BC 465 Lecture 5

Overview & Cellular Roles of Microtubules

• Third major cytoskeletal filament class discussed (after actin & intermediate filaments).
• Span entire cell; undergo dramatic re-arrangements (e.g., interphase array → mitotic spindle).
• Key, exclusive property: dynamic instability (iterative growth ⟷ shortening at ends).
  • Generates pushing/pulling forces required for many functions.
• Functional highlights
  • Provide internal skeleton; set overall cell polarity & geometry.
  • Polarised tracks for vesicle/organelle transport via kinesin (＋) & dynein (−) motors.
  • Shape organelles (stretch ER, position Golgi, etc.).
  • Core of cilia/flagella; dynein-driven beating.
  • Build mitotic spindle; segregate chromosomes.
  • Neurons: drive neurite extension in development; remain very stable in mature axons for long-range signalling.

Structural Organization of the Polymer

• Hollow tube, outer diameter ≈ $25\,\text{nm}$ (largest cytoskeletal filament); actin = $7\,\text{nm}$.
• Usually $13$ parallel protofilaments rolled into a cylinder; gives a long seam visible by EM.
• Polar filament
  • Plus end: β-tubulin exposed; highly dynamic.
  • Minus end: α-tubulin exposed; relatively stable or capped in cells.

Tubulin Dimer – the Minimal Subunit

• Heterodimer = α-tubulin + β-tubulin.
  • Each ≈ $55\,\text{kDa}$ → dimer $\approx 110\,\text{kDa}$.
  • Encoded by distinct but highly homologous genes; very similar 3-D folds.
  • Affinity within dimer extremely high (dissociation constant in low picomolar range) → always dimeric in vitro & in vivo.
• Head-to-tail incorporation only → inherent polarity.

Nucleotide Binding

• Both subunits bind GTP.
  • α-tubulin GTP is trapped (non-hydrolysable, non-exchangeable) – structural only.
  • β-tubulin GTP: exchangeable & hydrolysable → central to dynamics.
• Conditions required for polymerisation in vitro: tubulin dimers + GTP + moderate heat.

Spontaneous Polymerisation Kinetics

• Classic sigmoidal curve (polymer mass vs time):
  1. Nucleation (lag, stochastic, rate-limiting).
  2. Rapid elongation.
  3. Steady-state pseudo-equilibrium (growth of some filaments balanced by shortening of others).
• EM shows de-novo nucleation proceeds via a flat sheet that later rolls into a closed tube; explains longitudinal seam.

Cellular Nucleation – MTOCs & γ-TuRC

• Microtubule Organising Centers (MTOCs) provide pre-assembled nuclei.
  • Animal cells: centrosome = pericentriolar matrix (PCM) + core pair of centrioles.
• Centrioles:
  • Nine triplet microtubule array; extremely stable & length-restricted.
  • Surprisingly not the direct nucleators – removal does not abolish nucleation/organisation.
• Actual nucleator: γ-tubulin ring complex (γ-TuRC) embedded in PCM.
  • Contains scaffold (“fish-and-chips cone”) of accessory proteins + ring of $13$ γ-tubulins templating a perfect microtubule start.
  • Cryo-EM: conical structure; recent work shows nucleation efficiency increases when an unknown factor “tightens” spacing between γ-tubulins.
• Polarity organisation established at MTOC
  • Interphase somatic cells: minus ends clustered at centrosome, plus ends radiate outwards.
  • Mitotic spindle: two centrosomes → bipolar array; plus ends face chromosomes.
  • Neurons: minus ends near soma, plus ends along axon/dendrite shafts.

Lattice Damage & In-situ Repair (GTP Islands)

• Manuel Théry lab (≈ 2020) showed microtubule shafts can incur mechanical damage (bending at membranes, cross-overs, laser nicks, flow-induced shear).
• Damaged protofilaments lose subunits → gap enlarges if insult persists.
• Free GTP-tubulin dimers fill the gap within the lattice (middle insertion) → “GTP speckles/islands”.
  • Experiment: Rhodamine (red) microtubules + GFP-tubulin bath; laser pulse → green spot at damage site & green elongation at plus end.
  • GTP islands render region more stable (mechanistic link to dynamic instability revisited later).
• Exception to textbook rule that dimers add only at filament ends.

