Microtubules

αβ-Tubulin Heterodimer

• Each monomer ≈ 55 kDa → dimer ≈ 110 kDa

• Highly conserved proteins across eukaryotes

α-tubulin GTP binding site

• Contains non-exchangeable GTP

• Buried at α–β interface

• NEVER hydrolysed → purely structural

β-tubulin GTP binding site

• Contains exchangeable GTP

• Located at outer surface

• Hydrolysed after incorporation into microtubule

GTP-tubulin

• conformational site

• straight

• stable (favours assembly)

GDP-tubulin

• conformational site

• curved

• unstable (favours disassembly)

Longitudinal Interactions

α–β interactions along protofilament

Lateral Interactions

α–α and β–β between adjacent protofilaments

The Seam

• One discontinuity in lattice

• α contacts β instead of α–α / β–β

• Structural “weak point” in microtubule

Helical Arrangement

• Not perfectly symmetrical cylinder

• Slight helical pitch due to protofilament staggering

Why Polarity Exists

Head-to-tail assembly of dimers

Directional Growth due to polarity

• (+) end grows faster (lower Cc)

• (–) end grows slower (higher Cc)

Motor Protein Directionality due to polarity

• Kinesins → move toward (+) end

• Dyneins → move toward (–) end

Why Spontaneous Nucleation is Unfavourable

• Requires formation of:

  • Stable oligomeric nucleus (~3–4 dimers)

• This is energetically unstable → rate-limiting step

γ-TuRC Mechanism

• Ring structure (~13 γ-tubulin molecules)

• Mimics microtubule geometry

• Provides template for protofilament assembly

Importance:

• Eliminates nucleation barrier

• Defines 13-protofilament structure

Orientation Control:

• (–) end anchored at γ-TuRC

• (+) end free → dynamic

Centrosome Cycle

Duplication

• Occurs before mitosis (S phase)

Function in Mitosis

• Forms spindle poles

• Organises bipolar spindle

Augmin-Mediated Branching

1. Augmin binds existing MT

2. Recruits γ-TuRC

3. New MT nucleates at angle

Importance

• Amplifies microtubule network

• Essential in mitotic spindle formation

microtubule dynamics parameters

• growth rate - polymerisation speed

• shrinkage rate - depolymerisation speed

• catastrophe frequency - growth —> shrink switch

• rescue frequency - shrink —> growth switch

Dynamic Instability = NON-EQUILIBRIUM PROCESS

• Requires continuous GTP hydrolysis

• Energy-driven behavior

GTP Cap Model

1. Addition

• GTP-β-tubulin adds to (+) end

2. Hydrolysis

• Triggered by:

  • Interaction with next dimer

• Produces:

  • GDP + Pi intermediate

3. Pi Release

• Slow step

• Leaves GDP-tubulin lattice

Structural Consequence of GTP cap model

• GDP-tubulin prefers curved conformation

• Lattice forces it to stay straight → “stored strain”

Catastrophe Mechanism

When GTP cap is lost:

• Protofilaments peel outward

• “Ram’s horn” structures form

• Rapid depolymerisation occurs

Rescue Mechanism

Possible explanations:

• GTP islands in lattice

• Slowing of depolymerisation

• Re-establishment of GTP cap

Critical Concentration (Cc)

• + end = lower Cc

• - end = higher Cc

Treadmilling

Occurs when:

• Tubulin concentration is:

  • Above Cc(+) but below Cc(–)

👉 Result:

• Growth at (+) end

• Shrinkage at (–) end

Stabilising MAPs (MAP2, Tau)

• Bind along microtubule sides

• Reduce protofilament peeling

• Increase lattice stability

Tau Protein

• Stabilises axonal microtubules

• Maintains neuron polarity

Tau protein disease mechanism

• Hyperphosphorylation → reduced binding

• Aggregation → neurofibrillary tangles

• Leads to:

  • Axonal collapse

  • Transport failure

+TIP Proteins

• EB1 / EB3

• XMAP215

• CLASP

EB1 / EB3

Binding Site

• GDP-Pi region near + end

Mechanism

• Recognises specific lattice conformation

• Promotes:

  • GTP hydrolysis

  • Catastrophe

XMAP215

• Acts like a “polymerase”

• Delivers tubulin dimers to + end

CLASP

• Stabilises curved protofilaments

• Prevents transition to catastrophe

Kinesin-13 depolymerising protein

• Binds ends (not for transport)

• Induces curvature

• Promotes dimer dissociation

Stathmin (Op18) depolymerising protein

1. Sequesters free tubulin dimers

2. Promotes curved GDP state

Net effect: inhibits assembly

Severing Protein: Katanin

1. Binds microtubule lattice

2. Forms hexameric ring

3. Uses ATP hydrolysis

4. Extracts tubulin dimers

Consequences

• Creates new (+) and (–) ends

• Can:

