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）：该项目的主要目标是开发新的方法来可视化和控制初级纤毛形成的早期阶段。我们的长期目标是开发基于定量图像的高内涵筛选，用于治疗由原发性纤毛功能障碍引起的人类疾病。大多数细胞都具有初级纤毛，这是一种从表面延伸出的细长突起的细胞器。长期以来被忽视的初级纤毛在过去十年中被认为是一种重要的蜂窝天线，可以对光、声音、气味、化学物质和机械刺激做出反应。无法正确形成或维持初级纤毛会导致许多人类疾病，包括多囊肾病、视网膜变性、一系列人类综合症（例如 Bardet-Biedl、Meckel、Alstrom、MORM 和 Joubert），并且最近与几种侵袭性癌症相关。目前研究纤毛发生的方法时空分辨率较差，并且对纤毛形成过程几乎无法控制。因此，标准方法并不稳健，并且如我们的初步数据所示，无法区分纤毛形成的极其重要的阶段 - 尤其是纤毛是在细胞内部还是在表面，因为 纤毛的位置将极大地影响其感知细胞外环境的能力。事实上，对纤毛发生及其通过囊泡运输进行重塑的动态过程的理解仍然处于初级阶段，尽管初级纤毛在大多数哺乳动物细胞中很重要并且存在，但实际上这些关键步骤还没有可视化。这些主要障碍阻碍了理解纤毛发生的细胞和分子机制以及识别影响它的药物的能力。为了克服这一长期存在的障碍，我们将利用我们在膜交通成像方面的专业知识来实现​​两个高度创新的具体目标。首先，我们将使用与超分辨率成像兼容的新型探针开发一种新的基于图像的早期纤毛发生屏幕。其次，我们将实施图案化微阵列和光遗传学方法来控制纤毛发生的地点和时间。我们将验证这些方法，并将其应用于检验关于外囊和磷酸肌醇在初级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 综合征是由于磷酸肌醇 5-磷酸酶功能障碍所致，而光遗传学将能够直接测试磷酸肌醇代谢如何影响纤毛发生。该工具包将 为研究早期纤毛发生奠定新的基础，并帮助为未来筛选纤毛疾病治疗方法奠定基础。