CeEMAP-95 Function in the Nematode, C. elegans

微管是真核细胞的重要结构成分。它们在有丝分裂和减数分裂期间负责染色体的精确分离，并负责几种膜结合细胞器和大分子复合物的细胞内运输。不完成核分裂会导致染色体错误分离，导致非整倍体，这可能对生物体造成严重后果。此外，细胞内运输的故障会导致严重的发育缺陷。微管相关蛋白（MAPs）对微管组装的调控对这些基本的生物过程至关重要。该项目涉及对一种新的微管相关蛋白EMAP的详细分子研究，EMAP最初在海胆中发现，最近在高等和低等真核生物中发现。从线虫（秀丽隐杆线虫）到人类的高度序列保守性强调了EMAP的基本重要性。此外，人类EMAP-1基因与Usher综合征1a位点相对应，这是一种导致深度耳聋和失明的疾病。海胆EMAP对微管组装有独特的影响。在EMAP作用下组装的微管略长，但明显更具活力，很少发生抢救事件。因为EMAP是一个丰富的有丝分裂装置的组成部分，EMAP可能调节微管组装动力学在早期胚胎细胞周期。EMAP的不同寻常之处在于，它与与mrna亚群、聚(a)结合蛋白和核糖体相关的微管共同纯化。该项目的实验利用了Suprenant博士在EMAP的生化和分子表征方面的经验，以及在线虫C. elegans中操纵EMAP功能的能力。这些实验旨在确定线虫EMAP同源物CeEMAP-95是否是真正的微管结合蛋白，并鉴定线虫中依赖CeEMAP-95的过程。与海胆的EMAP类似，蠕虫的CeEMAP-95可能在胚胎发生过程中参与调节微管组装，或者可能通过特定发育决定因素的定位参与细胞不对称的产生。该项目的早期阶段将包括产生特异性抗体、CeEMAP-95突变等位基因和重组CeEMAP-95构建体，这些构建体将为继续研究这一有趣的蛋白质家族提供必要的分子试剂。此外，还将鉴定和表征蠕虫体内的其他微管相关蛋白，最终目的是确定这些蛋白是独立、协调还是与蠕虫的EMAP并行作用。具体而言，将解决以下问题：1，CeEMAP-95是否定位于蠕虫中富含微管的细胞类型？2、CeEMAP-95是微管装配动力学的调节剂吗？3、CeEMAP-95基因功能丧失是否影响胚胎细胞分裂或神经细胞功能？4、秀丽隐杆线虫中还存在哪些其他必需的微管结合蛋白？这项工作的长期目标是确定需要EMAP的细胞功能，并了解这些过程如何影响多细胞生物的发育。这将通过生物化学、细胞和分子生物学以及遗传学的结合来实现，包括体外EMAP功能的生化论证和体内功能的遗传鉴定。

Microtubules are critical structural components of eukaryotic cells. They are responsible for the accurate segregation of chromosomes during mitosis and meiosis, and for the intracellular transport of several membrane-bounded organelles and macromolecular complexes. Failure to complete nuclear division results in chromosome missegregation, resulting in aneuploidy, which can have drastic consequences for the organism. In addition, malfunctions in intracellular transport can lead to serious developmental defects. The regulation of microtubule assembly by microtubule-associated proteins (MAPs) is critical to these fundamental biological processes.This project involves a detailed molecular study of a novel microtubule-associated protein, EMAP, first identified in sea urchins and more recently discovered in higher and lower eukaryotes. The fundamental importance of EMAP is underscored by the high degree of sequence conservation from the nematode (C. elegans) to humans. In addition, the gene for human EMAP-1 maps to the Usher syndrome 1a locus, a disease that results in profound deafness and blindness. Sea urchin EMAP has unique effects on microtubule assembly. Microtubules assembled in the presence of EMAP are slightly longer, but significantly more dynamic, with infrequent rescue events. Because EMAP is an abundant component of the mitotic apparatus, EMAP may regulate microtubule assembly dynamics during the early embryonic cell cycles. EMAP is also unusual in that it co-purifies with microtubules that are associated with a subset of mRNAs, poly(A)-binding proteins and ribosomes. The experiments in this project take advantage of Dr. Suprenant's experience in the biochemical and molecular characterization of EMAP and the ability to manipulate EMAP function in the nematode, C. elegans. These experiments are designed to determine whether the C. elegans EMAP homologue, CeEMAP-95, is a bona fide microtubule-binding protein and to identify CeEMAP-95-dependent processes in the worm. Similar to sea urchin EMAP, worm CeEMAP-95 may be involved in regulating microtubule assembly during embryogenesis or perhaps in the generation of cellular asymmetry through the localization of specific developmental determinants. The early stages of this project will involve the generation of specific antibodies, CeEMAP-95 mutant alleles, and recombinant CeEMAP-95 constructs that will provide essential molecular reagents for the continuing study of this interesting protein family. Also, additional microtubule-associated proteins in the worm will be identified and characterized, with the eventual goal of determining whether these proteins act independently, coordinately or in parallel with worm EMAP. Specifically, the following questions will be addressed: 1, is CeEMAP-95 localized to microtubule-rich cell types in the worm? 2, is CeEMAP-95 a regulator of microtubule assembly dynamics? 3, does the loss of function of the CeEMAP-95 gene affect embryonic cell division or neuronal cell function? and 4, what other essential microtubule-binding proteins are present in C. elegans? The long term goal of this work is to identify the cellular functions that require EMAP and to understand how these processes affect the development of a multicellular organism. This will be done through the combination of biochemistry, cellular and molecular biology, and genetics, with both the biochemical demonstration of EMAP's function in vitro and the genetic identification of its function in vivo.