Introduction to the Cytoskeleton: Microtubules

Course Coverage

• Series topics on the cytoskeleton include:
    - Microtubules
    - Actin
    - Intermediate filaments
    - The cytoskeleton in mitosis
    - The cytoskeleton in cell migration
    - Methods for investigating protein function with a focus on the cytoskeleton

Learning Outcomes

By the end of this series, students will:
  - Describe the three types of filaments in the cytoskeleton, including:
    - Composition and properties
    - Polymerization and regulation of dynamics
    - Functions
  - Understand how each filament's functions relate to its unique physical properties
  - Be able to design experiments to investigate cytoskeleton function

Reading Resources

• Recommended Texts:
    - Alberts, Molecular Biology of the Cell
      - Chapter 16: The Cytoskeleton
      - Chapter 17: The Cell Cycle

Definition of the Cytoskeleton

• Definition: "Cytoskeleton" is derived from "cyto" meaning cell, and "skeleton" indicating the structural framework of the cell.
    - Composed of a network of filaments extending throughout the cytoplasm and nucleus
    - Consists of polymers of subunit proteins
    - Provides essential functions to the cell, including:
      - Shape
      - Strength
      - Organisation
      - Movement

Components of the Cytoskeleton

• The cytoskeleton is comprised of three filament types:
    - Microtubules
    - Actin
    - Intermediate Filaments

Co-existence of Cytoskeletal Filaments in Mammalian Cells

• Each type of filament exists in mammalian cells and plays specific roles.

• Distinct structures and functions due to different subunit proteins

• Each filament forms its individual network typically in the cytoplasm, enhancing cellular functions and survival

Properties of Cytoskeletal Filaments

• Subunits associate via multiple non-covalent bonds, leading to:
    - Increased filament strength
    - Rapid disassembly when necessary by disrupting non-covalent bonds

Organization of Filaments

• Different organizations of filaments exist in various cell types or at different times within the same cell

• Purpose:
    - Alter structural properties and locations of filaments
    - Change overall cell properties and functions

Cytoskeletal Organization in Different Cell Types

• Differences observed in fibroblasts versus epithelial cells
    - Migrating Fibroblast Characteristics: Notable cytoskeletal arrangement allowing movement
    - Epithelial Cell Characteristics: Features like microvilli, actin terminal web, adherens junctions, and the basal lamina

Changes in Cytoskeletal Organization During Mitosis

• Significant reorganization of cytoskeletal components occurs during mitosis, indicating their importance in cell division

Dynamic Nature of Cytoskeletal Filaments

• Cytoskeletal filaments are highly dynamic, facilitating rapid changes in organization
    - Achieved through processes of depolymerization and repolymerization
    - Adaptation to environmental changes and cellular demands
    - Regulated by various accessory proteins, especially for actin and microtubules

Regulation of Cytoskeletal Polymerization

• Polymerization of cytoskeletal filaments is regulated by:
    - Binding of accessory proteins (particularly for actin and microtubules)
    - Post-translational modifications, primarily phosphorylation

Microtubules Structure

• Composition: Microtubules are polymers formed from globular alpha and beta tubulin dimers

• 13 protofilaments come together to form a hollow tube structure

• Multiple interactions contribute to their rigidity, making them tough to bend
  
  

Polarity of Microtubules

• Microtubules exhibit polarity:
    - Plus end (+): Pointing towards the cell's periphery
    - Minus end (−): Anchored towards the cell center

• Important note: The terms ‘plus’ and ‘minus’ refer to structural orientation, not charge

Centrosome and Microtubule Organization

• Microtubules are nucleated from a microtubule-organizing center known as the centrosome, which consists of centrioles and pericentriolar material
  
  

Dynamic Instability of Microtubules

• Exhibits dynamic instability: microtubules can grow or shrink rapidly
    - Growth and shrinkage occur mainly at the plus end; the minus end is anchored
  
  

Mechanism of Dynamic Instability

• Powered by GTP hydrolysis:
    - Tubulin dimers added in GTP-bound state at (+) end
    - GTP hydrolysis to GDP triggers a conformational change that weakens the subunit bonds, facilitating depolymerization

Catastrophe and Rescue Dynamics

• Catastrophe: Occurs when GTP is hydrolyzed faster than dimer addition, destabilizing the plus end and causing rapid depolymerization

• Rescue: Happens when dimer addition surpasses GTP hydrolysis rate, restoring the GTP cap and allowing growth to resume

Constant Turnover and Adaptability

• Dynamic instability allows cellular responses and microtubule organization flexibility

• Comparable to treadmilling in actin filaments

Factors Determining Microtubule Dynamics

• Microtubule growth and shrinkage depend on:
    - Stochastic (random) processes that lead to growth-shrinkage transitions
    - Proteins regulating dynamics by promoting either catastrophe or rescue events

Microtubule Associated Proteins (MAPs)

• MAPs bind to microtubules and regulate their organization and dynamics:
    - Some MAPs stabilize filaments, promoting growth and reducing shrinkage
    - Others destabilize filaments, encouraging shrinkage

Examples of Microtubule Associated Proteins

• Stathmin: Binds subunits, preventing assembly

• Kinesin-13: Promotes catastrophe and disassembly

• Y-TURC: Nucleates assembly at the minus end

• XMAP215: Stabilizes and accelerates assembly at the plus ends

• Katanin: Severing microtubules

• Tau: Stabilizes microtubules and enhances axonal transport

• MAP2, Plectin: Additional proteins linking microtubules to intermediate filaments

Motor Proteins Related to Microtubules

• Microtubule-based motor proteins function like molecular motors: comparable to myosin with actin

• Two main families of motor proteins:
    - Kinesins: Move toward the plus end (periphery)
    - Dyneins: Move toward the minus end (center)

Mechanism of Motor Protein Movement

• Motor protein movement is ATP hydrolysis-dependent, driving conformational changes in the protein structure, allowing movement along microtubules

Cargo Transported by Motor Proteins

• Motor proteins transport various cargoes, including:
    - Membrane-bound organelles/vesicles identified through membrane receptor proteins
    - Other microtubules during mitosis to facilitate sliding past each other

Functions of Microtubules

• Microtubules and their associated motor proteins serve critical functions within the cell:
    - Determine intracellular organization and movement
    - Position organelles such as Golgi and ER
    - Facilitate intracellular vesicle trafficking
    - Maintain cell polarity
    - Form cilia and flagella
    - Construct the mitotic spindle during mitosis

Cell Polarity

• Many cells display polarity wherein different plasma membrane regions exhibit distinct protein composition and functions
    - Microtubule orientation relative to cell polarity is critical for determining cell functions
    - Dyneins and kinesins provide directional transport of intracellular molecules

Examples of Cell Polarisation

• Secretory vesicles targeted to specific membranes
    - Epithelial cell organization
    - Centrosome positioning anterior to nucleus
    - Microtubule (+) ends attached to the plasma membrane at the cell's front

Structure of Motile Cilia and Flagella

• Adapted microtubules form structure of motile cilia and flagella seen in specific cell types (e.g., airway epithelium and sperm cells)

Summary of Microtubules

• Microtubules are polar polymers of alpha/beta-tubulin dimers radiating outward from the cell center

• Exhibit dynamic instability, driven by GTP hydrolysis regulating their stability

• Motor proteins facilitate cargo movement along microtubules, integral for proper cellular function and organization