12. Microtubule Structure, Dynamics, and Molecular Motors

Course Overview and Introduction to Chapter 18

Topics in Chemical Foundations and Cell Biology

  • Chemical Foundation

  • Protein Structure, Function & Regulations

  • Gene to Protein

  • Protein Targeting/Sorting

  • Vesicular Transport

  • Cell Signaling

  • Cytoskeleton - Actin

  • Molecular Motor - Myosin

  • Microtubules and Intermediate Filaments (IF)

  • Cell Cycle, Mitosis & Meiosis

  • Stem Cells

  • Cell Death (Apoptosis)

  • Cancer

  • Membrane Structure

  • Membrane Transport

  • ATP Synthase

Chapter 18 Introduction

  • This section covers sections 18.2 to 18.4.

  • Bio-Art Project: Extra credit opportunity. Participants must fill out the BioArt Survey by 4/24/264/24/26.

Classification of Cytoplasmic Filaments

Three Types of Cytoplasmic Filaments

  1. Actin Filaments (Microfilaments):     - Characterized as thin and flexible.     - Polar and dynamic structures.     - Associated with the Myosin motor.

  2. Microtubules:     - Rigid, hollow tubes.     - Diameter: 25 nm25\,nm.     - Polar and dynamic structures.     - Associated with Kinesin and Dynein motors.

  3. Intermediate Filaments (IF):     - Structure varies, generally providing mechanical strength.

Microtubule Organizing Centers (MTOC)

Assembly and Distribution

Microtubules are assembled from the Microtubule Organizing Center (MTOC). The location and organization of the MTOC vary depending on the cell type:

  • Centrosome: The primary MTOC in many animal cells. Microtubules radiate outward from the centrosome, with the minus end ($-$) at the MTOC and the plus end ($+$) extending toward the periphery.

  • Spindle Poles: Formed during cell division (mitosis/meiosis) when the centrosome divides into two ($2$) poles to organize the spindle apparatus.

  • Neurons:     - Axon: Features highly organized microtubules that transport materials between the cell body and axon terminal.     - Dendrite: Contains microtubules oriented in various directions.

  • Cilia and Flagella: Nucleated from a Basal Body MTOC.

Scientific Experiments: Proving the MTOC Function

Experimental Evidence for Growth from MTOC

To determine where microtubules originate, scientists perform experiments using specific drugs:

  • Colchicine: A drug that binds to tubulin dimers and blocks polymer formation.     - Result: This drug effectively stops microtubule assembly, which can be used to treat the pain associated with gout by blocking the inflammatory response involving microfilaments/microtubules.

  • Procedure:     - Take a cultured cell and treat it with Colchicine to disassemble existing microtubules.     - Washed: Once the drug is washed away, microtubule assembly resumes.     - Observation: In cultured cells, microtubules are seen starting to grow from a specific region (the MTOC) and continuing outward.

  • Control: Always maintain an untreated cultured cell as a control to compare the normal microtubule distribution against treated/recovered cells.

Anatomy of the Centrosome

Structural Components

The centrosome is the main MTOC and consists of several specialized structures:

  • Centrioles: A pair of centrioles (Mother and Daughter centrioles) oriented at a 90∘90^{\circ} angle to each other.     - Mother Centriole: Characterized by associated proteins including Distal and Subdistal appendages.

  • Pericentriolar Material (PCM): A dense matrix of proteins surrounding the centrioles.

  • γ\gamma-Tubulin Ring Complex (γ\gamma-TuRC): These serve as the actual nucleating sites for microtubule growth. Microtubules are buried within the pericentriolar space/material at their origins.

The γ\gamma-Tubulin Ring Complex (γ\gamma-TuRC)

Nucleation and Stability

  • Function: The γ\gamma-TuRC nucleates microtubule assembly. It provides a template that determines the architecture of the microtubule.

  • Structural Components:     - α\alpha-tubulin and β\beta-tubulin dimers form the microtubule wall.     - γ\gamma-tubulin forms the ring at the base.     - Accessory Proteins: Help stabilize and grow the microtubule.

  • The Microtubule "Seam": A specific longitudinal line where the tubulin dimers meet as the tube closes.

  • Key Point: In cells, the minus ends ($-$) of microtubules are stabilized by either γ\gamma-tubulin ring complexes or a basal body. Consequently, microtubule dynamics in vivo are largely limited to the plus ($+$) ends.

Dynamic Instability of Microtubules

Observation in Vivo

Microtubules are not static; they grow and shrink dynamically. Fluorescently marked tubulin allows for the visualization of this process:

  • Example Timestamps:     - 0:000:00: Microtubule (A) is longer.     - 0:270:27: Microtubule (A) remains similar, while (B) might change.     - 3:513:51: Microtubule (A) is long; Microtubule (B) is shorter.

  • Microtubules can either grow or shrink from the plus ($+$) end.

Molecular Mechanism of Instability

Electron microscopy reveals structural differences during assembly and disassembly:

  • Growing Microtubule: Features blunted or slightly curved ends as dimers are added.

  • Shrinking Microtubule (Catastrophe): The end appears frayed or opened up as individual protofilaments peel away; it is not individual dimers falling off, but the whole structure breaking.

