Microtubule Cytoskeleton Notes

The Microtubule Cytoskeleton

Subunit Addition to Polymers

• During the elongation/growth phase, subunits are added to the ends of polymers. This addition is:

  • Independent of subunit concentration (at the end)

  • Proportional to subunit concentration (overall)

• The number of subunits added per second increases with higher concentrations of free subunits.

• In a closed system with a fixed pool of subunits, subunit incorporation leads to a decrease in the concentration of free subunits.

Critical Concentration

1. The rate of addition (K_{on}) is proportional to the concentration of monomer (C).

2. The off rate (K_{off}) is independent of monomer concentration.

3. As subunits incorporate into the polymer, the concentration of the free pool decreases.

4. The concentration of free monomer where the On and Off reactions are equivalent is the Critical Concentration (C_c).

   • At the critical concentration, there is no net increase in polymer.

Subunit Incorporation and Conformational Change

• Subunits undergo a conformational change when incorporated into the filament.

• Distinctions arise from:

  1. Structural polarity

  2. Conformational change

Critical Concentration of a Simple Polymer System

• Elongation Rate vs. Monomer Concentration

  • Above 2 uM: Both ends grow

  • At 2uM: Constant length with subunit exchange at each end

  • Below 2 uM: Both ends shrink

  • C_c of plus and minus end for G-Actin = 2 uM

• Actin and tubulin bind and hydrolyze NTPs

NTP Binding and Critical Concentration

• NTP binding changes the C_c of each end :

  1. NTP-bound subunits have a higher affinity for their neighbors.

  2. Subunit incorporation stimulates NTP hydrolysis.

  3. Cc (minus end) > Cc (plus end)

Properties Dictating Filament Dynamics

1. Polarity and conformational changes

   • Properties of the subunits dictate dynamic properties of the filaments they make up.

2. Nucleotide Binding and Hydrolysis

   • The filament is like the GAP.

   • Actin and (beta)-tubulin are like enzymes.

   • Confers different rates of growth to two ends of a filament.

   • Confers different critical concentrations to the two ends of a filament.

Actin Filament Treadmilling

• Actin filaments treadmill when free monomer concentrations are between the critical concentrations for each end.

  • C_c (minus) = 0.7 mM

  • C_c (plus) = 0.1 mM

Sarcomere Structure and Muscle Contraction

• Thin filaments (actin) and thick filaments (myosin) in a sarcomere.

• Sliding filament model proposed by Hugh Huxley in 1953.

• Combined light microscopy and electron microscopy to study the phenomenon of muscle contraction.

Cell Division and Microscopy

• Shinya Inoue applied polarized light microscopy to study the phenomenon of cell division in LIVING cells in the 1950s.

  • Pollen cells from Easter lily

• Addition of the drug colchicine made the spindle disassemble.

  • Inoue S. Experimental Cell Research, 1952

  • Aronson and Inoue, Journal of Cell Biology, 1970

Identifying Spindle Filament Proteins

• How would you approach identifying the protein that makes the spindle filaments in dividing cells?

Microtubule Subunit

• An obligatory alpha/beta tubulin heterodimer

  • alpha-tubulin

  • beta-tubulin

• Protofilament = a chain of tubulin heterodimers

• The lumen is the inner space of the microtubule.

Microtubule Structure

• A microtubule is a polymer of a/b tubulin heterodimers.

• A protofilament = a/b heterodimers arranged end to end.

• 13 Protofilaments make up the microtubule.

• A microtubule has POLARITY

  • Plus end = b tubulin (faster growing)

  • Minus end = a tubulin (slower growing)

• Tubulin is a GTPase (binds to and hydrolyzes GTP)

• End on interactions and lateral interactions occur

Steady-State Dynamics of Microtubules

• Bulk assays versus individual polymers At steady state (59 uM)

• Mitchison and Kirschner, Nature, 1984

Dynamic Instability vs. Treadmilling

• Steady-state behavior:

  • Bulk polymer

  • Individual Microtubules

  • Individual Actin Filaments

Dynamic Instability Parameters

• Dynamic Instability is Defined by Four Parameters

  1. Polymerization Rate (microns/minute)

  2. Depolymerization Rate (microns/minute)

  3. Catastrophe Frequency (catastrophe events/minute)

  4. Rescue Frequency (rescue events/minute)

GTP Cap and Microtubule Dynamics

• A growing microtubule has a GTP cap (The b-tubulin subunit(s) at the end are bound to GTP).

• A shrinking microtubule has lost its GTP cap (The b-tubulin subunit(s) at the end are bound to GDP).

GTP Hydrolysis and Subunit Conformation

• Incorporation induces GTP hydrolysis by beta-tubulin.

• GTP hydrolysis changes subunit conformation and weakens bond in the polymer

  • Polymerizing Microtubules are Straight

  • Depolymerizing Microtubules are Curled and Peeling

Microtubule Structure and Properties

• Microtubules

  • Outer diameter of 25 nm, they are much more rigid than actin filaments.

  • Microtubules are long and straight and typically have one end attached to a single microtubule-organizing center (MTOC) called a centrosome.

Specialized Microtubule Structures

• 9+2 arrangement in flagella and cilia.

• Triplet arrangements.

Microtubule Nucleation

• A pair of specialized microtubule-containing structures called centrioles that lie at 90 degrees to one another

• Centrioles recruit the PCM (pericentriolar material) that includes nucleating factors like gamma tubulin ring complex g-TuRC Microtubules

Gamma Tubulin Ring Complex

• The gamma tubulin ring complex acts as a microtubule template to nucleate microtubules

Mitotic Spindle Assembly

• Classical search and capture model

Centrosomes and Microtubules

• Searchers: Centrosomes and their microtubules

  • nucleating sites (y-tubulin ring complexes)

Kinetochores

• And Capture: Kinetochores

  • Assembles in Mitosis at the Centromere

  • Includes:

    • Outer kinetochore

    • Inner kinetochore

    • Inner centromere

Spindle Assembly: Classical Search and Capture

• Textbook spindle assembly:

  • Centrosomes

  • g-tubulin

  • Microtubules

  • Kinetochores (KTs)

  • Searchers Capturers

  • Metaphase Spindle

Biased vs. Unbiased Search and Capture

• Biased Search and Capture

• Unbiased Search and Capture

• What could bias this????

Arp2/3 Complex

• Branching actin nucleation is mediated by the Arp2/3 complex

Microtubule Nucleation

• Branching microtubule (MT) nucleation is mediated by g-TuRC and the Augmin complex

Branching Nucleation

• High spatio-temporal visualization of branching nucleation in living cells

  • g-tubulin Tubulin

Spindle Assembly and Microtubule Lifetimes

• Spindle assembly: Birth and death in the lifetimes of spindle MTs.

• Augmin complex binding g-TuRC binding Daughter MT nucleation

  • ~15 sec ~15 sec START ~30 sec FINISH

  • Non-kinetochore microtubule

    • Lifetime: ~30-60 seconds

  • Kinetochore microtubule

    • Lifetime: ~7-15 minutes

Spindle Assembly via Branching-Biased Search and Capture

• Centrosomes Kinetochores

• Searchers Capturers

• Metaphase Spindle

• MT branches (from founder KT-MTs)

  • Kinetochore biased: KT-MTs live long enough to support branches

  • Spatially biased: nucleated closer to the KT target than centrosomal MTs

  • Directionally biased: born oriented at the KT versus random orientation from centrosomes

Metaphase Extract and Spindle Formation

• Metaphase extract

• No Centrosomes

• No Kinetochores

• “Bead” spindle

• Text book spindle assembly: BS?

• X.