p27(Kip1) and Retinal Attachment

p27(Kip1) 和视网膜附着

• 批准号: 7366889
• 负责人: Dennis Michael Defoe
• 金额: $ 21.3万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7366889
• 关键词: Ablation; Actins; Address; Adhesions; Adult; Animals; Apical; Area; Binding Sites; Biological; Biological Assay; Breeding; CDKN1B gene; Cell Cycle; Cell Cycle Proteins; Cell Cycle Regulation; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Complex; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Cytoplasm; Cytoskeleton; DNA; Data; Defect; Development; Disruption; Dysplasia; Electrons; Engineering; Epithelial; Epithelial Cells; Epithelium; Event; Excision; Exhibits; Gene Targeting; Generations; Genes; Genetic; Goals; Hand; Knock-in Mouse; Knockout Mice; Light; Maintenance; Methods; Microscopic; Molecular; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Neural Retina; Nuclear; Nuclear Export; Phenotype; Phosphorylation; Phosphorylation Site; Photoreceptors; Play; Point Mutation; Process; Property; Protein Export Pathway; Proteins; Regulator Genes; Research; Retinal; Retinal Detachment; Retinal Dysplasia; Role; Signal Transduction; Site; Structure of retinal pigment epithelium; System; Testing; Tissues; Tyrosinase related protein-1; Vision; Western Blotting; base; bcl-1 Genes; cellular microvillus; cyclin-dependent kinase inhibitor 1B; density; gastrointestinal microvillus; human TYRP1 protein; inhibitor/antagonist; mutant; p27 Cell Cycle Protein; p27 Enzyme Inhibitor; promoter; protein expression; protein function; recombinase; research study; rho GTP-Binding Proteins

DESCRIPTION (provided by applicant): The proposed research has as its overall goal to define mechanisms that underlie normal retinal attachment, with the aim that better methods of restoring the interaction between the neural retina and its supporting retinal pigment epithelium (RPE) may be developed, and thus loss of visual function resulting from retinal detachment ameliorated. Little is known about how the structural components of the RPE-neural retina interface are generated, in terms of specific signaling events and their sequence of activation in development. The proposed experiments focus specifically on the role of the epithelium, and will make use of a strain of mice that is deficient in the cell cycle regulatory protein p27(Kip1). Aside from retinal proliferative defects and limited foci of retinal dysplasia, these animals exhibit broad areas of retinal detachment, evident as a loss of interdigitation between photoreceptor outer segments and RPE microvilli. Aim 1 will test the hypothesis that p27(Kip1) plays a role in establishment and maintenance of the RPE-neural retina interface that is independent of its involvement in epithelial cell cycle regulation. For these experiments, two strains of knock-in mice will be examined using established light and electron microscopic methods. The first strain (p27CK-) contains point mutations in the p27(Kip1) binding sites for cyclins and cyclin-dependent kinase (CDK), resulting in a protein that is unable to act as a cell cycle inhibitor, but nevertheless retains its cytoskeleton regulatory properties. In the second strain (p27S10A), the major phosphorylation site regulating p27(Kip1) protein export to the cytoplasm has been altered, yielding a protein that can carry out its function as a negative regulator of cell proliferation within the nucleus but is unable to influence the actin cytoskeleton. The possibility that the p27(S10A) mutation causes the induction of retinal detachment, and that p27CK- rescues detachment resulting from p27(Kip1) gene ablation will be examined. Finally, the possibility that retinal detachment is due to increased RPE cellular or nuclear density per se will be examined by crossing p27(Kip1)-null mice with mice lacking the positive cell cycle regulatory gene cyclin D1. Aim 2 will examine whether the retinal detachment we observe in p27(Kip1)-null mice results, at least in part, from a loss of protein function specifically within the epithelium. To test this, the Cre-lox system for conditional gene targeting will be implemented to ablate the p27(Kip1) gene selectively in RPE cells.

描述（由申请人提供）：拟议研究的总体目标是定义正常视网膜附着的机制，目的是开发更好的方法来恢复神经视网膜与其支持性视网膜色素上皮（RPE）之间的相互作用，从而改善因视网膜脱离导致的视觉功能丧失。关于 RPE-神经视网膜界面的结构成分是如何产生的、具体的信号事件及其在发育过程中的激活顺序，人们知之甚少。拟议的实验特别关注上皮的作用，并将利用缺乏细胞周期调节蛋白 p27（Kip1）的小鼠品系。除了视网膜增殖缺陷和有限的视网膜发育不良病灶外，这些动物还表现出大面积的视网膜脱离，明显表现为光感受器外节和 RPE 微绒毛之间的交叉缺失。目标 1 将检验以下假设：p27(Kip1) 在 RPE-神经视网膜界面的建立和维持中发挥作用，该作用独立于其对上皮细胞周期调节的参与。对于这些实验，将使用已建立的光和电子显微镜方法检查两种基因敲入小鼠品系。第一个菌株 (p27CK-) 在 p27(Kip1) 细胞周期蛋白和细胞周期蛋白依赖性激酶 (CDK) 结合位点中含有点突变，导致蛋白质无法充当细胞周期抑制剂，但仍保留其细胞骨架调节作用特性。在第二种菌株 (p27S10A) 中，调节 p27(Kip1) 蛋白输出到细胞质的主要磷酸化位点已被改变，产生的蛋白可以发挥其作为细胞核内细胞增殖负调节因子的功能，但无法影响肌动蛋白细胞骨架。将检查p27(S10A)突变引起视网膜脱离的诱导以及p27CK-挽救由p27(Kip1)基因消融引起的脱离的可能性。最后，通过将 p27(Kip1) 缺失小鼠与缺乏阳性细胞周期调节基因细胞周期蛋白 D1 的小鼠杂交来检查视网膜脱离是否是由于 RPE 细胞或核密度本身增加所致。目标 2 将检查我们在 p27(Kip1) 缺失小鼠中观察到的视网膜脱离是否至少部分是由于上皮内特定的蛋白质功能丧失所致。为了测试这一点，将采用用于条件基因靶向的 Cre-lox 系统来选择性地消除 RPE 细胞中的 p27(Kip1) 基因。

项目成果

Corneal endothelial cells possess an elaborate multipolar shape to maximize the basolateral to apical membrane area.

角膜内皮细胞具有复杂的多极形状，以最大化基底外侧至顶膜面积。

• 发表时间: 2016
• 期刊: Molecular vision
• 影响因子: 2.2
• 作者: Harrison,TheresaA;He,Zhiguo;Boggs,Kristin;Thuret,Gilles;Liu,Hong-Xiang;Defoe,DennisM
• 通讯作者: Defoe,DennisM