Dynamic Instability – Definition & Terminology

• Individual microtubule alternates between phases:
  • Growth (polymerisation).
  • Rapid shortening (depolymerisation).
• Stochastic transitions
  • Catastrophe: growth → shortening.
  • Rescue: shortening → growth.
• Life-history plots show saw-tooth profiles; at population level total polymer mass can appear constant (steady state).

Molecular Mechanism

1. Free dimer carries GTP on β-tubulin.
   • GDP will rapidly exchange to GTP due to high cellular $[\text{GTP}] \gg [\text{GDP}]$; no dedicated GEF required.
2. Incorporation into lattice accelerates GTP hydrolysis.
3. Post-hydrolysis β-tubulin undergoes pronounced bend/kink ⇒ GDP-tubulin is energetically unfavourable inside straight lattice.
4. As long as a terminal layer of GTP-tubulin (“GTP cap”) exists, bent GDP subunits are mechanically constrained.
5. If hydrolysis catches up before new GTP dimers add, cap is lost ⇒ constrained protofilaments peel outward → catastrophe.
   • Depolymerisation speed ≈ $100\times$ polymerisation rate.
6. Depolymerisation releases stored lattice energy; measured force output $\approx 30\text{–}60\,\text{pN}$ (≈ $10\times$ kinesin stall force) – drives chromosome movement, etc.
7. Rescue occurs when a new GTP cap re-forms (high free tubulin, embedded GTP islands, or unknown factors).

Cap Size Determinants

$\text{Cap size} \propto \frac{\text{Addition rate}}{\text{Hydrolysis rate}}$

• Hydrolysis rate in lattice ≈ constant.
• Addition rate ∝ free GTP-tubulin concentration.
  • High tubulin → stable, growth-biased.
  • Dilution → frequent catastrophes.

Visual Hallmarks (EM & Light Microscopy)

• Growing ends: straight or slight sheet protrusion; sometimes flat sheet visible before curling around.
• Shrinking ends: curled “ram’s horns”; peeled protofilament ribbons may detach forming inside-out rings (used to test lumen-binding proteins).

Biological & Experimental Implications

• Plus-end dynamics harnessed for cellular mechanics (spindle positioning, neurite outgrowth).
• Minus ends generally capped in vivo; their dynamics comparatively understudied (“black box”), though they can depolymerise slowly upon tubulin dilution.
• Dynamic instability entirely reconfigures microtubule network during cell-cycle transitions (interphase → mitosis).
• In vitro, pure tubulin recapitulates full dynamic instability cycle; addition of MAPs or lattice repair modifies catastrophe/rescue frequencies.

Key Numbers, Constants & Equations

• Outer diameter: $25\,\text{nm}$ (vs actin $7\,\text{nm}$).
• Typical protofilament count: $13$.
• Tubulin dimer mass: $\sim110\,\text{kDa}$.
• Dimer dissociation constant: $K_D \approx \text{low picomolar}$.
• Depolymerisation rate: $\sim 100\times$ growth rate.
• Force from one depolymerising end: $30\text{–}60\,\text{pN}$.
• γ-TuRC subunits: $13$ γ-tubulins per complex.

Outstanding Questions & Recent Discoveries

• Identity of factor(s) that tighten γ-TuRC conformation for maximal nucleation.
• Detailed biochemical pathway for lattice repair → how GTP islands trigger rescue.
• Predictive markers for catastrophe: recent preprint suggests “micro-pause” precedes shortening.
• Mechanistic understanding of minus-end dynamics; many cellular minus ends are capped by complexes (e.g., CAMSAP/Patronin family).
• Role of luminal microtubule-binding proteins revealed via inside-out rings.