Lecture 14

• Cytoskeleton

  Comprises three main types of protein polymers - actin, microtubules, and intermediate filaments - each playing crucial roles in cellular structure and function
• Microtubules

  • Polymers made from αβ-tubulin dimers
  • Dimers align head-to-tail to form protofilaments
  • Usually thirteen protofilaments laterally associate to form a hollow tube with a lumen at the center
• Dynamic instability

  Microtubules exhibit alternating phases of growth and shrinkage, particularly at the plus end
• GTP hydrolysis

  α-Tubulin and β-tubulin bind GTP, but only the GTP on β-tubulin is hydrolyzed to GDP after incorporation into the microtubule lattice
• Microtubule Associated Proteins (MAPs)

  • Key proteins like EB-1 and γ-tubulin play essential roles in microtubule functions
  • γ-Tubulin is important in nucleating microtubules at the microtubule organizing center (MTOC)
  • EB-1 binds to growing microtubule plus ends, regulating their dynamics and interactions
• Motor Proteins

  • Kinesin generally moves toward the microtubule plus end, involved in organelle transport and chromosome segregation
  • Dynein moves toward the minus end, crucial for retrograde transport from the periphery back to the cell center
• Cells often utilize both kinesin and dynein

  For transporting vesicles and organelles, leading to complex bi-directional movement along microtubules
• Cell Shape and Motility

  • The growth of microtubules toward the cell cortex and their interactions with the plasma membrane influence cell shape and are critical for determining sites of new growth during cell movement and division
• Chromosome Segregation

  • During mitosis, microtubules form the spindle apparatus, which is essential for the proper segregation of chromosomes to daughter cells
• Dynamic Instability

  • Microtubule assembly is facilitated when the microtubule is straight
  • GTP hydrolysis to GDP on β-tubulin leads to a conformational change that strains the microtubule lattice, causing protofilaments to splay outward during depolymerization
  • When polymerizing, this splaying is restrained by the presence of a GTP-tubulin cap at the growing ends, which, when lost, leads to rapid, catastrophic depolymerization
• Microtubule Organizing Centers (MTOCs)

  • The major MTOC in animal cells is the centrosome, composed of two centrioles surrounded by pericentriolar material
  • This matrix is crucial for the nucleation of microtubules, primarily due to the presence of γ-tubulin ring complexes (γ-TURCs) which initiate microtubule growth
• Tubulin Isoforms


  • α and β Tubulin: Primary components of microtubules, essential for their polymer structure
  • γ Tubulin: Found in γ-TURCs at the MTOCs, vital for microtubule nucleation
  • δ, ε, ζ, and η tubulin: Play specialized roles in the structure of centrioles, basal bodies, or other cellular structures
  • FtsZ: A bacterial relative of tubulin that forms polymers crucial for cytokinesis
• Motor Proteins

  • Kinesin typically moves towards the microtubule plus end, facilitating the outward transport of organelles like the ER and vesicles towards the plasma membrane
  • Dynein moves towards the minus end, directing organelles such as the Golgi apparatus and vesicles towards the cell center
• Drugs influencing microtubules

  Agents like nocodazole and colcemid bind to tubulin dimers, preventing their polymerization and thereby providing tools to study microtubule functions and dynamics in cellular processes
• The use of fluorescently labeled tubulin allows for the observation of microtubule behavior in live cells, providing insights into their growth dynamics and the interactions of microtubule tips with cellular structures and membranes
• Kinesin Movement

  • Kinesin operates on a "hand-over-hand" mechanism, where one head binds to the microtubule while the other swings forward
  • This coordinated action allows kinesin to walk along a microtubule filament, carrying cargo such as vesicles or mitochondria over long distances
• Dynein Structure and Function

  • Dynein is structurally complex, with two heavy chains that contain ATPase activity and are responsible for its movement
  • The motor domains at the base of dynein are linked to cargo-binding domains at the tail, which interact with the dynactin complex to attach various cargoes
  • Dynein's power stroke involves conformational changes in its ATPase domains, driving the motor along the microtubule
• Cells often use both kinesin and dynein simultaneously

  To facilitate bi-directional transport along microtubules, crucial for processes such as organelle positioning and vesicle transport in neurons
• Advanced imaging techniques, including live-cell imaging with fluorescently labeled motor proteins (such as EB-1 tagged with GFP to visualize growing microtubule ends), are crucial for studying motor protein dynamics and understanding their roles in cellular transport
• Clinical and Research Implications

  • Compounds like nocodazole and colcemid, which disrupt microtubule dynamics by binding to tubulin and inhibiting its polymerization, are used to study the role of microtubules and motor proteins in cellular processes
  • Understanding the mechanisms of motor proteins is fundamental in areas like neurobiology, where axonal transport is critical, and in cancer research, where alterations in cellular transport can affect cell division and tumor progression