RUI: Tubulin Folding and Assembly in Chlamydomonas

真核细胞含有复杂的内部结构，称为微管，微管是中空的管状微管蛋白聚合物，参与决定细胞的形状、组织、有丝分裂和减数分裂和运动。新微管蛋白亚基的产生始于编码α和β微管蛋白的mRNAs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单位池的一部分。这种翻译和二聚化途径涉及一种特殊的蛋白质，称为分子伴侣，它有助于微管蛋白亚基的成熟。一个关键的参与者是一个被称为CCT的大型多聚体复合体(用于含有TCP-1的伴侣蛋白)。CCT已经在多种细胞类型和酵母中进行了生物化学和遗传学研究；然而，对于折叠路径如何在细胞质的复杂环境中进行空间组织，或者它如何通过二聚体的可获得性在调节微管组装中发挥作用，人们知之甚少。在这个项目中，Johnson博士将进行实验，以定位CCT和单细胞绿藻衣藻中微管蛋白折叠路径的其他组件。衣藻是研究包括鞭毛运动在内的多种重要细胞特征的重要模式生物。它的一对长的鞭状鞭毛从每个细胞的顶端细胞质延伸出来，以波浪形、桨状的拍打推动细胞通过其水环境。每个鞭毛都是一个复杂的微型机器，包含一束专门的微管，这些微管由同样专门的动力蛋白分子马达驱动；这种复杂的微管和马达的纵向阵列被称为轴丝，通常是鞭毛的特征。这些鞭毛可以试验性地从衣藻细胞中移除，这种移除会触发鞭毛再生过程，在这个过程中，每个替换结构都会在90分钟内产生与细胞体本身一样长的替换结构。这个过程可以在实验室中方便地诱导，包括新的微管蛋白在细胞体内的大量翻译，微管蛋白二聚体进入和通过鞭毛空间，以及二聚体聚合成细长结构远端(远)端的新生微管。正是在这种空间分离的合成、运输和组装的背景下，约翰逊博士将研究微管蛋白折叠途径的组织。约翰逊博士将结合分子和生物化学方法，确定衣藻CCT亚单位编码基因的特征，并研究它们在鞭毛再生过程中的表达。针对这些亚单位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，本科生将广泛参与研究过程。这项研究为深入了解活细胞中微管蛋白生物发生的空间组织提供了重要的机会。由于微管结构和细胞用来创造它们的过程在所有真核生物中都是高度保守的，这样的研究将有助于从总体上更好地理解微管生物学。

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.