Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model.

Abstract

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-1ysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold-induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca ++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca ++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca ++ resistant microtubules.Most eucaryotic cells contain a network of microtubules that extends throughout the cytoplasm, commonly referred to as the cytoplasmic microtubule complex (CMTC) (for review, see reference 9). The microtubules are not arranged randomly, but, rather, assembly occurs at specific times in the cell cycle by regulated polymerization of tubulin in association with the centrosome or microtubule-organlzing center (MTOC) and proceeds in an organized fashion to the cell periphery. This pattern can be observed in daughter cells following mitosis or in cells recovering from colcemid block (9). The factors responsible for the precise spatial distribution of microtubules and the intracellular location of these factors remain to be elucidated. With respect to the polymerization process, there are essentially two major intracellular levels at which the spatial organization and extent of microtubule assembly can be determined; the organizing center and the microtubule subunit protein tubulin and its associated proteins.Ultrastructural studies of MTOCs have shown that they consist of one or two pairs of centrioles with microtubules radiating from an amorphous pericentriolar material. The latter may contain initiating factors required for assembly (4,21,50). Although the precise molecular composition of MTOCs re-

Effects of intracellular ph on the mitotic apparatus and mitotic stage in the sand dollar egg

“…It is known that the stability of microtubules and critical concentration for microtubule assembly are maximal at slightly acidic pH using purified brain and egg tubulin [Olmsted and Borisy, 1975;Regula et al, 1981;Suprenant, 1989;Simons et al, 1992;Tiwari and Suprenant, 1994]. Moreover, Deery and Brinkley [1983] reported that at a slightly acidified condition, microtubules assembled from endogeneous tubulin in the cell which was lysed and pretreated with colcemid. However, slightly alkaline pH, but not acidic pH, favored microtubule assembly in the crude extracts of eggs or oocytes [Suprenant and Marsh, 1987;Suprenant, 1989Suprenant, , 1991Simons et al, 1992;Tiwari and Suprenant, 1994].…”

Recurring views on the structure and function of the cytoskeleton: A 300-Year Epic

“…In turn, this material amply justifies the term MTOC, since it was shown that such condensations not only seem to anchor the proximal ends of microtubules in certain cytoplasmic locations, but also act as sites for the initiation of microtubule assembly, which then proceeds by incorporating tubulin subunits preferentially at the distal ends [Inoué et al, 1974;Weisenberg and Rosenfeld, 1975;Brinkley et al, 1981;Deery and Brinkley, 1983;Mitchison and Kirschner, 1984;Soltys and Borisy, 1985;Rodionov et al, 1999]. Similarly, the basal bodies at the roots of cilia and flagella not only help to hold in place the proximal ends of the vibratile organelles, but also nucleate the assembly of tubulin upon themselves [Snell et al, 1974;Rosenbaum et al, 1975].…”

Is intracellular pH a clock for mitosis?

“…The disagreement between these values has been considered in relation to the nucleation potential of microtubule organizing centers like centrosomes [6], suggesting that pH i regulates the nucleation potential of microtubule organizing centers [7,8]. Experiments have also shown that ionic concentrations play an important role in microtubule polymerization [9].…”