Actin protrustions, oocytes: Myosin VIIA and the actin cytoskeleton

肌动蛋白突起，卵母细胞：肌球蛋白VIIA和肌动蛋白细胞骨架

基本信息

批准号：
8100317
负责人：
Jennifer Leigh Sallee
金额：
$ 5.13万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8100317
关键词：
Actins Address Adult Affinity Chromatography Alleles Auditory Binding Biochemical Biological Biological Assay Biological Models Biological Process Blindness Cell-Cell Adhesion Cells Cellular Morphology Code Complement Complex Confocal Microscopy Cytoskeleton Defect Disease Drosophila genus Embryonic Development Employee Genes Genetic Genetic Epistasis Germ Lines Hair Hair Cells Hearing Homologous Gene Human Hybrids In Vitro Infertility Inherited Investigation Labyrinth Maintenance Molecular Morphology Mus Mutation Myosin ATPase Neoplasm Metastasis Nerve Degeneration Oocytes Oogenesis Organ Ovary Phenotype Play Point Mutation Process Proteins Role Series Severities Structure Syndrome Techniques Transgenic Organisms Usher Syndrome Yeasts Zebrafish base cell type cellular microvillus congenital deafness deafness design dimer egg fly genetic analysis genetic manipulation human disease in vivo insight mutant novel prospective protein complex public health relevance research study yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): The actin cytoskeleton plays a key role in diverse biological processes and when perturbed cause human disease and suffering. One such example is Usher type 1 syndrome, a condition characterized by congenital deafness and blindness. Myosin VIIA has been identified as a gene responsible for human Usher syndrome 1B and non-syndromic deafness. In Drosophila, Myosin VIIA is coded by the locus crinkled (ck). Severe mutations of ck/MyoVIIA in flies are lethal or "semi-lethal", with only a small fraction of homozygous flies reaching adulthood. These escapers show defects in actin rich structures such as bristles, epidermal hairs, and in the fly's hearing organ. In addition, severe ck mutant allele flies are infertile. To fully understand how ck contributes to biological functions like bristle and hair formation, further investigation is needed on how the forces it produces are transmitted to the appropriate structure or cargo. Specific Aim 1 will employee two approaches designed to identify proteins interacting with ck/MyoVIIA, yeast 2-hybrid screens and tandem affinity purification from transgenic flies. Interactions between identified proteins and ck/MyoVIIA will be confirmed with in vitro binding assays, in vivo co-localization studies, and genetic manipulation in transgenic flies. With the observation that severe ck mutants that survive to adulthood are infertile, we propose a novel role for ck/MyoVIIA in actin rich structure in oogenesis which is likely distinct from the bristle and hearing phenotypes based on the cell types in the egg chambers. Specific Aim 1 will explore the role of ck/MyoVIIA in oogenesis and early embryogenesis using germ line clones homozygous for an allelic series of mutant ck/MyoVIIA ranging from truncated protein to single point mutations. The different ck alleles display different severities of phenotypes regarding the number of escapers and the bristle and hair formation. It is expected to see a range of phenotypes here as well. The morphology of egg chambers will be characterized using confocal microscopy, focusing on irregularities in actin rich structures such as ring canals, microvilli, and cell-cell adhesions. Together these aims will provide insight into how protein complexes are contributing to ck/Myosin VIIA function in actin protrusions and oogenesis. PUBLIC HEALTH RELEVANCE: Usher Syndrome, a leading cause of inherited deafness and blindness, is associated with defects in inner-ear hair cells caused by mutations in several proteins, including Myosin VIIA. Myosin VIIA is essential for proper formation of the actin-based hair cells; however the exact molecular mechanism is unknown. This project will examine proteins in complex with Myosin VIIA and their requirement for normal actin-based protrusion formation and function.

描述（由申请人提供）：肌动蛋白细胞骨架在多种生物过程中发挥着关键作用，当受到干扰时会导致人类疾病和痛苦。 Usher 1 型综合症就是一个这样的例子，这是一种以先天性耳聋和失明为特征的疾病。肌球蛋白 VIIA 已被确定为导致人类亚瑟综合征 1B 和非综合征性耳聋的基因。在果蝇中，肌球蛋白 VIIA 由 crinkled 基因座 (ck) 编码。果蝇中 ck/MyoVIIA 的严重突变是致死或“半致死”的，只有一小部分纯合果蝇达到成年。这些逃逸者在富含肌动蛋白的结构（如刚毛、表皮毛发和果蝇的听觉器官）中表现出缺陷。此外，严重的ck突变等位基因果蝇是不育的。为了充分了解 ck 如何促进鬃毛和毛发形成等生物功能，需要进一步研究它产生的力如何传递到适当的结构或货物。具体目标 1 将采用两种方法来识别与 ck/MyoVIIA 相互作用的蛋白质、酵母 2 杂交筛选和转基因果蝇的串联亲和纯化。已鉴定的蛋白质和 ck/MyoVIIA 之间的相互作用将通过体外结合测定、体内共定位研究和转基因果蝇的遗传操作来证实。通过观察存活到成年的严重 ck 突变体是不育的，我们提出 ck/MyoVIIA 在卵子发生中富含肌动蛋白的结构中的新作用，这可能与基于卵室中细胞类型的刚毛和听力表型不同。具体目标 1 将使用一系列等位基因突变 ck/MyoVIIA（从截短蛋白到单点突变）纯合的种系克隆来探索 ck/MyoVIIA 在卵子发生和早期胚胎发生中的作用。不同的 ck 等位基因在逃逸者数量以及鬃毛和毛发形成方面表现出不同严重程度的表型。预计这里也会出现一系列的表型。将使用共聚焦显微镜来表征卵室的形态，重点关注富含肌动蛋白的结构的不规则性，例如环管、微绒毛和细胞间粘附。这些目标共同将深入了解蛋白质复合物如何促进肌动蛋白突出和卵子发生中的 ck/肌球蛋白 VIIA 功能。公共健康相关性：亚瑟综合症是遗传性耳聋和失明的主要原因，它与多种蛋白质（包括肌球蛋白 VIIA）突变引起的内耳毛细胞缺陷有关。肌球蛋白VIIA对于基于肌动蛋白的毛细胞的正确形成至关重要；然而确切的分子机制尚不清楚。该项目将检查与肌球蛋白 VIIA 复合的蛋白质及其对正常肌动蛋白突起形成和功能的要求。