The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells.

We used live cell imaging to compare the fate of human nontransformed (RPE-1) and cancer (HeLa, U2OS) cells as they entered mitosis in nocodazole or taxol. In the same field, and in either drug, a cell in all lines could die in mitosis, exit mitosis and die within 10 h, or exit mitosis and survive > or =10 h. Relative to RPE-1 cells, significantly fewer HeLa or U2OS cells survived mitosis or remained viable after mitosis: in nocodazole concentrations that inhibit spindle microtubule assembly, or in 500 nM taxol, 30% and 27% of RPE-1 cells, respectively, died in or within 10 h of exiting mitosis while 90% and 49% of U2OS and 78% and 81% of HeLa died. This was even true for clinically relevant taxol concentrations (5 nM) which killed 93% and 46%, respectively, of HeLa and U2OS cells in mitosis or within 10 h of escaping mitosis, compared to 1% of RPE-1 cells. Together these data imply that studies using HeLa or U2OS cells, harvested after a prolonged block in mitosis with nocodazole or taxol, are significantly contaminated with dead or dying cells. We also found that the relationship between the duration of mitosis and survival is drug and cell type specific and that lethality is related to the cell type and drug used to prevent satisfaction of the kinetochore attachment checkpoint. Finally, work with a pan-caspase inhibitor suggests that the primary apoptotic pathway triggered by nocodazole during mitosis in RPE-1 cells is not active in U2OS cells. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.

Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.

In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast , and likely Drosophila, this occurs through an "adaptation" process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, we conducted fluorescence microscopy studies on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. We find that in the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. We also find that cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, our data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis.

Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium.

Overexpression of dynein fragments in Dictyostelium induces the movement of the entire interphase microtubule array. Centrosomes in these cells circulate through the cytoplasm at rates between 0.4 and 2.5 microm/s and are trailed by a comet-tail like arrangement of the microtubule array. Previous work suggested that these cells use a dynein-mediated pulling mechanism to generate this dramatic movement and that similar forces are at work to maintain the interphase MTOC position in wild-type cells. In the present study, we address the nature of the forces used to produce microtubule movement. We have used a laser microbeam to sever the connection between the motile centrosomes and trailing microtubules, demonstrating that the major force for such motility results from a pushing on the microtubules. We eliminate the possibility that microtubule assembly/disassembly reactions are significant contributors to this motility and suggest that the cell cortex figures prominently in locating force-producing molecules. Our findings indicate that interphase microtubules in Dictyostelium are subject to both dynein- and kinesin-like forces and that these act in concert to maintain centrosome position in the cell and to support the radial character of the microtubule network.

Deconstructing the centriole: structure and number control.

Centrioles are very small microtubule-based organelles essential for centrosome, cilia and flagella assembly, which are involved in a variety of cellular and developmental processes. Although the centriole was first described almost a century ago, the knowledge on its assembly mechanism remains poor. In the past decade, forefront functional studies have provided important data on the different players involved in centriole biogenesis. Centriole research has now started to profit from highly sensitive structural, imaging, and biochemical techniques that are unveiling how those players contribute to assemble such a small and complex structure. We will review those studies and discuss how this field will increasingly benefit from the newborn and exciting era of super resolution analyses.

Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy.

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.

Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments.

To determine why the duration of mitosis (DM) is less in Taxol than in nocodazole or Eg5 inhibitors we studied the relationship between Taxol concentration, the DM, and the mitotic checkpoint. We found that unlike for other spindle poisons, in Taxol the DM becomes progressively shorter as the concentration surpasses approximately 0.5 microM. Studies on RPE1 and PtK2 expressing GFP/cyclin B or YFP/Mad2 revealed that cells ultimately satisfy the checkpoint in Taxol and do so faster at concentrations >0.5 microM. Inhibiting the aurora-B kinase in Taxol-treated RPE1 cells accelerates checkpoint satisfaction by stabilizing syntelic kinetochore attachments and reduces the DM to approximately 1.5 h regardless of drug concentration. A similar stabilization of syntelic attachments by Taxol itself appears responsible for accelerated checkpoint satisfaction at concentrations >0.5 microM. Our results provide a novel conceptual framework for how Taxol prolongs mitosis and caution against using it in checkpoint studies. They also offer an explanation for why some cells are more sensitive to lower versus higher Taxol concentrations.

Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied.

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37 degrees C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.