New Toolkit for Imaging and Controlling Early Ciliogenesis

描述(由申请人提供)：这个项目的主要目标是开发新的使能方法，以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图像的高含量定量屏幕，用于治疗由原发纤毛功能障碍引起的人类疾病。大多数细胞都有一个初级纤毛，这是一个细胞器，从表面伸出一个细长的突起。长期以来一直被忽视的是，在过去的十年里，初级纤毛被认为是一种重要的细胞天线，对光、声音、气味、化学物质和机械刺激做出反应。不能正确地形成或维持原发纤毛导致许多人类疾病，包括多囊肾病、视网膜变性、一系列人类综合征(例如Bardet-Biedl、Meckel、Alstrom、MORM和Joubert)，最近还与几种侵袭性癌症有关。目前研究纤毛发生的方法具有较低的时空分辨率，对纤毛的形成过程缺乏可控性。因此，标准的方法并不可靠，而且，正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--最明显的是纤毛是在细胞内部还是在细胞表面，因为 纤毛的位置将极大地影响其感知细胞外环境的能力。目前，对于纤毛发生的动态过程以及囊泡运输对纤毛的重塑的了解还处于起步阶段，尽管初级纤毛在大多数哺乳动物细胞中的重要性和存在，但这些关键步骤几乎没有可视化。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纤毛发生的药物的能力。为了克服这一长期存在的障碍，我们将在两个高度创新的具体目标上利用我们在膜交通成像方面的专业知识。首先，我们将开发一种新的基于图像的早期纤毛发生屏幕，使用与超分辨率成像兼容的新型探针。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，并将它们应用于测试有关胞囊和肌醇磷脂在初级纤毛出现中的作用的假说。后者与疾病高度相关，因为MORM和Joubert综合征是由于肌醇磷脂5-磷酸酶功能障碍引起的，光遗传学将使直接测试肌醇磷脂代谢如何影响纤毛发生成为可能。此工具包将 为研究早期纤毛发生奠定了新的基础，并为未来睫状体疾病治疗药物的筛选奠定了基础。

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.