M7L2 - Microtubules

Microtubule Structure

• Comprised of 13 protofilaments

• Arrayed circularly to form a tube wall

• They’re staggered to resemble a spiral 

What are the basic subunits of each protofilament (microtubule structure)

Dimers of alpha and beta tubulin proteins

What are the GTP-binding properties of α- and β-tubulin subunits?

• Both α- and β-tubulin bind GTP.

• α-tubulin: GTP is tightly bound

  • never hydrolyzed

  • does not exchange with free nucleotides.

• β-tubulin: GTP is loosely bound

  • hydrolyzed to GDP

  • exchanged for GTP in the cytosol.

How are tubulin subunits added and removed during microtubule assembly?

• α- and β-tubulin subunits are added/removed as dimers.

• αβ–GTP dimers have a higher affinity for the growing microtubule (more stable).

• αβ–GDP dimers have a lower affinity and tend to dissociate from the filament.

Microtubule Polarity

• They’re polar so the two ends have different characteristics and dynamics

  • (+) end = fast growing 

  • (-) = slow growing 

• Within the dimers

  • the beta-subunit is closer to (+)

  • the alpha-subunit is closer to (-)

Microtubule Dynamics

• Dimers with αβ–GTP are added to (+) end

  • Rescue phase 

• Dimers with αβ–GDP are released from shrinking filament

  • Catastrophe

• GTP hydrolysis occurs within polymerized microtubule

  • Most of it consists of dimers containing αβ–GDP

• (+) has GTP cap (unhydrolyzed) which favours growth 

  • αβ–GTP dimers have a 4x slower disassociation rate in comparison to αβ–GdP

  • They thus have higher affinity for their neighbours and stay together 

• (+) end has dynamic instability

  • Oscillates between growth or shortening

  • High [GTP-tubulin] = polymerization

  • Low [GTP-tubulin] = depolymerization

EB1 Protein (Microtubule)

• Plus-end binding protein

• Prevents premature catastrophes

• Acts as positive regulator of microtubule growth 

MAPs - Microtubule Associated Proteins

Proteins controlling the assembly and disassembly of microtubules 

MAPs - Microtubule Associated Proteins (Function)

• Interconnect microtubules to form bundles 

• Inc stability 

• alter rigidity 

• influence assembly rate 

MAPs - Microtubule Associated Proteins (Two Groups) 

1. Those that stabilize microtubules (Ex. Tau and EB1)

2. Those that destabilize microtubules (Ex. catastrophin)

Microtubule Nucleation 

• Starting off growth 

• Involves γ‐tubulin which is present in smaller amounts in the cell compared to alpha/beta tubulin

• Helps form γ‐tubulin ring complex (γ-TuRC)

  • Nucleates at (-) end of a new microtubule 

  • Forms a template for the growing (+) end

• γ-TuRC acts as a cap of the (-) end while microtubule growth occurs at (+) end

MTOC (Microtubule Organizing Center)

• A specific location inside the cell where microtubule nucleation occurs 

• In animal cells, the MTOC is centrosome (red dot)

  • Located near nucleus

MTOC: Centrosome and yTuRC

• Consists of 2 cylindrical structures called centrioles (inside centrosome which is in green) 

• Also has pericentriolar material (PCM) containing many γ‐TuRC complexes (red rings on green ball)

• (-) end of microtubules are nucleated at the γ‐TuRC

• (+) end are directed towards the cell periphery (shown as +)

MTOC role in Mitosis

• The MTOC (centrosome) organizes microtubules that form the mitotic spindle.

• The spindle’s microtubules attach to chromosomes to separate replicated sister chromatids.

• Centrosomes are duplicated before mitosis, creating two MTOCs that move apart to opposite poles.

• Microtubules nucleate from the γ-TuRC complexes at each MTOC, with plus ends growing outward.

