MCB 2210 L22: Microtubules

Microtubule assembly

Tubulin heterodimer = subunit

• α-tubulin

• β-tubulin

Microtubule = polymer

• ~ 24 nm in diameter

Structure of Microtubules

• Microtubules = tubes = tubular polymers

  • αβ tubulin heterodimers (encoded by 2 genes) = ALWAYS STUCK TOGETHER (under normal conditions)

    • α-tubulin = DOES NOT hydrolyze GTP (binds irreversibly)

    • β-tubulin = DOES hydrolyze GTP

  • Tube forms form from base (vertically)

    • Protofilament = single linear chain of tubulin heterodimers (lateral interactions = cylinder)

      • 13 protofilaments = 1 microtubule

      • Polar → MT = polar

• (-) end = slow growing = ringed with α-tubulin

  • IN VIVO = Usually capped + embedded in MTOC/centrosome

• (+) end = fast growing = ringed with β-tubulin

  • Dynamics primarily @ (+) end

What type of imaging is used to image microtubules?

Cryo-EM: proteins = frozen in liquid nitrogen vapor

• 2D snapshots in electron microscope

What is a common source of pure tubulin?

Pig brains (high concentration of MTs → tubulin in nervous tissue)

How can MT dynamics be controlled in vitro?

What are the 2 methods to study microtubule dynamics?

IN VITRO → Δ Temperature

• Polymerization @ 37°C

• Depolymerization @ 4°C

1. Scattering of light

   1. Polymer = scatters more light than heterodimer (larger)

2. Fluorescent tag (rhodamine/fluorescein) → tubulin → visualize MT incorporation

Describe the 3 steps of MT assembly kinetics (IN VITRO)

1. Nucleation

   1. Oligomers = stable seed/nucleus → used to initiate

      1. Slow lag phase

2. Elongation = fast addition of heterodimers

3. Steady state = when [tubulin heterodimer] = Cc

   1. Rate of elongation = rate of shrinkage → TREADMILLS

Critical concentration concept on a graph

Critical concentration = [free tubulin] in equilibrium w/ [MT] = TREADMILLINg

[Tubulin] < Cc → no polymerization

[Tubulin] > Cc → polymerization

UNTIL [Tubulin] = Cc → steady state = TREADMILLING

Explain the 2 differences between the (+) and (-) ends of MTs

1. Polymerization rate + Critical concentration

   1. (+) end = grows faster, ↓ Cc

   2. (-) end = grows slower, ↑ Cc

2. Treadmilling

   1. Constant length

   2. (+) end = growing

   3. (-) end = shrinking

Describe the role of nucleotides in MT dynamics

1. GTP-bound αβ tubulin heterodimers add @ (+) end

2. Tubulin dimer incorporates into MT

3. β-tubulin hydrolyzes GTP → GDP

   1. GTP Cap @ (+) end of MT w/ GTP-β-tubulin

   2. Rest of MT = GDP-β-tubulin

At what concentrations of free tubulin will GTP cap be lost?

[Tubulin] ~ Cc → rate of polymerization slows → GTP cap lost → exposes GDP-tubulin @ end

• GTP hydrolysis

• GTP dissociation from MT (+) end

Explain the role of nucleotides and GTP cap in MT disassembly

GTP cap → MTs elongate

• GTP-tubulin = makes lateral interactions w/ protofilaments (maintain cylindrical structure)

Loss of GTP cap → GDP cap → MTs = destabilized

• GDP-tubulin = weakens lateral interactions w/ protofilaments →

  • Protofilament splaying = lateral interactions BREAK → curl outward → rapid depolymerization + shrinkage

    • Can be completely disassembled

• Catastrophe = MT disassembly

• Rescue = MT regrowth

Explain the formation of a GTP cap

GTP cap = rate of polymerization > rate of hydrolysis

Loss of GTP cap = rate of polymerization < rate of hydrolysis

• Rate of addition of GTP = too slow → GDP-β-tubulin → weakened lateral interactions → protofilament splaying → catastrophe

Explain how protofilament splaying works with tubulin heterodimers as GTP-β-tubulin is hydrolyzed to GDP-β-tubulin.

