MicroTubules

Role of Cytoskeleton

• Scaffolding that provide structural support

• Network of tracks to direct movement of materials

• A force generating apparatus for movement and contraction

• A framework for positioning various organelles within the cell

• A essential component of the cell division machinery

Location of cytoskeleton elements

• MF surrounds membrane in epithelial cells

  • MT and IF is on the basal side

• MF is condensed on the leading edge of a migrating cell

• IF surround nuclear envelope

• MT protrudes out of nucleus

Structure of MT

 

MT are straight, hollow cylinders of varied length made of longitudinal arrays of protofilaments

Protofilaments

• Made of 13 subunits

• Heterodimer of tubulin

• Alternates between alpha-tubulin and beta-tubulin

• They bind non-covalently to form an alpha/beta-heterodimer and doesn't normally dissociate

• Alpha/beta subunits have similar 3D structure but only 40% a.a identity

• Alpha tubulin is permanently in GTP bound state

• Beta tubulin can be bound to either GDP or GTP

Polarity

• All dimers in MT are orientated the same way causing protofilaments to have an inherent polarity

• Plus end and Minus end

MT Singlets, Doublets and Triplets

• Cytoplasmic MT are simple tubes with 13 protofilaments

• Some axonemal MT form doublet or triplet MTs

  • Doublets and triplets have 1 13-protofilaments and 1-2 incomplete rings with 10-12 protofilaments

• Doublets is found in cilia and flagella

• Triplets is found in basal bodies and centrioles

Dynamic Instability

• Rapid interconversion between growing and shrinking state

  • Never stagnant

Steps of Growth and Shrinkage

1. Growing tip contains GTP bound beta-tubulin

2. Hydrolysis of GTP as the tube zips up

3. GDP bound Beta-tubulin at the end

4. Strain resulting from GDP Beta-tubulin subunits at the plus end is released as the protofilament curls outward and undergoes catastrophic shrinkage

   1. Hydrolysis of GTP changes conformation of subunits, forces protofilament into a curved shape and is less able to pack into MT wall

MTOC

• MTOC is site of MT assembly nucleation and acts as an anchor for the minus end

• During interphase, cells have centrosome (MTOC) near the nucleus

• Centrosome is a major microtubule nucleation site

  • Pair of centrioles

  • Pericentriolar material surrounds the centrioles

  • Gamma-tubulin ring complexes is the site of nucleation

    • Within the PCM of the centrosome

Centrioles

• A tubule is a complete protofilament, B/C are incomplete protofilaments

• Centrioles are orientated at right angle to each other

• Accessory protein recruit the pericentriolar material where MT nucleation takes place

• Involve in basal body formation for cilia/flagella

• Cells without centrioles have poorly organize mitotic spindles but still divide

MT Polarity in non-dividing cells

• MT orientation may vary with a particular cell's function

• In axons, plus end is away from MTOC

• In dendrites plus/minus ends are staggerred

• Epithelial cells, plus end is on apical side, minus end on basal side

MT Polarity Dividing cells

• MTOC influences the number of MTs in a cell

• High MT-nucleating activity occurs during prophase and metaphase

• Minus end at MTOC, Plus end attaches to chromosome

Role of Gamma-TuRCS

• Nucleate the assembly of new MTs away from the centrosome, extends the plus end

• Loss of gamma-TuRCs prevent cell from nucleating MTs. Extends the plus end

• Loss of gamma-TuRC prevents a cell from nucleating MT

Gamma-tubulin ring complex

• Gamma tubulin found a lower level than alpha and beta tubulin

  • Associated with a number of accessory proteins in the gamma-TuRCs

• 13 gamma tubulins per turn and binds to the minus end of alpha/beta-tubulin dimers

• Determines the polarity of the microtubule and caps the minus ends

  • Prevents the loss of gain of tubulin subunits

• MT nucleation can also occur at non-centrosome sites that recruit the y-TuRC

Kinetics of MT Assembly in Vitro

• MT form by addition of tubulin dimers at the ends

• Reversible polymerization occurs in the presence of GTP and Mg2+

Nucleation

• Dimers aggregate into oligomers and serve as nuclei for new MT to grow

• MT formation is slow at first, referred as the lag phase, due to the slow process of nucleation

Elongation

• Addition of more subunits at either end

• Much faster than the lag phase

Plateau phase

• Concentration of tubulin becomes limiting

• Rate of assembly is balanced by disassembl

Critical Concentration

The concentration of both rates of assembly and disassembly is equal

• MTs grow when concentration exceeds the critical concentration and disassembled when concentration is below

• The plus/minus ends are chemically different

  • Plus end grows faster than the minus

  • Minus is anchored to centrosomes

MT Stability Regulation

tightly regulated in cells by a many Microtubule-Binding Proteins (MAP)

• Cells tightly regulate the assembly and structure of microtubules by using MAP

  • Some MAP use ATP to drive vesicle or organelle transport or generate sliding forces between MT

  • MT-stabilizing/bundling proteins

  • Plus-End Tubulin Interacting proteins (+/- TIP)

  • Microtubule-Destabilizing/Severing Protein

 

 

Stabilizing/Bundling Proteins

• Bind at regular intervals along the MT wall, can increase stability and affect that density MTs

• Tau causes MTs to form tight bundles in axons

• MAP2 promotes the formation of looser bundles in dendrites

• 1 region of the protein binds to the MT wall and the other extends at a right angle of the MT and allows for interaction with other proteins

  • Length of extended arm controls the spacing of MTs in the bundle

• Mutation in Tau is associated with neurodegenerative diseases

MT plus-end tracking proteins can bind to dynamic plus ends of MT

• MTs are too unstable to remain intact for long periods of time and will de-polymerize unless they are stabilized in some way

• +TIPs can stabilize MT by capturing the growing plus end and protect from catastrophic subunit loss

• EB1 associates with GTP-Tubulin at plus end to stabilize MT

MT Destabilizing/Severing Proteins promote depolarization of MT

• Stathmin/Op 18

  • Bin to tubulin heterodimers and prevents their polymerization

• Catastrophins (kinesins)

  • Act at the ends of MT, promote the peeling of subunits from the ends

• Proteins like katanins sever Mt