Microtubule Toxins: Cholchicine 

• Useful in lab to arrest the cell cycle 

• Ex. cholchicine 

  • Derived from meadow saffron 

  • Inhibits polymerization 

  • Binds and stabilizes αβ‐tubulin dimers

  • Prevents addition/loss of tubulin dimers

  • Arrests cells in metaphase without chromatid seperation

Microtubule Toxins: Taxol

• Useful in lab to arrest the cell cycle 

• Taxol Function

  • Binds to β‐tubulin to increase affinity for (+) end

  • Prevents depolymerization 

  • Prevents assembly of mitotic spindle to inhibit mitosis

• Used in cancer treatment

• Hard to synthesize in lab so it’s derived from pacific yew tree

Kinesin Motor Protein

• (+) directed transport on microtubules, so towards cell periphery away from MTOC

• Tetrameric complex made of 2 heavy chains and 2 light chains 

• The globular heads (motor domains) cyclically bind to microtubules 

  • Generates movement through ATP hydrolysis 

• The tails determine specificity of cargo binding

  • The tails are highly variable

Kinesin Mechanochemical Cycle

• The lagging head is bound to ATP

• The leading head is bound to ADP

• ATP kinesin has a higher affinity for the microtubule than ADP bound kinesin

• The ATPase motor lagging head hydrolyzes ATP to ADP + Pi

  • Reduces affinity of lagging head for microtubule

• ADP is exchanged for ATP in leading head

  • Increases affinity of leading head

• The binding of ATP induces conformational change causing lagging head to swing in front to another microtubule binding site

• This resets cycle to the top

How does kinesin move along microtubules?

• Kinesin moves in a “hand-over-hand” fashion.

• It has two motor heads (domains), and one is always attached to the microtubule.

• The two heads work in a coordinated cycle, each in a complementary stage of ATP binding or hydrolysis.

In-vitro assays for kinesin movement

• Nomarski Microscope

  • Following plastic beads tethered to kinesin

  • The track is anchored to the dick made from purified tubulin 

• Gliding mobility assay

  • kinesin are tethered to a glass slide at their cargo (Tail) ends

  • They can then move fluorescently labeled microtubules added to solution above slide

Dynein

• (-) directed, moving towards MTOC 

• 2 main forms: Cytoplasmic and Axonemal 

• Has 2 heavy chains and a variety of intermediate and light chains

Two forms of dynein

• Cytoplasmic

  • Associated with microtubules 

  • Direct movement of organelles and vesicles in cytoplasm

• Axonemal 

  • Found in structures powering movement of whole cells

  • Ex. cilia or flagella 

How does dynein move cargo along microtubules?

• Movement is powered by a power stroke in the linker arm (near the cargo attachment site).

• In a dynein dimer, the two motor units alternate power strokes, producing continuous movement.

Describe the steps of dynein’s ATP-driven power stroke

• ATP binding releases motor head group from microtubule

• ATP hydrolysis creates dynein-ADP+Pi that can now attach to the microtubule

• The release of Pi powers the power-stroke of the liner

  • Pulls the cargo 

• Each power stroke, the cargo moves towards the (-) end by 8mm

Bidirectional Vesicle Movement: Neural Cells 

• Microtubules span the axons of neural cells 

• The (-) ends are anchored to MTOC 

• The (+) ends extend along the axons towards synapse cell membrane 

• Vesicles with NTs are carried from cell body to synapse along microtubules 

Microtubule Tug of War

• Model describing the movement of proteins if they’re bidirectionally transported

• The final direction of movement is the winner of this ‘battle’ 

• There are regulatory proteins controlling direction in response to cell signals 

Change of Direction (Microtubule Transport) Application: Melanosomes in Fish

• Melanosomes: Pigment-filled organelles 

• Movement of it changes skin cells in response to behavioural signalling 

• This movement is done by molecular motors carrying it to the cell periphery or center 

  • Dynein: Move towards (-) end MTOC 

  • Kinesin: Move towards cell periphery (+) end

• Dispersion to periphery = cell appears darker 

• Concentrated in middle = Cell appears lighter 

• This is controlled by signals using cAMP as a secondary messenger