GTP → GDP = Δ conformation → protofilament curves → stress → catastrophe

Explain the steps of dynamic instability

Individual MTs = dynamic instability

Dynamic instability = individual MTs = rapidly Δ length

• [Tubulin] ~ Cc → some MTs grow, others shrink

1. Rapid growth w/ GTP-capped (+) end

   1. Rate of polymerization > rate of hydrolysis

2. Catastrophe = loss of GTP cap → rapid shrinkage

   1. Rate of polymerization < rate of hydrolysis

3. Rescue = regain of GTP cap → rapid growth

   1. Rate of polymerization > rate of hydrolysis

4. Cycle repeats

CHANGE IS RANDOM result of conversions b/w GTP→ GDP @ (+) end

Steady state = total amount of polymer = constant, any individual MT can be elongating/shortening

Describe how a graph of microtubule length of an individual MT vs Time can demonstrate dynamic instability

Curve increasing = MT Assembly

Peak = Catastrophe

Curve decreasing = MT disassembly

Valley = Rescue

What two types of protiens is commonly used to visualize dynamic instability IN VITRO?

EB1 = (+) end tracking protein (+TIPs) → binds to GTP cap of MTs

• Detects presence/absence of GTP cap

• Tagged with GFP/fluorescent marker to visualize

Microtubule-Associated Proteins (MAPs) = interact w/ MT ends

• Tagged w/ fluorescent markers to visualize MT dynamics

What are 3 ways that cells regulate MT dynamics?

Generally: [Tubulin] > Cc → MT polymerization = favored

• MT DO NOT form randomly in cells

• Kinetic barrier to nucleation = Unfavorable process of oligomeric “seed”/nucleus” formation from tubulin heterodimers

• Microtubule Associated Proteins (MAPs)

  • Regulate MT assembly in cells

• Special Nucleation Sites = allow immediate MT polymerization (eliminate lag phase)

  • Cell regulates where + when MT polymerizes

Explain 2 methods of how localization of (+) and (-) ends of MTOC can be proven in an experiment

1. Δ temperature

   1. Depolymerization @ 4°C

   2. Polymerization @ 37°C

   3. Observe new MTS growing from centrosome → return of normal MT distribution

2. Adding/washing away drug

   1. MT destabilizing drug = depolymerization

   2. Washing drug away = polymerization

   3. Observe new MTS growing from centrosome → return of normal MT distribution

Describe 3 types of drugs that affect MT polymerization

• Taxol = MT stabilizing drug → prevents disassembly

  • Blocks mitotic spindle assembly

  • Antineoplastic (anti-cancer) drug

• Colchicine = caps (+) end of microtubules → prevent elongation → GTP cap lost → MT depolymerization

  • REVERSIBLE

• Nocodazole = binds to β-tubulin → MT depolymerization

  • REVERSIBLE

  • Antineoplastic (anti-cancer) drug

What happens to MTs when nocodazole or colchicine is removed?

MT regrowth from MTOC

Explain parts of the centrosome

Centrosome = 2 centrioles + Pericentriolar Material (PCM)

• PCM holds γ-TuRC → MT (-) ends embedded in PCM → (+) ends face outward into cytoplasm

What protein anchors MTs @ the MTOC/centrosome?

γ-TuRC = cone-shaped protein complex = nucleation template for MT growth @ PCM of centrosome/MTOC

What is the microtubule nucleator used in cells?

• γ-tubulin = microtubule nucleator

• Part of γ-tubulin ring complex (γ-TuRC)

• Purified γ-TuRC nucleates assembly of pure tubulin

  • Mechanism not well understood

• MTs NOT treadmilling in interphase cells

  • (-) ends bound to MTOCs/centrosomes

  • Dynamic instability

Describe major functions of microtubules

• Cell polarity

• Membrane trafficking

• Mitosis

• Special functions in flagella + nerve axons

• Drugs → Δ MT assembly → treat diseases (Ex. cancer)