H3 Toth- Cytoskeleton I - microtubules

describe the 3 kinds of cytoskeleton

• actin (micro) filaments- all eukaryotes, polar, use ATP, dynamic, 7nm

• tubulin microtubules- all eukaryotes, polar, use GTP, dynamic, 25nm

• intermediate filaments (multiple kinds)- only animals, apolar, don’t use ATP/GTP, less dynamic, 10nm

describe intermediate filament structure and function (give examples)

• apolar (the two ends have the same properties)

• long strands with globular C and N termini assemble into coiled-coil dimers

• dimers assemble antiparallel into tetramers

• these line up into smaller subunits, which anneal into long filaments

• eg. keratin, vimentin, desmin, lamin

• mainly used for structural support, determining and maintaining the cell and nucleus shape (so they aren’t needed in plants)

• this is because they have a high tensile strength and are resistant to compression and bending

• eg. lamins form a lattice structure which covers the inner side of the nuclear envelope as a site for chromatin anchorage

describe the structure of microtubules

• tubulin monomers are heterodimers of two subunits: alpha and beta

• the heterodimers assemble end to end, so that the alpha and beta subunits alternate, into long strands called protofilaments

• 13 of these protofilaments assemble into a hollow cylinder

• the plus end is the beta end, and the minus end is the alpha end

• each of the subunits has a GTP-binding site, but only beta-tubulin can hydrolyse it- the GTP bound to alpha-tubulin lends stability and proper folding

decribe microtubule formation, polymerisation and depolymerisation

• self-assembly from scratch is difficult because 13 subunits need to interact laterally, which is energetically unfavourable

• in cells gamma-tubulin ring complexes are used in initiation to act as structural templates for the alpha-tubulin of the heterodimers to bind (at the minus end)

• these complexes are anchored at particular organelles

• tubulin monomers, bound to GTP, are added to the plus end

• the GTP provides strong lateral interactions for stability

• once a monomer associates, it triggers GTP hydrolysis by the beta-tubulin subunit

• this is very slow, so new subunits can incorporate before GTP hydrolysis is achieved, creating a GTP cap at the plus end

• the GDP-bound heterodimer has a different conformation, which can induce dissociation if the GTP cap isn’t maintained (when the hydrolysis rate > polymerisation rate), resulting in shrinkage, and vice versa

• this is dynamic instability, which can result in complete depolymerisation if there are no GTP-bound dimers to rescue the growth

what is the function and organisation of microtubules in plant cells?

• in young cells microtubules radiate out from the nucleus

• in larger growing cells (interphase) they associate and run parallel with the membrane

• these guide cellulose deposition in the correct orientation

• enzyme complexes in the plasma membrane extrude cellulose polymers as they travel along parallel membrane anchored microtubules

what is the function and organisation of microtubules in animal cells?

• microtubules radiate out from the centrosome (the microtubule organising centre, MTOC), which contains a pair of centrioles- the centrosome has many gamma-tubulin ring complexes for initiation

• most microtubules constantly polymerise and depolymerise out from the centrosome due to dynamic instability

• this is because hitting the membrane perpendicularly causes the loss of the GTP cap and results in depolymerisation

• we believe this functions to help the cell to explore and sense changes in the plasma membrane

• microtubules also act as tracks for motor proteins to move organelles and transport vesicles around the cell eg. the endomembrane system

• the spindle fibres used in mitosis are microtubules

• (eukaryotic) cilia contain microtubules in a 9+2 formation for movement eg. sperm, and signalling/sensing eg. rod cells

how is microtubule growth regulated?

most of the time dynamic instability isn’t observed because of protein action:

• microtubule associated proteins (MAPs) stabilise the plus end to promote growth

• catastrophins destabilise the plus end to promote depolymerisation

how are organelles moved around the cell by microtubules?

motor proteins bind to vesicles and organelles and move along the microtubules

• kinesins move towards the plus end (outwards)- eg. for COP-I vesicles golgi → ER

  • these are dimers with a cargo-binding tail domain, a stalk and a two head domain, which moves along microtubules in 8nm steps, each using one ATP (binding and hydrolysis cause conformational changes for moving the heads forwards)

• dyneins move towards the minus end (inwards)- eg. for COP-II vesicles ER → golgi

  • these are more complicated, sometimes involving accessory proteins for binding, but using ATP to make irregular movements

how are microtubules organised in eukaryotic cilia and how do they bend?

• the basal body (originating from centrioles, but not in plants) extrudes microtubules in a 9+2 arrangement

• 9 doublets (13 protofilament cylindrical A microtubule + 11 protofilament attached B microtubule) are arranged in a ring, with 2 singlets in the centre

• the doublets are connected by nexin proteins and have radial spokes towards the centre

• the microtubules are always enclosed by the plasma membrane

• ciliary dynein (a motor protein complex) moves along the doublet protofilaments due to ATP

• this would cause the filaments to slide, but due to the nexin linking proteins and controlled activity, this causes bending

• in short cilia this produces a force perpendicular to the long axis, but in long cilia this symmetric beat produces a parallel force (in a sinusoidal wave)

compare bacterial and eukaryotic cilia proteins, positions and movement