Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Abstract

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 ,uM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

“…To avoid the confounding effects that can be produced by the activity of these drugs in the other mitotic phases, control and CIT-K-depleted HeLa cells were synchronized and treated with the drugs during telophase, that is, after the genetic material had already been segregated by spindle microtubules. The drugs were used at a low concentration, which is known to specifically affect dynamic microtubules, 32,33 Conversely, we found that nocodazole treatment rescues the cytokinesis delay phenotype induced by CIT-K overexpression that we previously described 23 ( Figure 2f). Altogether, these results indicate that CIT-K regulates abscission by increasing the stability of midbody microtubules and that differential stability of dynamic microtubules during cytokinesis may determine the sensitivity of cells to CIT-K loss.…”

C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes

“…Role of the cytoskeleton network The involvement of the cytoskeleton network in C-peptide endocytosis was studied in HAEC with cytochalasin D (30 mmol/l) and nocodazole (10 mmol/l), which act by inducing depolymerisation of actin filaments and microtubules, respectively [25,26]. As shown in Fig.…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…Migratory cells form a stable sub-population of microtubules at the leading edge to reorient the microtubule organizing center (MTOC) between the nucleus and the leading edge to create a polarized microtubule array that directs vesicular trafficking and maintains cell shape [9,10,19]. Additionally, disruption of the microtubule network by nocodazole-induced microtubule catastrophe or taxol-mediated microtubule stabilization, results in a depolarized cell morphology and a dramatic reduction in both the rate and directionality of migration [2,20]. Conversely, release from the stabilizing effects of microtubule antagonists has been shown to induce microtubule regrowth toward the membrane ruffle, activation of Rac1 and the formation of filopodia and lamellipodia [2,19,21].…”

“…For example, nocodazole-induced microtubule depolymerisation leads to an increase in the number and size of focal adhesions at both the leading and trailing edge, anchoring the cell to the ECM to the point where it becomes immobilized [19]. Rescue of microtubule growth following catastrophe shows a resurgence in targeting events of focal adhesions by microtubule plus ends, resulting in focal adhesion dissolution and cell migration [2,19,20]. Microtubule plus ends have been shown to be differentially regulated depending on their subcellular location within the migrating cell.…”