Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody.

Abstract

A lysed cell system was developed to determine whether tubulin antibody can block the nucleation of exogenous tubulin at kinetochores and centrosomes .Mitotic PtK2 cells were pretreated with colcemid to remove all endogenous microtubules and were lysed with Triton X-100 in PIPES-EGTA-Mg" buffer . This procedure left centrosomes, chromosomes, and kinetochores intact as determined by electron microscopy of thin-sectioned cells. Exposure of the lysed cells to phosphocellulose-purified tubulin dimers at 37°C in the presence of 1 mM GTP resulted in site-specific nucleation of microtubules at centrosomes and kinetochores . Treatment of the lysed cell preparations with tubulin antibody before subsequent exposure to the exogenous tubulin resulted in almost complete blockage of microtubule nucleation, especially at kinetochores. Pretreatment of the lysed cell preparations with control antibody or buffer without antibody had no effect on the ability of centrosomes and kinetochores to initiate microtubule assembly. The implications of these results with respect to the molecular composition of centrosomes and kinetochores are discussed .KEY WORDS Centrosomes " kinetochores microtubule nucleation " tubulin antibody electron microscopy During mitosis, spindle microtubule assembly occurs at specific nucleating sites such as the kinetochore on the chromosomes and the spindle poles (for reviews, see references 1, 15, 17) . Such regions, known widely as microtubule organizing centers (MTOCs) (20), have been described extensively but relatively little is known of their molecular composition or the way in which they initiate the assembly of tubulin into microtubules. Several cytochemical studies have indicated that kinetochores, centrioles (basal bodies), and related MTOCs contain RNA which may be involved in the nucleation of microtubule assembly (2,13, 19,30 could be detected by immunoperoxidase staining with tubulin antibodies. Moreover, it was proposed that tubulin was an intrinsic protein of the MTOC and was essential for the initiation of microtubule polymerization at these sites. To further evaluate the existence of tubulin in MTOCs and to test the notion that intrinsic tubulin of kinetochores is essential for microtubule polymerization, we investigated the assembly of purified 6S bovine brain tubulin at kinetochores and centrioles of lysed mitotic cells before and after exposure to tubulin antibodies . MATERIALS AND METHODSTubulin Purification and Characterization J. CELL BIOLOGY © The Rockefeller University Press -0021-9525/79/08/0585/07 $1 .00Microtubule protein was prepared from bovine brain by three cycles of polymerization-depolymerization according to the method of Borisy et al. (5) . The microtubule pellet obtained was resuspended in cold buffer A

Contribution of microtubule growth polarity and flux to spindle assembly and functioning in plant cells

“…During spindle assembly in some lower plants, ␥-tubulin extends from the polar regions to the metaphase plate, which suggests that microtubule nucleation sites translocate towards the chromosomes (Brown et al, 2004). In mammalian cells also, microtubules are nucleated at chromosomes in vitro and in vivo (Telzer et al, 1975;Pepper and Brinkley, 1979;Bergen et al, 1980;Mitchison and Kirschner, 1985). Recently, chromosome-originated kinetochore fibre-formation has been observed in vivo in centrosomal Drosophila S2 cells both during prometaphase and metaphase, suggesting the persistent capacity of kinetochores to nucleate microtubules in vivo (Maiato et al, 2004).…”

Interplay Between Spindle Architecture and Function

“…Early studies from Brinkley and McIntosh described MTs polymerizing from kinetochores, but until more recently this was not demonstrated in live cells (Khodjakov et al, 2003;Maiato et al, 2004;Pepper and Brinkley, 1979;Snyder and McIntosh, 1975). While the molecular mechanism of MT nucleation at the kinetochore is unknown, there is evidence that this process, like chromatin-mediated spindle assembly, requires the small GTPase Ran (Torosantucci et al, 2008;Tulu et al, 2006).…”

51‐kd protein, a component of microtubule‐organizing granules in the mitotic apparatus involved in aster formation in vitro

“…Isolated centrosomes radially nucleate microtubules forming asterlike structures [McGill and Brinkley, 1975;Snyder and McIntosh, 1975;Weisenberg and Rosenfeld, 1975;Gould and Borisy, 1977;Pepper and Brinkley, 1979;Telzer and Rosenbaum, 1979;Bergen et al, 19801. On the other hand, the basal body, having the same structure as the centriole, nucleates microtubules in vitro at either end of it in a template manner [Snell et al, 19741.…”