Cloning & in vivo role of flagellar protein Kinase A

克隆

• 批准号: 6954782
• 负责人: DAVID R HOWARD
• 金额: $ 18.52万
• 依托单位: UNIVERSITY OF WISCONSIN LA CROSSE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954782
• 关键词: Chlamydomonas; RNA interference; SDS polyacrylamide gel electrophoresis; biological signal transduction; biotechnology; cilium /flagellum motility; cyclic AMP; electroporation; mass spectrometry; molecular cloning; plant proteins; polymerase chain reaction; protein kinase A; protein localization; protein structure function; transmission electron microscopy; western blottings

Numerous diseases are caused by defective cilia and/or flagella, including primary ciliary dyskinesia, polycystic kidney disease, lateralization defects, Bardet-Biedl syndrome, retinal degeneration, and male infertility. All of these diseases can be caused by a variety of molecular defects, some of which have recently been uncovered and some of which remain unknown. Progress in understanding cilia and flagella in health is largely due to the knowledge gained through basic science done in the model organism Chlamydomonas reinhardtii. The regulation of motility in cilia and flagella remains relatively poorly understood, both at a basic level and in the relationship to disease. This proposal is focused on determining the identity and the in vivo role of flagellar cAMP dependent protein kinase (PKA) in Chlamydomonas. Abundant evidence indicates that PKA regulates flagellar motility in both Chlamydomonas and humans. In Chlamydomonas, it has been shown that PKA slows the activity of dynein motors in vitro, but the in vivo role of PKA in flagella is not known. In addition, the PKA protein or gene has not been positively identified or characterized. In Aim 1, flagellar PKA proteins will be identified in an affinity-based approach using the same tools that were used to show PKA's function in vitro. This method allows the discoveries to be immediately related to previously established in vitro data. Identified PKA(s) will be partially sequence using tandem MS, and their full sequence located. A gene exists in the Chlamydomonas database that could possibly be PKA, but its predicted protein sequence does not clearly classify it as a PKA. Based on our preliminary data, there seems to be multiple PKAs in flagella. In Aim 2, cDNAs coding for flagellar PKA will be cloned and used to determine the number of different PKA transcripts that are made. In Aim 3, the in vivo role of flagellar PKA will be determined by disrupting its function, either by electroporatirig cells with biotin-PKI to inhibit kinase activity or by RNA-mediated interference (RNAi). Swimming velocity, phototaxis ability, and beat waveform parameters will then be measured. Together, this project will allow data about the in vitro control of dynein activity to be related to changes in flagellar beating in vivo. In addition the project will provide critical information about a key component of the pathway that regulates flagellar notility. An integral component of this project is the involvement of undergraduates in all aspects of the work.

许多疾病是由纤毛和/或鞭毛缺陷引起的，包括原发性睫状运动障碍，多囊性肾脏疾病，横向缺陷，Bardet-Biedl综合征，视网膜变性和男性不孕症。所有这些疾病都可能是由多种分子缺陷引起的，其中一些缺陷最近被发现，其中一些仍然未知。理解纤毛和鞭毛在健康方面的进展很大程度上是由于模型有机体中的基础科学获得的知识。在基本水平和与疾病的关系中，纤毛和鞭毛运动中运动的调节仍然相对较低。该建议的重点是确定鞭毛cAPP依赖性蛋白激酶（PKA）在衣原体中的身份和体内作用。大量证据表明，PKA调节衣原体和人类的鞭毛运动。在 衣原体，已经表明，PKA在体外减慢了Dynein Motor的活性，但是PKA在鞭毛中的体内作用尚不清楚。另外，尚未对PKA蛋白或基因进行积极鉴定或表征。在AIM 1中，将使用用于在体外显示PKA功能的工具的基于亲和力的方法中识别鞭毛PKA蛋白。这种方法允许发现与先前建立的体外数据立即相关。鉴定的PKA（S）将使用Tandem MS及其完整序列进行部分序列。可以是PKA的衣原体数据库中存在一个基因，但其预测的蛋白质序列并未明确将其归类为PKA。根据我们的初步数据，鞭毛中似乎有多个PKA。在AIM 2中，编码鞭毛PKA的cDNA将会 克隆并用于确定所制作的不同PKA转录本的数量。在AIM 3中，鞭毛PKA的体内作用将通过使用生物素-PKI的电载纤维细胞破坏其功能来确定其功能以抑制激酶活性或通过RNA介导的干扰（RNAI）。然后将测量游泳速度，光疗能力和击败波形参数。该项目一起将允许有关动力蛋白活性体外控制的数据，与体内鞭毛跳动的变化有关。此外，该项目将提供有关调节鞭毛通知性的路径关键组成部分的关键信息。一个积分 该项目的组成部分是本科生参与工作的各个方面。