Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:676202 |
| Date | January 2013 |
| Creators | Lynch, Eric Michael |
| Contributors | Sawin, Ken ; Allshire, Robin |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12224 |
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