New Toolkit for Imaging and Controlling Early Ciliogenesis

用于成像和控制早期纤毛发生的新工具包

• 批准号: 8621325
• 负责人: Derek K. Toomre
• 金额: $ 24.98万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621325
• 关键词: Acute; Affect; Biological Assay; Blindness; Brain; Breast; Cell Shape; Cell surface; Cells; Chemicals; Cilia; Data; Diabetes Mellitus; Disease; Environment; Foundations; Functional disorder; Future; Geometry; Goals; Hair; Heart Diseases; Hereditary Disease; Human; Human Genetics; Huntington Disease; Image; Imagery; Joubert syndrome; Kidney Diseases; Life; Light; Link; Location; Malignant Neoplasms; Mammalian Cell; Mechanics; Membrane; Membrane Protein Traffic; Metabolism; Methodology; Methods; Microscope; Molecular; Obesity; Odors; Optics; Organelles; Pancreas; Pattern; Pharmaceutical Preparations; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Physiological; Polycystic Kidney Diseases; Positioning Attribute; Process; Resolution; Retinal Degeneration; Role; Shapes; Staging; Stimulus; Structure; Surface; Syndrome; Testing; Therapeutic; Time; Total Internal Reflection Fluorescent; Translating; base; ciliopathy; cilium biogenesis; congenital heart disorder; deafness; design; extracellular; human disease; innovation; interest; novel; optogenetics; public health relevance; screening; sensor; sound; tool; trafficking; tumorigenesis

DESCRIPTION (provided by applicant): The major objective of this project is to develop new enabling methods to visualize and control early stages of primary cilia formation. Our long-term goal is to develop quantitative image-based high content screens for therapeutics to human diseases resulting from primary cilium dysfunction. Most cells possess a primary cilium, an organelle that extends as a single long slender protrusion from the surface. Long ignored, in the last decade the primary cilium has been recognized as an essential cellular antenna that responds to light, sound, odors, chemicals and mechanical stimuli. The inability to correctly form or maintain a primary cilium causes numerous human diseases including polycystic kidney disease, retinal degeneration, a gamut of human syndromes (e.g. Bardet- Biedl, Meckel, Alstrom, MORM and Joubert) and has recently been correlated with several aggressive cancers. Current methods to study ciliogenesis have poor spatial-temporal resolution and afford little control over the process of cilia formation. Per consequence, standard methods are not robust and, as shown in our preliminary data, fail to discriminate between extremely important stages of cilia formation - most notably if the cilium is inside the cell or on the surface, as the location of the cilia will greatly affect its ability to sense the extracellular environment. Indee, the understanding of the dynamic process of ciliogenesis and its remodeling by vesicular traffic is still rudimentary and despite the importance and occurrence of primary cilia in most mammalian cells there is practically no visualization of these key steps. These major roadblocks hamper the ability to understand the cellular and molecular mechanisms of ciliogenesis and identify drugs that influence it. To overcome this longstanding barrier we will leverage our expertise in imaging of membrane traffic in two highly innovative Specific Aims. Firstly, we will develop a new image-based screen of early ciliogenesis using novel probes compatible with super-resolution imaging. Secondly, we will implement patterned microarrays and optogenetics approaches to control where and when ciliogenesis occurs. We will validate the methods and apply them to test hypotheses about roles of the exocyst and phosphoinositides in the emergence of the primary cilium. The latter is highly relevant to disease as MORM and Joubert syndromes are due to dysfunction of a phosphoinositides 5-phosphatase and optogenetics will enable directly testing of how phosphoinositides metabolism affects ciliogenesis. This toolkit will establish a new foundation to study early ciliogenesis and help set the stage for future screening of therapeutics to ciliary diseases.

描述（由申请人提供）：该项目的主要目的是开发新的启示方法，以可视化和控制主要纤毛形成的早期阶段。我们的长期目标是开发基于原发性纤毛功能障碍引起的人类疾病的基于定量图像的高素质筛选。大多数细胞具有原发性纤毛，这是一个细长的细长延伸的细胞器，从表面延伸。长期以来一直被忽略，在过去的十年中，原发性纤毛被认为是对光，声音，气味，化学物质和机械刺激的反应的必需细胞天线。无法正确形成或维持原发性纤毛会导致许多人类疾病，包括多囊性肾脏疾病，视网膜变性，人类综合征的一系列（例如Bardet-Biedl，Meckel，Meckel，Meckel，Alstrom，Morm和Joubert），并且最近与几种侵略性癌症有关。当前研究纤毛生成的方法的时空分辨率较差，几乎无法控制纤毛形成过程。根据结果​​，标准方法不健壮，如我们的初步数据所示，无法区分纤毛形成的极为重要的阶段 - 最值得注意的是，如果纤毛在细胞内或表面，则为 纤毛的位置将极大地影响其感知细胞外环境的能力。 Indee，对纤毛生成的动态过程的理解及其通过囊泡交通进行重塑仍然是基本的，尽管在大多数哺乳动物细胞中，原发性纤毛的重要性和发生的重要性实际上没有可视化这些关键步骤。这些主要障碍阻碍了了解纤毛生成的细胞和分子机制并鉴定影响它的药物的能力。为了克服这个长期存在的障碍，我们将利用我们在两个高度创新的特定目标中对膜交通进行成像的专业知识。首先，我们将使用与超分辨率成像兼容的新型探针来开发一个新的基于图像的纤毛生成屏幕。其次，我们将实施模式的微阵列和光遗传学方法来控制纤毛发生的何时何时发生。我们将验证这些方法并将其应用于对胞外肌瘤和磷酸肌醇在原发性纤毛出现中的作用的假设。后者与疾病高度相关，因为MORM和JOUBERT综合征是由于5-磷酸酶的磷酸肌醇功能障碍，并且光遗传学将可以直接测试磷酸固醇的代谢如何影响纤毛生成。这个工具包将 建立一个新的基础来研究早期的纤毛生成，并为将来筛查纤毛疾病的治疗疗法奠定了基础。