Rieder
Conly L.
Rieder
Conly L.
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ArticleCells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments(Rockefeller University Press, 2009-08) Yang, Zhenye ; Kenny, Alison E. ; Brito, Daniela A. ; Rieder, Conly L.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 ~0.5 µM. 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 µM. Inhibiting the aurora-B kinase in Taxol-treated RPE1 cells accelerates checkpoint satisfaction by stabilizing syntelic kinetochore attachments and reduces the DM to ~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 µM. 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.
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PreprintKinetochore fiber formation in animal somatic cells : dueling mechanisms come to a draw( 2005-09-12) Rieder, Conly L.The attachment to and movement of a chromosome on the mitotic spindle is 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, there are only three possible ways to form a K-fiber: either it a) grows from the pole until it contacts the kinetochore; b) grows directly from the kinetochore; or c) it forms as a result of an interaction between the pole and the chromosome. Since Schrader’s time it has been firmly established that K-fibers in centrosome-containing animal somatic cells form as kinetochores capture MTs growing from the spindle pole (route a). It is now similarly clear that in cells lacking centrosomes, including plants and many animal oocytes, K-fibers “self-assemble” from MTs generated by the chromosomes (route b). Can animal somatic cells form K-fibers in the absence of centrosomes by the “self-assembly” pathway? In 2000 the answer to this question was shown to be a resounding “yes”. With this result, the next question became whether the presence of a centrosome normally suppresses K-fiber self-assembly, or if this route works concurrently with centrosome-mediated K-fiber formation. This question, too, has recently been answered: observations on untreated live animal cells expressing GFP-tagged tubulin clearly show that kinetochores can nucleate the formation of their associated MTs in the presence of functional centrosomes. The concurrent operation of these two “dueling” routes for forming K-fibers in animals helps explain why the attachment of kinetochores and the maturation of K-fibers occur as quickly as it does on all chromosomes within a cell.
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PreprintImaging the division process in living tissue culture cells( 2005-07-29) Khodjakov, Alexey ; Rieder, Conly L.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. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy.
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PreprintLaser microsurgery in the GFP era : a cell biologist's perspective( 2007-06) Magidson, Valentin ; Loncarek, Jadranka ; Hergert, Polla ; Rieder, Conly L. ; Khodjakov, AlexeyModern 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.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter we review some of the applications for laser microsurgery at the subcellular level, and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our lab. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope or software, but instead only in defining creative questions and in visualizing the target to be destroyed.
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ArticleMicrotubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied(Rockefeller University Press, 2008-08-18) Brito, Daniela A. ; Yang, Zhenye ; Rieder, Conly L.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°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.
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PreprintExtra centrosomes and/or chromosomes prolong mitosis in human cells( 2008-04-04) Yang, Zhenye ; Loncarek, Jadranka ; Khodjakov, Alexey ; Rieder, Conly L.Using laser microsurgery and cell fusion we have explored how additional centrosomes and/or chromosomes influence the duration of mitosis in human cells. We find that doubling the chromosome number adds ~10 minutes to a 20 minute division while doubling the number of centrosomes adds ~30 minutes more, and extra centrosomes and/or chromosomes prolong mitosis by delaying satisfaction of the spindle assembly checkpoint. Thus mitosis can be prolonged by non genetic means and extra chromosomes and centrosomes likely contribute to the elevated mitotic index seen in many tumors.