The broad objectives of this proposal are to determine how entry and exit from mitosis is controlled, how centrosomes and kinetochores function and interact to form the spindle, and how the forces for chromosome motion & positioning are generated & regulated.
In Aim #1 time-lapse video lightmicroscopy (VLM), GFP-imaging and 3-D electron microscopy (3D-EM) will be used to: a) test the hypothesis that microtubulesare involved in the checkpoint regulating progression through prophase in vertebrates; & to determine b) if prophase cells, induced by hypothermia to revert to 62, fail to re-enter mitosis after 24 hrs because they lack cyclin A or Cdc25; & c) the etiology of chromosome segregation abnormalities in such revertants when induced to re-enter mitosis after 16 hrs in G2.
In Aim #2 we will use cell fusion, GFP-imaging, 3D EM and antibody injection to evaluate the hypotheses that in vertebrate cells: a) the numbers & length of astral microtubules, formed by centrosomes not associated with chromosomes, changes during prometaphase; b) that separating asters interact during prometaphase in the absence of associated chromosomes; c) that the acentrosomal pathway for spindleassembly is a constitutive component of mitosis; & d) that centrosomes &/or centrioles are required for the completion of cytokinesis.
In Aim #3 we will use antibody injection, VLM and 3D-EM to test the hypotheses that: a) the kinetochore associated motor responsiblefor fast poleward motion during kinetochore attachment is no longer functional during anaphase, and b) that ZW-10 is involved in kinetochore re-orientation during mitosis & meiosis in Drosophila.
In Aim #4 we will use laser microsurgery to determine: a) if """"""""polar winds"""""""" are present during meiosis & mitosis in Drosophilaspermatocytes & neuroblasts& if so whether they are present in neuroblasts lacking functional Klp38b &/orNod proteins. We will also test the hypotheses that: b) destroying part of one kinetochore does not inhibit chromosome congression in vertebrates, & c) that a chromosome, bioriented on a spindle formed from two poles that nucleate vastly different numbers of microtubules, congresses closer to the weak spindle pole. Finally, in Aim #5 we will use laser microsurgery and GFP-imagingto test the hypotheses: a) that the mitotic arrest induced in vertebrate cells by nocodazole or Taxol is due to kinetochores; b) that C-anaphase in these cells requires a functional centrosome; c) that C-anaphase is correlated temporally with the destruction of cyclin B; & d) that Drosophila spermatocytes lack a spindle assembly checkpoint. The knowledgeobtained from these studies is required to better understand the etiology of various birth defect syndromes and cancers, to design new therapeutic strategies for the control of cell proliferation, and for the treatment of other disease states involvingmicrotubules including arthritis, metastasis and Alzheimer's.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Deatherage, James F
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Wadsworth Center
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Lee, Kyunghee; Kenny, Alison E; Rieder, Conly L (2011) Caspase activity is not required for the mitotic checkpoint or mitotic slippage in human cells. Mol Biol Cell 22:2470-9
Yang, Zhenye; Kenny, Alison E; Brito, Daniela A et al. (2009) Cells satisfy the mitotic checkpoint in Taxol, and do so faster in concentrations that stabilize syntelic attachments. J Cell Biol 186:675-84
Rieder, Conly L; Medema, René H (2009) No way out for tumor cells. Cancer Cell 16:274-5
Khodjakov, Alexey; Rieder, Conly L (2009) The nature of cell-cycle checkpoints: facts and fallacies. J Biol 8:88
Brito, Daniela A; Rieder, Conly L (2009) The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells. Cell Motil Cytoskeleton 66:437-47
DiMaio, Michael A; Mikhailov, Alexei; Rieder, Conly L et al. (2009) The small organic compound HMN-176 delays satisfaction of the spindle assembly checkpoint by inhibiting centrosome-dependent microtubule nucleation. Mol Cancer Ther 8:592-601
Khodjakov, Alexey; Rieder, Conly L (2009) Mitosis: too much of a good thing (can be bad). Curr Biol 19:R1032-4
Brito, Daniela A; Yang, Zhenye; Rieder, Conly L (2008) Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied. J Cell Biol 182:623-9
Yang, Zhenye; Loncarek, Jadranka; Khodjakov, Alexey et al. (2008) Extra centrosomes and/or chromosomes prolong mitosis in human cells. Nat Cell Biol 10:748-51
Mikhailov, Alexei; Patel, Daksha; McCance, Dennis J et al. (2007) The G2 p38-mediated stress-activated checkpoint pathway becomes attenuated in transformed cells. Curr Biol 17:2162-8

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