  • Promote disassembly OR

  • Enable new growth sites

Microtubules in Cilia & Flagella

Axoneme: 9+2 arrangement

• 9 outer doublets

• 2 central singlets

Microtubules in basal body

• Derived from centriole

• Triplet MT structure

Microtubule-Based Transport

Kinesin

• Moves toward (+) end

• Transports vesicles outward

Dynein

• Moves toward (–) end

• Transports cargo inward

Mechanism

• ATP-dependent “walking”

• Step size ≈ 8 nm (one tubulin dimer)

Colchicine

Mechanism

• Binds free tubulin dimers

• Prevents polymerisation

Effect

• Depletes available tubulin

• Causes MT disassembly

• Prevent dynamic instability

• → Block mitosis → cell death

Taxol (Paclitaxel)

Mechanism

• Binds β-tubulin in microtubule

• Stabilises GDP-tubulin

Effect

• Prevents depolymerisation

• Blocks mitotic spindle function

• Prevent dynamic instability

• → Block mitosis → cell death

Tubular shape

~25nm

Composition and structure

• composed of α- and β-tubulin heterodimers, each with a molecular weight of approximately 55 kDa.

• The α- and β-tubulin dimers assemble into hollow, tube-like cylinders, forming protofilaments that stack side by side to create the microtubule wall.

• The structural polarity of microtubules is significant; subunits are preferentially added at the (+) end, where β-tubulin is exposed.

Types of microtubules

• Singlet Microtubules: Composed of 13 protofilaments, forming a simple tube structure.

• Doublet Microtubules: Formed by an additional set of 10 protofilaments that fuse to a singlet microtubule, creating a second tubule.

• Triplet Microtubules: Created by attaching another 10 protofilaments to the doublet, resulting in a three-tubule structure.

structural support function

• Microtubules provide mechanical support to the cell, maintaining its shape and integrity.

• They are crucial for the organization of the cytoplasm and the positioning of organelles.

Role in cell division

• Microtubules are essential during mitosis, forming the mitotic spindle that separates chromosomes into daughter cells.

• They ensure accurate distribution of genetic material during cell division.

Intracellular transport function

Microtubules serve as tracks for the movement of organelles and vesicles within the cell, facilitated by motor proteins such as kinesins and dyneins.

Dynamics

• Microtubules undergo dynamic instability, characterized by phases of growth and shrinkage, which is crucial for their function.

• GTP bound to α-tubulin is trapped and not hydrolyzed, while GTP on β-tubulin can be hydrolyzed to GDP, influencing stability.

Microtubule-Associated Proteins (MAPs)

• MAPs regulate microtubule stability and organization, influencing their dynamics and interactions with other cellular components.

• Examples include tau proteins, which stabilize microtubules in neurons, and can be implicated in neurodegenerative diseases when dysregulated.

Microtubule Motor Proteins

• Motor proteins such as kinesins and dyneins 'walk' along microtubules, transporting cellular cargo.

• Kinesins typically move towards the (+) end, while dyneins move towards the (-) end, demonstrating the polarity of microtubules.

Microtubule Organizing Centres (MTOCs)

• MTOCs are structures that nucleate and organize microtubules, ensuring proper assembly and orientation within the cell.

• The (-) ends of microtubules are anchored to MTOCs, preventing disassembly and stabilizing the microtubule network.

Centrosomes as MTOCs

• In animal cells, the centrosome is the primary MTOC, consisting of a pair of centrioles surrounded by pericentriolar material.

• Centrosomes play a critical role in organizing the microtubule network during cell division and maintaining cell shape.

Importance of MTOCs in Microtubule Assembly

• Spontaneous nucleation of microtubules is energetically unfavorable; thus, MTOCs are essential for efficient microtubule assembly in vivo.

• MTOCs facilitate the rapid assembly of microtubules in response to cellular needs, such as during mitosis or intracellular transport.

Overview of Centrosomes

• Centrosomes are cellular structures that play a crucial role in organizing microtubules and are composed of a pair of centrioles arranged orthogonally, surrounded by pericentriolar material.

• Each centriole measures approximately 0.5μm in length and 0.2μm in diameter, consisting of nine triplet microtubules, which are highly organized and stable structures.

• The centrioles do not directly nucleate the cytoplasmic microtubule array; instead, this function is performed by factors in the pericentriolar material.

Centrioles and Microtubule Nucleation

• The γ-tubulin ring complex (γ-TuRC) is a critical component located in the pericentriolar material

• γ-TuRC acts as a helical template that binds αβ-tubulin dimers, facilitating the formation of new microtubules, with the (−) end associated with γ-TuRC and the (+) end free for further assembly.