  • GTP-Cap Theory:     - GTP-β\beta-tubulin: The active form that provides stability to the microtubule end.     - Assembly: When sub-units are added faster than GTP is hydrolyzed, a "GTP cap" forms, allowing further growth.     - Disassembly: When GTP is hydrolyzed to GDP (GDP−β−tubulinGDP-\beta-tubulin), the tubulin dimers become inactive/unstable.     - Catastrophe: Loss of the GTP cap leading to rapid shrinkage.     - Rescue: Regaining the GTP cap, allowing the microtubule to start growing again.

Microtubule Associated Proteins (MAPs)

Structural and Stability Proteins

MAPs bind to the surface of microtubules to regulate their stability and organization.

  1. MAP2: Binds sideways along the microtubule to provide structural stability. It features long projections that help space microtubules apart (25 nm25\,nm).

  2. Tau: Similar to MAP2 but has shorter projections, resulting in more closely packed microtubules (25 nm25\,nm).     - Pathology: Hyper-phosphorylated Tau increases microtubule rigidity or causes them to detach and form plaques, which are linked to neurodegenerative diseases.

Plus-End Tracking Proteins ($+$TIPs)

  • Stabilizers:     - XMAP215 (Xenopus MAP): Binds to the plus end to promote growth.     - CLASP: Helps stabilize the ends.     - TOG Domains: Specifically bind to α/β\alpha/\beta-tubulin to make the protein/microtubule more stable.     - Note: Overexpression of these stabilizers is often a cancer marker.

  • Destabilizers:     - Kinesin-13: Unlike other kinesins, it does not have motor function. It uses energy from ATP to break the microtubule ends.     - Stathmin: Promotes the breakdown of microtubules into dimers.

Molecular Motors of Microtubules

Overview of Motors

Microtubules serve as a "roadmap" for intracellular transport. There are two primary motor families:

  1. Kinesin: Moves primarily toward the plus ($+$) end (periphery).

  2. Dynein: Moves toward the minus ($-$) end (center/MTOC).

Organelle Transport and Directionality

Organelles are moved in specific directions depending on the motor used:

  • Kinesin (Outward/Plus-End): Transports the Endoplasmic Reticulum (ER), Secretory vesicles, Early endosomes, Lysosomes, and Mitochondria.

  • Dynein (Inward/Minus-End): Transports the Golgi apparatus (kept near the MTOC), COPII vesicles, ERGIC compartment (ER-Golgi Intermediate Compartment), Late endosomes, and Pigment granules.

Structure and Mechanism of Kinesin-1

Molecular Structure

Kinesin-1 is a dimer consisting of:

  • Head: Contains the ATP binding domain and the microtubule-binding sites.

  • Linker: Connects the head to the stalk.

  • Stalk: A coiled-coil region that provides flexibility.

  • Tail: Contains the light chains which bind to the vesicle/cargo receptor.

Walking Mechanism

Kinesin moves via a "hand-over-hand" mechanism:

  1. Activation: Inactive kinesin is folded (head-to-tail interaction). It becomes active upon binding its cargo.

  2. Step 1: One head binds the microtubule and releases ADP.

  3. Step 2 (Power Stroke): ATP binds to the leading head. This binding causes the neck linker to swing forward, throwing the second (trailing) head forward.

  4. Step 3: The now-leading head binds to the microtubule 16 nm16\,nm in front of its previous position (effective step size of the cargo is 8 nm8\,nm per ATP).

  5. Step 4: ATP hydrolysis in the trailing head and phosphate ($P_i$) release prepares the cycle to repeat.

Structure and Mechanism of Dynein

Molecular Structure

Dynein is a much larger and more complex motor than kinesin:

  • Stem: Binds to cargo or intermediate/light chains.

  • Head: Contains a ring of six ($6$) AAA ATPase domains.

  • Stalk: Extends from the head and contains the microtubule-binding domain.

Walking Mechanism

  • Power Stroke: The power stroke in dynein is triggered by the release of inorganic phosphate ($P_i$) following ATP hydrolysis (ATP→ADP+PiATP \rightarrow ADP + P_i).

  • Process: During the pre-stroke (ATP-bound or ADP-Pi state), the linker is cocked. Upon $P_i$ release (post-stroke), the linker moves, pushing the stem/cargo forward toward the minus end.

  • Visual: The motor heads appear to "march" or step toward the $(-)$ end as ATP is processed.

Specialized Transport: Melanophores

Skin Color Regulation

Melanophores are specialized cells containing melanosomes (pigment granules). Transport is regulated by hormones like adrenaline:

  • Dispersion (Skin darkens): Melanosomes are moved toward the plus ($+$) ends of microtubules by Kinesin-2.

  • Aggregation (Skin lightens): Melanosomes are moved toward the minus ($-$) ends (centrosome) by Dynein.

Summary Recap Questions

  • Which drug inhibits microtubule assembly? (Answer: Colchicine)

  • Which end is attached to the pericentriolar space? (Answer: Minus end)

  • Which end is growing or shrinking in vivo? (Answer: Plus end)

  • What are the two types of motors on microtubules? (Answer: Kinesin and Dynein)

  • Which motor is plus-end directed? (Answer: Kinesin)

  • How does ATP provide the power stroke in Kinesin-1? (Answer: Binding of ATP to the head)

  • How does ATP provide the power stroke in Dynein? (Answer: Release of PiP_i following hydrolysis)

  • What does melanosome aggregation vs. dispersion suggest? (Answer: Coordination between different motor types for physiological responses)