Lecture 11: Cytoskeleton (microtubules)

Cytoskeleton Function

• maintains cell shape, organization, provides support for internal/external movement

• proteins:

  • actin

  • microtubules

Cytoskeletal Filaments

• microfilaments

  • composed of actin

• microtubules

  • composed of tubulin

• intermediate filaments

  • composed of various proteins; desmin, lamin, keratin

Microtubules Structure

• polymers of the protein tubulin

  • tubulin subunit is a heterodimer formed from two closely related globular proteins called ⍺-tubulin and β-tubulin

• hollow tubes

• have polarity due to the orientation of the subunits

  • ⍺-tubulin end = - end

    • grows and shrinks less rapidly

  • β-tubulin = + end

    • grows and shrinks more rapidly

• built from 13 parallel protofilaments, each composed of ⍺β-tubulin heterodimers stacked head to tail, then folded into a tube

  • helical microtubule lattice make them stiff and hard to bend.

  • helix forms from a slight stagger in the protofilament lateral contacts

Microtubule Growth

• rapid growth occurs by the addition of tubulin dimers at the ends

• 3 phases:

  • lag phase

  • elongation phase

  • plateau phase

• dynamic instability

Lag phase

• nucleation

• process in which several tubulin molecules interact to form a microtubule seed

• assemble into protofilaments

  • addition of GTP-tubulin to + end of a protofilament causes the end to grow in a linear conformation that assembles into the cylindrical wall of the microtubule

• nucleation is slow, and depends on the y-tubulin ring complex

Elongation Phase

• microtubules lengthen by adding GTP-bound tubulin dimers, forming a stable GTP-cap at the plus-end, allowing them to grow and probe the cell

• growing microtubules

Plateau Phase

• the final stage of in vitro microtubule assembly, where the concentration of free tubulin subunits reaches a stable point, and the overall polymer mass stops changing significantly

• microtubules with subunits coming on and off

Dynamic Instability

• process which individual microtubules alternate between cycles of growth and shrinkage

Catastrophe

• change from growth to shrinkage

Rescue

• change from shrinkage to growth

Protofilament Shape

• GTP hydrolysis after assemble changes the conformation of the subunits and tends to force the protofilament into a curved shape that is less able to pack into the microtubule wall

Microtubule Organizing Centre

• MTOC

• where microtubules are generally nucleated from

  • where y-tubulin is most enriched

• most animals possess a single, well-defined MTOC: centrosome

Centrosome

• single, well defined MTOC of most animals

• composed of 2 centrioles and surrounded by a dense mass of protein

  • pericenrtiolar material

• unless the cell is dividing, y-tubulin is only found in the centromere

Microtubule Associated Proteins

• MAPs

• bind and stabilize microtubules

  • Map2 and Tau works to se the spacing of the microtubule bundles

  • because of this, Tau mutations cause neurodegenerative diseases like Alzheimer’s

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Microtubules Orientation

• axon microtubules have uniform orientation

• dendrite/others have mixed orientations

  • dendrites also contain organelles, unlike axons

Microtubule Motors

• 2 types:

  • kinesins

  • dyneins

• move vesicles/organelles in the secretory pathway

Kinesins

• move towards the + end of microtubules

  • motor domain which splits ATP and converts the energy into motion

  • cargo-binding domain connects to the object being moved

• small size

• regular movements

• capable of transporting mitochondria/other organelles to the periphery

• must be inhibited for - end transport

  • kinesin-binding proteins prevent kinesin-microtubule binding

Dyenin

• cytoplasmic dynein moves towards the - end

• ATPase domain body

  • ATP binding changes conformation structure to dissociate microtubule binding

• tail which binds to cargo

• large size

• irregular movements

• mediates rapid movement of melanosomes

  • melanosome: organelle which synthesizes and stores melanin

  • coordinated movement because microtubules are uniformly polarized

Axonal Organelle Transport

• neurons transport vesicles with neurotransmitters to the synapse

• a vesicle contains both types of motor proteins

  • motor proteins can be inactivated to get them back

Cilia/Flagella

• hairlike appendages with a bundle of microtubules at their core

• flagella: found on sperm and many protozoa

  • have an undulating motion

• cillia: beat with a whiplike motion

• axoneme: core of both, composed of microtubules and their associated proteins

Axonemal dyenin

• bends the axoneme which moves the cilium and flagellum