Probing the cAMP signaling microdomain of the primary cilium

DESCRIPTION（由申请人提供）：初级纤毛是一个孤立的细胞器，当间期细胞的母中心粒迁移到细胞表面并推出质膜的延伸时形成。这种高度浓缩的非运动结构（存在于几乎所有细胞中，包括神经元）是“纤毛病”一类不断扩大的疾病的核心角色。引起纤毛结构或功能缺陷的突变可引起一系列临床疾病，包括orofaciodigital、Bardet-Biedl、Meckel和Jouber综合征、肾病和多囊肾病。虽然这些是不同的疾病实体，但许多纤毛病具有重叠的表型特征，如严重的颅面畸形、肾和肝囊肿、多指畸形、肥胖以及出生时的神经、感觉和认知障碍。发育过程中重要的信号通路成分（如Hedgehog、Wnt、平面细胞极性）以初级纤毛为基础。然而，现在很明显，这个微小的细胞器也含有环AMP （cAMP）信号通路的许多成分，并且有强有力的证据（尽管是间接的）表明，这个经典的第二信使可以与初级纤毛中的其他途径和蛋白质交叉并修饰。在这个探索性/发展性项目中，我们将考虑初级纤毛代表一个独特的亚细胞cAMP /蛋白激酶a （PKA）信号室的假设。为了克服使用这种非常小的细胞器的挑战，我们组装了一组光学指示器和遗传编码工具来监测和调节纤毛基质内的cAMP/PKA信号。在我们的第一个具体目标中，我们建议完善和扩展这些工具，并评估它们在选择性增强或抑制初级纤毛cAMP信号和PKA活性方面的功效。我们将进一步测试它们修饰纤毛内PKA靶蛋白磷酸化的能力。在我们的第二个具体目标中，作为我们工具箱实用性概念的证明，我们建议研究纤毛内cAMP如何影响已知在某种程度上与cAMP途径和初级纤毛相关的三个生物过程：(i)纤毛伸长过程；（ii）神经元树突树枝化的纤毛依赖性变化；（iii） Hedgehog信号通路的调节。

DESCRIPTION (provided by applicant): The primary cilium is a solitary organelle that is formed when the mother centriole of an interphase cell migrates to the cell surface and pushes out an extension of the plasma membrane. This highly condensed, non-motile structure (present on nearly all cells, including neurons) is the central player in an expanding class of diseases known as "ciliopathies". Mutations that cause structural or functional defects in the cilium give rise to a range of clinical disorders that include orofaciodigital, Bardet-Biedl, Meckel and Jouber syndromes, nephronopthisis, and polycystic kidney diseases. Although these are distinct disease entities, many ciliopathies share overlapping phenotypic features, such as severe craniofacial malformations, kidney and liver cysts, polydactyly, obesity, and neurological, sensory, and cognitive impairment at birth. Components of signaling pathways prominent in development (e.g. Hedgehog, Wnt, planar cell polarity) use the primary cilium as their home base. However, it's now apparent that this tiny organelle also harbors many of the constituents of the cyclic AMP (cAMP) signaling circuit, and there is strong, albeit indirect, evidence that thi classical second messenger can intersect with, and modify, other pathways and proteins within the primary cilium. In this Exploratory/Developmental project we will consider the hypothesis that the primary cilium represents a distinct subcellular cAMP / protein kinase A (PKA) signaling compartment. In order to overcome the challenges of working with this very small organelle, we have assembled a panel of optical indicators and genetically encoded tools to monitor and tune the cAMP/PKA signal within the ciliary matrix. In our first Specific Aim, we propose to refine and expand these tools and assess their efficacy in selectively enhancing or inhibiting cAMP signaling and PKA activity in the primary cilium. We will further test their ability to modify the phosphorylation of putative PKA target proteins resident within the cilium. In our second Specific Aim, as proof of concept of the utility of our toolbox, we propose to investigate how intraciliary cAMP affects three biological processes that are known to be in some way linked to both the cAMP pathway and to the primary cilium: (i) the process of ciliary elongation; (ii) cilium-dependent changes in arborization of neuronal dendrites; and (iii) modulation of the Hedgehog signaling pathway.