Tubulin dynamics in cultured mammalian cells.

Abstract

Bovine neurotubulin has been labeled with dichlorotriazinyl-aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cellcycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (~3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF-tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.The organization and apparent function of microtubules in cultured mammalian cells vary markedly as the cell cycle proceeds from interphase to mitosis and back to interphase again. Structural studies with polarization optics, immunofluorescence, and electron microscopy have shown that the mitotic spindle is an active structure in which microtubules exhibit rather rapid changes in arrangement and length (l 1-13, 22, 23, 27, 3 l, 38). The function of mitotic microtubules has been the subject of a great deal of debate for many years (reviewed in references 11 and 27), but it is clear that they are involved in the movement of chromosomes. Interphase cytoskeletal microtubules appear comparatively static with changes in arrangement and length occurring slowly (15). Although their function is also the subject of debate, they may be involved in the specification of cell polarity, shape, and cytoplasmic organization (3,22,28,37,44).To understand the different behaviors of microtubules in mitotic and interphase cells, it may be useful to investigate the dynamic relationship between tubulin and microtubules in vivo. Studying this relationship in detail requires a method for viewing tubulin in both polymer and dimer form in living cells. In principle, microinjection of fluorescently labeled tubulin followed by fluorescence microscopy provides such a method. In practice it is a useful method only if the labeled tubulin retains the functional characteristics of the native protein (17,18,36,40). Recent work supports the belief that the behavior of tubulin labeled with dichlorotriazinyl-aminofluorescein (DTAF) is analogous to the behavior of unlabeled tubulin (15,19,...

MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

“…Our measured t 1/2 values for the slow phase of the recovery for prometaphase cells are similar to those reported previously for the K-fibers . Our values for the fast phase, however, are faster than those reported by others (Saxton et al, 1984;Zhai et al, 1995;Cimini et al, 2006). One possibility is that our fast phase actually represents the diffusion of unincorporated GFP-tubulin subunits into the bleach zone.…”

“…Our data support the model that MCAK perturbs spindle organization by acting preferentially on astral MTs and that it contributes to error correction by controlling MT dynamics in the K-fiber. Previous studies measured the dynamics of spindle MTs by using either FRAP or photoactivation Saxton et al, 1984;Mitchison, 1989;Zhai et al, 1995;WatermanStorer et al, 1998;Cimini et al, 2006). In most studies using FRAP, the data were fit to a single exponential that represented the turnover of the highly dynamic spindle MTs.…”

“…The spindle MTs or nonkinetochore MTs (nonkt-MTs) represent the bulk of the MT polymer in the spindle and are highly dynamic, with a half-time for recovery (t 1/2 ) of ϳ18 s (Saxton et al, 1984;Cimini et al, 2006). The kinetochore MTs (K-fibers) are organized into bundles of MTs that mediate the attachment of the chromosomes to the spindle (Rieder, 1982;McDonald et al, 1992;McIntosh et al, 2002).…”

“…The spindle is a macromolecular structure composed of a dynamic array of microtubules (MTs) and their associated proteins that is necessary for proper chromosome segregation (Compton, 2000;Walczak and Heald, 2008). At the onset of mitosis, there is an increase in MT turnover that allows for breakdown of the interphase array and assembly of the mitotic spindle (Saxton et al, 1984;. This change is correlated with an increased catastrophe frequency (transition from growth to shrinkage) and a decreased rescue frequency (transition from shrinkage to growth) (Belmont et al, 1990;Gliksman et al, 1992;Verde et al, 1992;Rusan et al, 2001).…”

Fueled by microtubules: Does tubulin dimer/polymer partitioning regulate intracellular metabolism?

“…Such a scenario seems unlikely given the 1-10 mM pool of ATP normally present in a cell [Beis and Newsholme, 1975]. About half of the cell's 20 lM tubulin is in polymer form [Zhai and Borisy, 1994;Holmfeldt et al, 2009] and this 10 lM polymer turns over with a half-time of about 10 min during interphase [Saxton et al, 1984]. Therefore, 5 lM of tubulin dimers depolymerizes and repolymerizes every 10 min, consuming 5 lM of GTP at the same time.…”