover.

End-Capping & Stabilizing Proteins

• Capping Protein (CapZ, gelsolin-family members, etc.)
  • Binds barbed end → halts further addition → shifts balance toward minus-end loss; indirectly promotes turnover.
• Tropomyosin
  • Coiled-coil dimer that decorates along F-actin helix; generally stabilizes filaments (important in muscle thin filaments and some cytoskeletal bundles).

Integrated Example: Lamellipodial Crawling Cycle

1. External cue activates WASP/WAVE → Arp2/3 bursts nucleate branched network at membrane.
2. Profilin-ATP-actin pool feeds rapid barbed-end elongation ⇒ protrusive force.
3. As filament ages, ATP→ADP conversion; minus ends enriched in ADP-actin.
4. Cofilin binds ADP segments → severing; barbed ends of new fragments may be re-used or capped.
5. Capping protein terminates older barbed ends, funneling monomers back into profilin cycle.
6. Continuous treadmilling drives membrane extension while recycling subunits (spatially restricted by localized phosphorylation cascades that activate/inactivate ABPs).

Dynamic Rearrangements During Cell Cycle

• Interphase: long radial microtubules, cortical/lamellipodial actin.
• Entry into mitosis: rapid MT depolymerization and re-assembly into bipolar spindle; actin reorganizes into short filaments under cortex for cytokinetic contractile ring.
• Exit mitosis: disassembly of spindle, re-growth of interphase arrays; demonstrates speed & plasticity of cytoskeleton.

Force-Driven Phenomena Illustrated in Videos

• Dynamic instability – EB-labelled microtubules “grow–shrink–grow” (green flares at ends).
• Neutrophil chasing Staphylococcus aureus – actin waves (pseudo-colored red) remodel every second to steer toward chemoattractant.
• Listeria monocytogenes “comet tails”
  • Bacterial surface protein ActA recruits Arp2/3 → actin rockets propel pathogen through cytosol and into neighboring cells.
  • Demonstrates magnitude of polymerization force (modeled biophysically).

Additional Biological Contexts for Actin

• Muscle contraction (actin + myosin II sliding).
• Cytokinetic ring constriction.
• Cell–ECM adhesion (stress fibers, focal adhesions).
• Intracellular trafficking – actin “back roads” vs. microtubule “highways.”
• Clathrin-mediated endocytosis – actin filaments assemble at pits to drive vesicle scission.
• Nuclear functions – G-actin modulates transcription factors; links cytoskeleton to gene expression.

Numbers & Trivia

• PubMed search (lecture date): $122{,}522$ papers cite “actin”; ≈100 new papers/week → field still rapidly expanding.
• Actin diameter $\approx 7\,\text{nm}$; microtubule $25\,\text{nm}$; intermediate filament $10\,\text{nm}$.
• One helical turn of F-actin = $75\,\text{nm}$ (≈ 37 subunits).
• Human cytoplasmic G-actin concentration can reach $\sim 200\,\mu M$, but only ≈60 % is polymerized thanks to thymosin sequestration.

Experimental & Practical Implications

• Seeding strategies overcome nucleation lag in vitro assays.
• Photoactivation / FRAP / single-molecule TIRF critical for measuring on/off kinetics.
• Pharmacological modulators (e.g., latrunculin binds G-actin; cytochalasin caps barbed ends) exploit principles discussed for research & potential therapeutics.
• Pathogen hijacking (Listeria, Shigella, vaccinia virus) underscores importance of host cytoskeletal regulation for infection control.

Ethical / Real-World Relevance

• Food safety: unpasteurized cheeses risk Listeria outbreaks due to actin-based motility enabling cell-to-cell spread.
• Cancer metastasis research targets actin regulators (e.g., Arp2/3, cofilin) to curb invasive migration.
• Neurodegenerative diseases (Alzheimer’s) linked to mis-regulated cofilin-actin rods; discovery by Jim Bamburg highlights bench-to-bedside trajectory.