RUI: Tubulin Folding and Assembly in Chlamydomonas

RUI：衣藻中微管蛋白的折叠和组装

• 批准号: 9982733
• 负责人: Karl Johnson
• 金额: $ 24.71万
• 依托单位: Haverford College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982733&HistoricalAwards=false
• 关键词: RUI; Tubulin; Folding; Assembly; Chlamydomonas

Eukaryotic cells contain elaborate internal structures called microtubules, which are hollow, pipe-like polymers of tubulin proteins involved in the determination of the cell's shape, organization, mitotitc and meiotic divisions and movement. The production of new tubulin subunits begins with the translation of alpha and beta tubulin-encoding mRNAs, followed by folding of the nascent polypeptide chains and formation of alpha-beta dimers. The dimers then become part of the pool of free subunits available for microtubule assembly. This translation and dimerization pathway involves special proteins called molecular chaperones that assist in the maturation of tubulin subunits. A key participant is a large multimeric complex called CCT (for Chaperonin Containing TCP- 1). CCT has been studied biochemically in a variety of cell types and genetically in yeast; however, relatively little is known about how the folding pathway is spatially organized within the complex environment of the cytoplasm, or how it may play a role in regulating microtubule assembly through dimer availability.In this project, Dr. Johnson will perform experiments directed at localizing CCT and other components of the tubulin folding pathway in the single-celled green alga Chlamydomonas. Chlamydomonas is an important model organism for the study of a variety of significant cellular features, including flagellar motility. Its pair of long, whip-like flagella extend from the apical cytoplasm of each cell and beat in wave-form, oar-like strokes to propel the cell though its aqueous environment. Each flagellum is a complex miniature machine containing a bundle of specialized microtubules that are animated by also-specialized dynein molecular motors; this complex longitudinal array of microtubules and motors is termed an axoneme and is characteristic of flagella in general. These flagella can be experimentally removed from the Chlamydomonas cells, and such removal results in the triggering of a flagellar regeneration process in which replacement structures, each as long as the cell body itself, are produced within 90 minutes. This process, which can be conveniently induced in the laboratory, involves massive translation of new tubulin proteins within the cell body, movement of tubulin dimers into and through the flagellar space and polymerization of the dimers into nascent microtubules at the distal (far) end of the elongating structure. It is within this context of spatially separated synthesis, transport and assembly that Dr. Johnson will investigate the organization of the tubulin folding pathway. Using a combination of molecular and biochemical approaches, Dr. Johnson will characterize genes encoding Chlamydomonas CCT subunits and study their expression during flagellar regeneration. Specific antibody probes to the subunits will be used to localize CCT within the cell using immunofluorescence and immunoEM techniques. These experiments will be carried out in a predominantly undergraduate institution and will involve extensive participation by undergraduates in the research process. This investigation offers significant opportunities for novel insight into the spatialorganization of tubulin biogenesis in the living cell. Because microtubule architectures,and the processes that cells use to create them, are highly conserved in all eukaryotes,such studies will lead to a better understanding of microtubule biology in general.

真核细胞含有称为微管的精细内部结构，微管是微管蛋白的中空管状聚合物，参与决定细胞的形状、组织、有丝分裂和减数分裂分裂和运动。 新微管蛋白亚基的产生始于编码α和β微管蛋白的mRNA的翻译，随后是新生多肽链的折叠和α-β二聚体的形成。然后二聚体成为可用于微管组装的游离亚基库的一部分。 这种翻译和二聚化途径涉及称为分子伴侣的特殊蛋白质，其有助于微管蛋白亚基的成熟。 一个关键的参与者是一个大的多聚体复合物称为CCT（伴侣蛋白包含TCP- 1）。 CCT已在多种细胞类型中进行了生物化学研究，并在酵母中进行了遗传研究;然而，对于折叠途径如何在细胞质复杂环境中进行空间组织，或者它如何通过二聚体的可用性在调节微管组装中发挥作用，人们知之甚少。在该项目中，约翰逊博士将在单细胞绿色衣原体中进行针对CCT和微管蛋白折叠途径其他组分的定位实验。 衣原体是一种重要的模式生物，用于研究各种重要的细胞特征，包括鞭毛运动。 它的一对长长的鞭状鞭毛从每个细胞的顶端细胞质延伸出来，以波浪状的桨状划动来推动细胞穿过水环境。 每个鞭毛都是一个复杂的微型机器，包含一束特殊的微管，这些微管由同样特殊的动力蛋白分子马达驱动;这种微管和马达的复杂纵向阵列被称为轴丝，通常是鞭毛的特征。 这些鞭毛可以通过实验从衣原体细胞中去除，并且这种去除导致鞭毛再生过程的触发，在该过程中，在90分钟内产生替换结构，每个替换结构与细胞体本身一样长。 这个过程，这可以方便地在实验室中诱导，涉及大量的翻译细胞体内的新的微管蛋白，微管蛋白二聚体的运动进入并通过鞭毛空间和聚合的二聚体成新生的微管在远端（远）端的延伸结构。 约翰逊博士将在空间分离的合成、运输和组装的背景下研究微管蛋白折叠途径的组织。 使用分子和生物化学方法的组合，约翰逊博士将表征编码衣原体CCT亚基的基因，并研究它们在鞭毛再生过程中的表达。亚基的特异性抗体探针将用于使用免疫荧光和免疫EM技术在细胞内定位CCT。这些实验将在一个以本科生为主的机构中进行，并将涉及本科生在研究过程中的广泛参与。 这项研究为深入了解活细胞中微管蛋白生物合成的空间组织提供了重要的机会。由于微管结构和细胞用于创建它们的过程在所有真核生物中都是高度保守的，因此这些研究将有助于更好地理解微管生物学。