FORMATION,INTERACTION & FUNCTION OF SPINDLE COMPONENTS

描述（申请人的描述）：本提案的广泛目标 是为了确定有丝分裂的进入和退出是如何控制的， 和动粒的作用和相互作用形成了纺锤体， 产生和调节染色体运动和定位。目标#1 延时视频光学显微镜（VLM），GFP成像和3-D电子 显微镜（3D-EM）将用于：a）检验微管 参与检查点调节进展，通过前期， 脊椎动物;并确定B）如果前期细胞，由低温诱导， 回复到G2，24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;和c）在这种细胞中染色体分离异常的病因学， 当在G2中诱导16小时后重新进入有丝分裂时的回复突变体。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 假设在脊椎动物细胞：a）数量和长度的星形 由与染色体无关的中心体形成的微管发生变化， B）在前中期， 缺乏相关染色体; c）无中心体途径 纺锤体组装是有丝分裂的组成部分;以及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来检验假设 认为：a）与动粒相关的马达负责快速向极运动 在后期期间，动粒附着不再起作用，和B） ZW- 10参与有丝分裂过程中动粒的重新定向， 果蝇的减数分裂在目标4中，我们将使用激光显微手术来确定： a）在果蝇减数分裂和有丝分裂期间是否存在“极风” 精母细胞和神经母细胞，如果是，它们是否存在于神经母细胞中 缺乏功能性Klp 3 Sb和/或Nod蛋白。我们还将测试 假设：B）破坏一个动粒的一部分不会抑制 脊椎动物中的染色体聚集，以及c）染色体，双定向在 一种由两极形成的纺锤体， 微管，大会更接近弱梭极。最后，在目标#5中，我们 将使用激光显微手术和GFP成像来测试假设：a） 在脊椎动物细胞中由诺考达唑或紫杉醇诱导的有丝分裂停滞是由于 着丝粒; B）这些细胞中C-后期需要功能性 c）C-后期与中心体的破坏在时间上相关， 细胞周期蛋白B;和d）果蝇精母细胞缺乏纺锤体装配 检查站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， 设计新的治疗策略来控制细胞增殖， 用于治疗涉及微管的其它疾病状态， 关节炎、转移和老年痴呆症。

DESCRIPTION (applicant's description): 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 and positioning are generated and regulated. In Aim #1 time-lapse video light microscopy (VLM), GFP-imaging and 3-D electron microscopy (3D-EM) will be used to: a) test the hypothesis that microtubules are involved in the checkpoint regulating progression through prophase in vertebrates; and to determine b) if prophase cells, induced by hypothermia to revert to G2, fail to re-enter mitosis after 24 hrs because they lack cyclin A or Cdc25; and 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 and 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 spindle assembly is a constitutive component of mitosis; and d) that centrosomes and/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 responsible for 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 and meiosis in Drosophila. In Aim #4 we will use laser microsurgery to determine: a) if "polar winds" are present during meiosis and mitosis in Drosophila spermatocytes and neuroblasts and if so whether they are present in neuroblasts lacking functional Klp3 Sb and/or Nod proteins. We will also test the hypotheses that: b) destroying part of one kinetochore does not inhibit chromosome congression in vertebrates, and 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-imaging to 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; and d) that Drosophila spermatocytes lack a spindle assembly checkpoint. The knowledge obtained 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 involving microtubules including arthritis, metastasis and Alzheimer' s.