Cytoskeleton and Microtubules Lecture Notes

Cytoskeletal Podocytopathy

• Cytoskeletal podocytopathy is a form of kidney disease.

• Caused by misregulation of actin filaments in the podocytes of the kidneys.

• Reference: He, Su & Zhang (2013) discusses potential medical treatments for cytoskeletal podocytopathy.

Lesson Objectives

• Describe the role of myosin in non-muscle cells.

• Describe the molecular structure and function of microtubules.

• Categorize microtubule-binding proteins by their function.

• Predict the pathology of disruptions to microtubule structure or microtubule-binding proteins.

Role of Myosin in Non-Muscle Cells

Adherens Junctions

• A band of actin microtubules encircles one pole of a cell.

• Connected via transmembrane cadherins to other cells.

• Myosin can pull on the adherens belt to:

  • Stretch epithelial cells (e.g., during embryogenesis).

  • Detect a loss of tension (e.g., from damage).

• Reference: Medical Cell Biology (2007).

Contractile Ring or Actomyosin Ring

• A loose formation of actin and myosin bands encircle the cell.

• Myosin operates through a hand-over-hand mechanism to contract the ring.

• Adapter proteins attach actin to the membrane.

• Reference: Kamasaki, Osumi & Mabuchi (2007).

Stress Fibers

• Organized like contractile bundles in muscle cells but anchored to the extracellular matrix by transmembrane adhesions.

• Contraction of bundles forces cytoplasm toward the leading edge of the cell.

  • Illustrated in Figure 17-39 in MCB.

Adherens Junctions in Drosophila

• Sen et al (2012) identified PATJ as an essential adherens tight junction protein for Drosophila growth.

• Mechanistic role of PATJ measured through mortality of different life stages of Drosophila larvae:

  • During embryonic development, adherens junctions establish the polarity of epithelial cells.

  • During larval growth, they maintain the integrity of the cuticle.

  • During pupation, they facilitate the mobility of epithelial cells for adult structures.

• Key roles of PATJ include:

  • Identifying polarity of actin filaments.

  • Anchoring actin filaments to transmembrane proteins.

  • Strengthening myosin crosslinking of actin filaments.

  • Assisting myosin movement along actin filaments.

• Data represented with graphs comparing PATJ mutants (black bars) and cadherin mutants (gray bars).

Functions of Microtubules

• Microtubules play critical roles in:

  1. Organization of intracellular organelles and transport of vesicles (motor proteins).

  2. Beating of cilia and flagella.

  3. Structure in nerve cells, red blood cells, and flagella.

  4. Alignment and separation of chromosomes during mitosis and meiosis.

Microtubule Structure

• Microtubules are built from a-tubulin (bound to GTP) and b-tubulin (bound to GDP) that dimerize to form protofilaments.

• Protofilaments can be organized into: - Singlets - Doublets - Triplets

• Microtubule organization centers (MTOCs):

  • g-TuRC acts as a nucleating hub for microtubules.

• Protofilaments grow more easily at the (+) end (b-tubulin), while the (-) end is anchored to g-TuRC at MTOC.

  • Figures 18-4 and 18-3 in MCB illustrate this structure.

Identifying Centrosomes in Mitosis

• To determine where centrosomes form during mitosis through immunofluorescence, target:

  • a-tubulin

  • b-tubulin

  • g-TuRC

  • MAP2

Microtubule Dynamics

• Microtubules exhibit dynamic instability, with rapid growth and collapse (illustrated in Figure 18-9 in MCB).

• Factors influencing dynamics:

  • Low temperatures facilitate depolymerization.

  • Tubulin concentration does not significantly influence the growth or disassembly of protofilaments.

• Microtubule-associated proteins (MAPs) regulate the dynamics of microtubules.

  • Created by Francesca Bonato.

Force and Cytoskeletal Filaments

• Discussion on force defined as the product of mass and acceleration:

  • Microtubules are expected to generate greater force than microfilaments because of their rapid assembly.

  • Assembling speed leads to greater potential force generation.

Microtubule-Associated Proteins (MAPs)

• Protofilaments show a slight curve at the (+) end.

• Increased curvature correlates with a higher rate of dissociation.

• Proteins maintaining protofilament straightness include:

  • GTP-b-tubulin

  • MAP2

  • Tau

  • XMAP215

  • CLASP

• Proteins enhancing protofilament curvature include:

  • Kinesin-13

  • Stathmin

  • EB1.

  • Illustrated in Figure 18-15 in MCB.

Microtubule-Based Motor Proteins

• Microtubules in neuron axons are very stable.

• The polarity of microtubules aids transport by motor proteins:

  • Kinesins move anterograde, toward the (+) end.

  • Dyneins move retrograde, toward the (-) end.

  • Reference: Leterrier, Dubey & Roy (2017).

Motor Kinesins and Dyneins

• Kinesins "walk" down microtubules using ATP as an energy source.

• Dyneins also utilize ATP to power movement along microtubules.

• In cilia and flagella, dyneins facilitate the sliding of microtubules in opposite directions.

• Microtubules utilized by motor proteins are often post-translationally modified for increased stability and binding capacity.

  • Illustrated in Figure 18-33 in MCB.

Consequence of Reduced Binding in Neurons

• Discussion about potential consequences of a disease reducing binding between kinesin-1 and tubulin in neurons.

Microtubule Disruption in Therapeutics

• Disruption of normal microtubule functions can sometimes provide therapeutic benefits.

• Example: Paclitaxel (Taxol) is a common chemotherapy drug that stabilizes GDP-tubulin.

  • It straightens protofilaments and prevents catastrophes.

  • This mechanism halts cell progression at the metaphase checkpoint, leading to apoptosis.

Summary of Lesson Objectives

• Lesson objectives reiterated:

  • Describe the role of myosin in non-muscle cells.

  • Describe the molecular structure and function of microtubules.

  • Categorize microtubule-binding proteins by their function.

  • Predict the pathology of disruptions to microtubule structure or microtubule-binding proteins.