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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03226

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Microtubule nucleation: -tubulin and beyond

¹ Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706, USA
² HHMI and Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA

View larger version (87K): [in a new window]   Fig. 1. Different types of microtubule array are organized by centrosomes during interphase and mitosis, as in these cultured Xenopus laevis kidney epithelial cells. Microtubules are shown in red, DNA in blue and the centrosomal protein -tubulin in green.

View larger version (17K): [in a new window]   Fig. 2. Microtubules are assembled from /ß-tubulin dimers, which associate head-to-tail to form linear protofilaments (boxed area). 12-15 protofilaments associate laterally to form the walls of the microtubule cylinder, which has a diameter of 25 nm. The GTP-binding pocket in -tubulin is covered by ß-tubulin at all times and is therefore not exchangeable (see text). By contrast, the nucleotide on ß-tubulin can be exchanged while the dimer is not part of a microtubule. GTP hydrolysis is coupled to the incorporation of the dimer into the microtubule, and its energy facilitates rapid microtubule disassembly. What triggers the transition between growth and shrinkage of the microtubule, or between its shrinkage and growth, is not yet understood. Tubulin subunits can add to both ends of the cylinder, but the plus end grows about three times faster than the minus end in vitro. Within the cell, almost all microtubule minus ends are anchored at MTOCs, leaving the plus ends to extend into the cell periphery. During rapid microtubule disassembly, protofilaments peel back from the microtubule wall. Disassembly is facilitated by tension introduced into the protofilament by a conformational change in ß-tubulin upon GTP hydrolysis, which occurs when the subunit is incorporated into the microtubule lattice.

View larger version (32K): [in a new window]   Fig. 3. Microtubule-organizing centers (MTOCs) in (A) animal cells, (B) budding yeast and (C) Dictyostelium. (A) The animal centrosome consists of a pair of centrioles surrounded by pericentriolar material (PCM). MTs are nucleated from -tubulin ring structures embedded in the PCM. (B) The yeast spindle-pole body (SPB) is a disk-like trilaminar plug that spans both nuclear membranes and organizes astral and spindle MTs on the cytoplasmic and nuclear sides of the nuclear envelope, respectively. (C) The slime mold (Dictyostelium discoideum) interphase centrosome consists of a three-layered core surrounded by a fibrous corona, from which MTs emanate.

View larger version (12K): [in a new window]   Fig. 4. A current model of the arrangement of subunits within the TuRC postulates that 6-7 TuSC subcomplexes are held together by the other Grip proteins, which together form the cap subunits. Dgp71WD, which is the only known TuRC subunits that lacks grip motifs (see below), is not required for the assembly of the TuRC, and might be attached peripherally.

View larger version (106K): [in a new window]   Fig. 5. Sequence alignments of five known grip-motif-containing Drosophila TuRC subunits (Dgrip75, Dgrip84, Dgrip91, Dgrip128 and Dgrip163) with Dgrip79, a protein identified in database searches on the basis of the presence of the grip motifs; it is not yet known whether Dgrip79 is a component of the TuRC. (A) An overall sequence alignment shows two regions of higher sequence similarity (orange), labeled `grip motif 1' and `grip motif 2', shared among the six grip-domain proteins. (Please note that the sequence of Dgrip163 was truncated except in regions of homology to the other grip proteins.) The number of amino acids for each of the proteins is indicated to the right of the alignments. (B,C) Sequence alignments of (B) grip motif 1 or (C) grip motif 2. Starting and ending amino acid numbers, respectively, are listed at the left and the right of the alignment for each protein. Shading indicates the degree of sequence conservation (see key). Sequence alignments were generated using the `T-coffee' algorithm (Notredame et al., 2000; Poirot et al., 2004).

View larger version (41K): [in a new window]   Fig. 6. Schematic diagrams of TuRC recruitment to interphase centrosomes, mitotic centrosomes during centrosome maturation, and to mitotic spindle MTs. (A) During interphase, centrosomal -tubulin is exchanged with the cytoplasmic pool. (B) Additional TuRC is recruited to centrosomes as cells enter mitosis in a process called centrosome maturation. During centrosome maturation, the interphase centrosome increases in size and recruits five times more -tubulin. Concomitantly, five times more MTs are nucleated by the mature centrosomes. (C) During mitosis, TuRC is recruited to centrosomes as well as to spindle MTs (indicated by arrows; notice that TuRC is not drawn to scale). How -tubulin is recruited to different sites and in a cell-cycle-dependent manner is only beginning to emerge. The mechanisms of TuRC recruitment to centrosomes during interphase and mitosis are probably mediated by distinct sets of proteins (e.g. Casenghi et al., 2003).

View larger version (26K): [in a new window]   Fig. 7. Two possibilities exist for the orientation of -tubulin (and consequently the TuRC) with respect to the long axis of the MT to which it is bound. (A) Adjacent -tubulin subunits make shoulder-to-shoulder contacts in the template model, as indicated by the red oval that marks the position of the GTP-binding pocket on -tubulin. (B) -tubulin subunits make head-to-tail contacts in the protofilament model.