FORMATION,INTERACTION & FUNCTION OF SPINDLE COMPONENTS

这项建议的主要目标是确定有丝分裂的进入和退出是如何控制的，中心体是如何控制的， 和动粒的功能和相互作用，以形成纺锤体，以及如何为染色体的运动和定位的力量 产生和调节。在目标#1中，时间推移视频光学显微镜（VLM），GFP成像和3-D电子 显微镜（3D-EM）将用于：a）检验微管参与检查点调节的假设 在脊椎动物中通过前期的进展;以及确定B）前期细胞是否由低温诱导恢复到 62，24小时后不能重新进入有丝分裂，因为它们缺乏细胞周期蛋白A或Cdc 25; 当在G2中16小时后被诱导重新进入有丝分裂时，这些回复突变体中的分离异常。在目标#2中，我们将使用 细胞融合、GFP成像、3D EM和抗体注射来评估脊椎动物细胞中的假设： 由与染色体无关的中心体形成的星形微管的数量和长度， 前中期; B）在没有相关染色体的情况下，在前中期期间，分离的紫苑相互作用; c） 纺锤体组装无中心体途径是有丝分裂的组成部分; d）中心体和/或 中心粒是完成胞质分裂所必需的。在目标#3中，我们将使用抗体注射，VLM和3D-EM， 检验以下假设：a）动粒相关运动负责动粒期间的快速向极运动 附着在后期不再起作用，和B）ZW-10参与后期的动粒重定向， 果蝇的有丝分裂和减数分裂在目标4中，我们将使用激光显微手术来确定：a）是否存在“极地风” 在果蝇精子细胞和成神经细胞的减数分裂和有丝分裂期间，如果是，它们是否存在于成神经细胞中 缺乏功能性Klp 38 b和/或Nod蛋白。我们还将检验以下假设：B）破坏一个 动粒不抑制脊椎动物的染色体聚集，c）染色体在纺锤体上双向取向 由两个极形成，这两个极成核的微管数量相差很大， 柱最后，在目标5中，我们将使用激光显微手术和GFP成像来验证假设：a）有丝分裂停滞 在脊椎动物细胞中由诺考达唑或紫杉醇诱导的C-后期是由于着丝粒; B）这些细胞中的C-后期需要一个 c）C-后期与细胞周期蛋白B的破坏在时间上相关; d）果蝇 精母细胞缺乏纺锤体组装检查点。从这些研究中获得的知识需要更好地 了解各种出生缺陷综合征和癌症的病因，为新生儿设计新的治疗策略。 控制细胞增殖，以及用于治疗涉及微管的其它疾病状态，包括关节炎， 转移和老年痴呆症

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.