Probing the cAMP signaling microdomain of the primary cilium

描述(申请人提供)：初级纤毛是间期细胞的母中心粒迁移到细胞表面并推出质膜延伸物时形成的孤立细胞器。这种高度浓缩的非运动性结构(存在于几乎所有细胞，包括神经元上)是一类不断扩大的疾病的中心参与者，这种疾病被称为“纤毛疾病”。导致纤毛结构或功能缺陷的突变会引起一系列临床疾病，包括口腔面-指综合征、Bardet-Biedl综合征、Meckel和Jouber综合征、肾结核和多囊肾病。虽然这些都是不同的疾病实体，但许多纤毛疾病都有重叠的表型特征，如严重的颅面畸形、肾和肝脏囊肿、多指畸形、肥胖以及出生时的神经、感觉和认知障碍。发育中重要的信号通路组件(如Hedgehog、Wnt、平面细胞极性)使用初级纤毛作为它们的基础。然而，现在很明显，这个微小的细胞器也含有环磷酸腺苷(CAMP)信号回路的许多成分，尽管是间接的，但有强有力的证据表明，这个经典的第二信使可以与初级纤毛内的其他途径和蛋白质相交并修改。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。为了克服使用这个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中的cAMP/PKA信号。在我们的第一个具体目标中，我们建议改进和扩展这些工具，并评估它们在选择性增强或抑制初级纤毛中cAMP信号和PKA活性方面的有效性。我们将进一步测试它们修改位于纤毛内的假定的PKA目标蛋白的磷酸化的能力。在我们的第二个具体目标中，作为我们工具箱的概念的证据，我们建议研究睫状肌内cAMP如何影响以某种方式与cAMP途径和初级纤毛相关联的三个生物过程：(I)纤毛延长的过程；(Ii)纤毛依赖的神经元树突树枝形成的变化；以及(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.