W22 Flashcards
Describe the structure of microtubules.
Consist of alpha- and beta-tubulin heterodimers. Both monomers are GTP binding, which is hydrolysable and exchangeable in beta-tubulin. Tubulin heterodimers stack ‘end on’ as protofilaments, 13 protofilaments typically assemble to form the cylindrical microtubule
Describe the structure of the centrosome.
Contains two centrioles at an angle to one another, composed of triplet microtubules. Only one microtubule is complete while the other two are incomplete grafted onto the side of each other. These triplet microtubules are found in nine sets that form a cart wheel structure anchored around a central set of nine coil-coil protein SAS-6 dimers joined in a ring structure. The two centrioles are surrounded by a matrix of proteins in pericentriolar material
Describe the structure of gamma-tubulin ring complexes (gamma-TuRCs).
Gamma-TuRCs are cone shaped structures held in a ring complex by a set of accessory proteins forming a cap that acts as a template to initiate formation of microtubule polymers
Describe the phases of microtubule growth.
A slow phase of unstable early assembly intermediates has energetics favouring disassembly over assembly but after a sufficiently large oligomer is formed by a variable number of steps, assembly is energetically favoured and elongation proceeds in the rapid phase
Describe the effect of a nucleator on microtubule growth.
Preformed nuclei allow microtubule growth to bypass the slow phase, providing spatial and temporal control over new microtubule growth. In bulk assays, the presence of a nucleator creates rapid microtubule polymerisation, bypassing the lag phase observed during spontaneous growth
Describe how gamma-TuRCs are formed.
Gamma-tubulin small complexes (gamma-TuSCs) are comprised of two gamma-tubulin bound to the accessory proteins GCP2 and GCP3. Seven gamma-TuSC assemble into a cone-shaped ring structure (gamma-TuRC) by binding additional accessory proteins GCP4-6. The gamma-TuRC is in a ‘lock washer’ spiral by which the first and last gamma-TuSC subunits slightly overlap each other. The overlap within the seven assembled gamma-TuSCs explains why there is a 13-fold symmetry of microtubules rather than 14-fold
Describe the microtubule seam.
The ‘lock washer’ spiral of gamma-TuRC leads to the formation of the microtubule seam by which the alpha- and beta-tubulin heterodimers in the first and last protofilaments are discontinuously aligned, providing binding sites for particular proteins
Describe attachment of gamma-tubulin complexes to different surfaces.
Attachment factors alter microtubule stability, anchoring and even direct branching. Gamma-TuSC and gamma-TuRC localisation can occur at the MTOC but also on the side of existing microtubules to create branches
Describe how the guanine nucleotide status of tubulin dictates structure of the microtubule.
GTP-tubulin aligns as straight protofilaments, upon GTP hydrolysis, the subunit interactions are weakened and the protofilaments curve away from one another, triggering depolymerisation
Describe how microtubule binding proteins affect stability.
Non-cargo transporting kinesin proteins (kinesin-13) regulate microtubule stability during mitosis by binding microtubule ends, destabilising them and increasing the frequency of catastrophes. XMAP215 is a protein that binds microtubule ends, suppressing the frequency of catastrophes and enhancing the growth rate
Describe how EB1 dimers provide microtubule function.
EB1 dimers bind to microtubules using calponin homology (CH) domains, they bind only to growing microtubules because it recognises the GTP cap. Many other proteins associate with microtubule plus-ends because they bind to EB1. Other +TIP binding proteins interact with EB1 via the EBH domain binding to SxIP motifs in other proteins, or the EEY/F motif in EB1 binding to CAP-Gly domains in other proteins. EB1 binds to many different proteins that have different functions such as regulating microtubule stability, and interactions with the cell cortex or kinetochores
Describe microtubule targeting agents.
Either stabilisers or destabilisers. Taxanes bind within the microtubule lumen to beta-tubulin and stabilises lateral contacts between subunits. Colchicine binds the intradimer alpha/beta tubulin interface and prevents the curved-to-straight transition in the tubulin heterodimer upon binding the microtubule. Vinca alkaloids bind at the interface between heterodimers, inducing a kink or causing assembly of aberrant ring-like structures