• The augmin complex, consisting of eight polypeptides, can bind to existing microtubules and recruit γ-TuRC to nucleate new microtubule assembly.

Characteristics of Microtubules

• Microtubules are dynamic structures that can rapidly assemble or disassemble at their ends, with lifetimes varying significantly based on cellular context.

• In mitotic cells, microtubules can last less than one minute, while in interphase animal cells, they typically last 5-10 minutes; however, they are more stable in axons and cilia/flagella.

• The dynamic properties of microtubules are crucial for their role in cellular organization and function.

Critical Concentration and Assembly Dynamics

• For microtubule assembly to occur, the concentration of αβ-tubulin must exceed a critical concentration (Cc), which differs for the (+) and (−) ends.

• At concentrations above Cc, tubulin dimers are added more rapidly to the (+) end, which is designated as the preferred end for assembly, leading to microtubule growth.

GTP Cap Model

• The GTP cap model explains the dynamic instability of microtubules, where GTP-tubulin can add to the growing end, stabilizing the microtubule.

• Once GTP is hydrolyzed to GDP, the stabilizing cap is lost, leading to microtubule shortening unless a new GTP cap is formed.

• The process of dynamic instability is characterized by alternating phases of growth and rapid shortening, with assembly rates differing significantly from disassembly rates.

Rescue Events and Microtubule Regulation

• Rescue events occur when GTP-β-tubulin 'islands' along the microtubule length pause disassembly and promote growth.

• The presence of microtubule-associated proteins (MAPs) can significantly influence microtubule dynamics, with some stabilizing and others destabilizing microtubules.

Role of Microtubule-Associated Proteins (MAPs)

• MAPs regulate microtubule dynamics through various mechanisms, including stabilization, destabilization, and alteration of growth properties.

• Examples of MAPs include MAP2, MAP4, and Tau, which stabilize microtubules, while others like Kinesin-13 promote disassembly.

Plus End Tracking Proteins (+TIPs)

• +TIPs, such as EB1, enhance microtubule dynamics by influencing assembly and disassembly at the (+) end.

• EB1 associates with the microtubule tip and induces a twist in tubulin subunits, which enhances GTP hydrolysis in the cap, potentially leading to increased rates of catastrophe.

Dynamic Instability and GTP Cap Model

• Dynamic instability refers to the rapid switching between growth and shrinkage of microtubules, which is crucial for their function in cellular processes.

• The GTP cap model explains how the addition of GTP-bound tubulin dimers stabilizes the growing end of microtubules, preventing depolymerization.

• When GTP is hydrolyzed to GDP, the stability of the microtubule decreases, leading to potential catastrophe and rapid disassembly.

• EB1 and EB3 proteins are key players in stabilizing the GTP cap at the growing ends of microtubules, promoting assembly.

Key Microtubule-Associated Proteins

• EB1 and EB3: These proteins associate with the growing ends of microtubules, remaining attached during growth but dissociating during pauses or shrinkage.

• XMAP15: Binds to the growing ends of microtubules, increasing local concentrations of tubulin dimers and enhancing assembly.

• CLASPs: Stabilize the growing ends of microtubules and suppress catastrophic disassembly events.

• Kinesin-13: Induces curvature in tubulin protofilaments, promoting the removal of terminal dimers and increasing catastrophe frequency.

Severing Proteins and Their Functions

• Microtubule-severing proteins, such as katanin, utilize ATP hydrolysis to disassemble microtubules by pulling subunits out, leading to destabilization.

• Katanin is notable for its six-membered ring structure that allows it to sever microtubules effectively.

• The exact roles of severing proteins in animal cells remain somewhat unclear, but they are essential for regulating microtubule dynamics.

MAP2 and Tau Proteins

• MAP2 and Tau are neuronal proteins with modular designs that interact with microtubules, influencing their spacing and stability.

• The first domain of these proteins binds to the negatively charged surface of microtubules, while the second domain projects outward, affecting microtubule organization.

• Tau is implicated in neurodegenerative disorders, known as Tauopathies, including Alzheimer's Disease, where mutations lead to decreased microtubule stability and increased aggregation.

Microtubule-Binding Drugs

• Microtubule-binding drugs target the conserved nature of tubulins, affecting their polymerization and depolymerization, which is crucial for cell division and other processes.

• Colchicine: Prevents tubulin polymerization by binding to dimers, leading to a reduction in microtubule dynamics, which is useful in treating gout by inhibiting white blood cell migration.

• Paclitaxel (Taxol): Stabilizes microtubules, preventing disassembly and halting cell division, making it effective in treating various cancers.