The microtubule cytoskeleton is required for a G2 cell cycle delay in cancer cells lacking stathmin and p53

Abstract

In several cancer cell lines, depleting the microtubule-destabilizing protein stathmin/oncoprotein18 leads to a G2 cell cycle delay and apoptosis. These phenotypes are observed only in synergy with low levels of p53, but the pathway(s) activated by stathmin depletion to delay the cell cycle are unknown. We found that stathmin depletion caused greater microtubule stability in synergy with loss of p53, measured by the levels of acetylated α-tubulin and the rate of centrosomal microtubule nucleation. Nocodazole or vinblastine-induced microtubule depolymerization abrogated the stathmin-depletion induced G2 delay, measured by the percentage of cells staining positive for several markers (TPX2, CDK1 with inhibitory phosphorylation), indicating that microtubules are required to lengthen G2. Live cell imaging showed that stathmin depletion increased time in G2 without an impact on the duration of mitosis, indicating that the longer interphase duration is not simply a consequence of a previous slowed mitosis. In contrast, stabilization of microtubules with paclitaxel (8 nM) slowed mitosis without lengthening the duration of interphase, demonstrating that increased microtubule stability alone is not sufficient to delay cells in G2.

Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1

“…Western blot for stathmin, reprobed for tubulin as a loading control. (See also Carney and Cassimeris, 2010; Carney et al. , 2012).…”

“…Additionally, we found that the delay induced by stathmin depletion could be abrogated by treatment with nocodazole to depolymerize MTs, suggesting that stathmin's role is carried out via MTs, a new function for the MT cytoskeleton in mitotic entry (Carney et al ., 2012). To determine the cause of delayed mitotic entry induced by stathmin depletion, we examined the signaling pathways that converge on cyclin B/CDK1 activation.…”

Cardiomyocyte proliferation contributes to heart growth in young humans

“…Calculating the number of cardiomyocytes generated based on cell cycle activity requires input of the incidence of a cell cycle marker (1), the duration of its presence (2), and the probability that the marker indicates division (3). We used the percentage of cardiomyocytes in M-phase, which shows conservation of regulatory mechanisms and duration (∼1.5 h) between species, tissues, and ages (34)(35)(36). To calculate the number of cardiomyocytes generated from M-phase activity, determined by H3P analysis, we used the previously determined duration of 1.8 ± 0.3 h in adult ventricular cardiomyocytes (21,37).…”

MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells

“…Mitotic entry was scored by the first image showing extensive cell rounding and is defined as the start time, whereas mitotic exit was scored as the last image showing the pinching-off of the two daughter cells (stop) (8,19,45). The duration time of mitosis (DTM) was measured for each mitotic cell in both conditions, thus the total sum of the DTM and the average DTM were calculated (error bars represent standard error; P Ͻ 0.005, Student's t-test).…”