Caspase activity is not required for the mitotic checkpoint or mitotic slippage in human cells.

Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells.

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints.

In this review, I stress the importance of direct data and accurate terminology when formulating and communicating conclusions on how the G2/M and metaphase/anaphase transitions are regulated. I argue that entry into mitosis (i.e.

P38 mitogen-activated protein kinase activity is required during mitosis for timely satisfaction of the mitotic checkpoint but not for the fidelity of chromosome segregation.

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by approximately 40% in nontransformed human RPE-1, approximately 80% in PtK2 (rat kangaroo), and approximately 25% in mouse cells, and this prolongation leads to an elevated mitotic index.

Mitosis: too much of a good thing (can be bad).

Recent studies reveal that the precise regulation of microtubule dynamics is essential for an error-free mitosis. Kinetochore microtubule attachments that are too stable increase the rate of chromosome mis-segregation, a leading cause of chromosomal instability in tumors.

The nature of cell-cycle checkpoints: facts and fallacies.

The concept of checkpoint controls revolutionized our understanding of the cell cycle. Here we revisit the defining features of checkpoints and argue that failure to properly appreciate the concept is leading to misinterpretation of experimental results. We illustrate, using the mitotic checkpoint, problems that can arise from a failure to respect strict definitions and precise terminology.

No way out for tumor cells.

In this issue of Cancer Cell, Huang et al. report that preventing exit from mitosis provides a very efficient strategy for killing cells. Although this is not an unexpected finding, it nevertheless provides a novel conceptual framework for augmenting the antimitotic strategies currently under development for fighting cancer.

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.

The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation.

HMN-176 is a potential new cancer therapeutic known to retard the proliferation of tumor cell lines. Here, we show that this compound inhibits meiotic spindle assembly in surf clam oocytes and delays satisfaction of the spindle assembly checkpoint in human somatic cells by inducing the formation of short and/or multipolar spindles. HMN-176 does not affect centrosome assembly, nuclear envelope breakdown, or other aspects of meiotic or mitotic progression, nor does it affect the kinetics of Spisula or mammalian microtubule (MT) assembly in vitro.

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.

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.

Extra centrosomes and/or chromosomes prolong mitosis in human cells.

Using laser microsurgery and cell fusion we have explored how additional centrosomes and/or chromosomes influence the duration of mitosis in human cells. We found that doubling the chromosome number added approximately 10 min to a 20 min division, whereas doubling the number of centrosomes added approximately 30 min more. Extra centrosomes and/or chromosomes prolong mitosis by delaying satisfaction of the spindle assembly checkpoint.

The G2 p38-mediated stress-activated checkpoint pathway becomes attenuated in transformed cells.

When human cells are stressed during G2, they are delayed from entering mitosis via a checkpoint mediated by the p38 kinase, and this delay can be modeled by the selective activation of p38 with anisomycin. Here, we report, on the basis of live-cell studies, that 75 nM anisomycin transiently (1 hr) activates p38 which, in turn, rapidly and completely blocks entry into mitosis for at least 4 hr in all primary, telomerase- or spontaneously immortalized (p53+ and pRB+) human cells. However, the same treatment does not delay entry into mitosis in cancer cells, or the delay in entering mitosis is shortened, even though it induces a similar transient and comparable (or stronger) activation of p38.

Mitch a rapidly evolving component of the Ndc80 kinetochore complex required for correct chromosome segregation in Drosophila.

We identified an essential kinetochore protein, Mitch, from a genetic screen in D. melanogaster. Mitch localizes to the kinetochore, and its targeting is independent of microtubules (MTs) and several other known kinetochore components.

The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis.

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear.

Laser microsurgery in the GFP era: a cell biologist's perspective.

Modern biology is based largely on a reductionistic "dissection" approach-most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.

Comment on "A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint".

Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the gamma-tubulin ring complex (gamma-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that gamma-TuRC proteins play molecular roles in SAC activation.

Kinetochore dynein is required for chromosome motion and congression independent of the spindle checkpoint.

During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6].

Extracellular signal-regulated kinase 1/2 activity is not required in mammalian cells during late G2 for timely entry into or exit from mitosis.

Extracellular signal-regulated kinase (ERK)1/2 activity is reported to be required in mammalian cells for timely entry into and exit from mitosis (i.e., the G2-mitosis [G2/M] and metaphase-anaphase [M/A] transitions).

Mammalian CLASP1 and CLASP2 cooperate to ensure mitotic fidelity by regulating spindle and kinetochore function.

CLASPs are widely conserved microtubule plus-end-tracking proteins with essential roles in the local regulation of microtubule dynamics. In yeast, Drosophila, and Xenopus, a single CLASP orthologue is present, which is required for mitotic spindle assembly by regulating microtubule dynamics at the kinetochore. In mammals, however, only CLASP1 has been directly implicated in cell division, despite the existence of a second paralogue, CLASP2, whose mitotic roles remain unknown.

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells.

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs).

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).

Imaging the division process in living tissue culture cells.

We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination.

Kinetochore fiber formation in animal somatic cells: dueling mechanisms come to a draw.

The attachment to and movement of a chromosome on the mitotic spindle are mediated by the formation of a bundle of microtubules (MTs) that tethers the kinetochore on the chromosome to a spindle pole. The origin of these "kinetochore fibers" (K fibers) has been investigated for over 125 years. As noted in 1944 by Schrader [Mitosis, Columbia University Press, New York, 110 pp.

The p38-mediated stress-activated checkpoint. A rapid response system for delaying progression through antephase and entry into mitosis.

Cells have evolved a number of control pathways that delay or prevent them from entering mitosis under conditions that can compromise genome integrity. One recently appreciated and versatile control pathway involves the p38 stress activated protein kinase. During late G2 p38 is rapidly activated by diverse stresses (topoisomerase II (topo II)) and histone deacetylase inhibitors, osmotic shock, microtubule disassembly, UV light, etc) via a number of different pathways.