Mechanisms of Morphogen Secretion in Visual System Development and Disease

描述(由申请人提供)：许多发育障碍和病理情况是由保守的细胞-细胞交流途径的缺陷引起的，例如由Hedgehog蛋白家族介导的那些。最近的研究证明，蛋白质输出机制在形成Hedgehog和其他信号分子在发育和疾病中的活性方面发挥了重要作用。刺猬可以顶部或底部释放，以大多聚体或小单体形式释放。在释放之前，它可以在长细胞延伸中穿过许多细胞直径，如细胞丝和轴突。刺猬输出和传播的潜在机制是复杂和尚未解决的，但涉及到许多细胞机制，这些机制出了问题，导致了病理后果。这项建议解决了刺猬生物学这个研究较少的领域。我们将讨论Hedgehog的输出机制，并特别关注一种新的轴突运输机制，该机制将Hedgehog运送到细胞器上的轴突终末，称为脂滴。因此，我们将确定在发育、再生和疾病中控制这一途径的新靶点。果蝇视觉系统提供了一个很好的机会来解开刺猬输出和传播的复杂性。在这个系统中，顶端刺猬的分泌物传播小眼发育的时间波，从而产生复眼；基础靶向、运输和从光感受器轴突终末释放触发脑内突触后神经元的分化。将Hedgehog分割为释放在光感受器神经元的相反两极，对于这些神经元的协调发展至关重要，这些神经元组装成一个精确的神经回路。Hedgehog是由N-末端和C-末端结构域组成的，它们通过自身催化的蛋白水解性切割而解离。N末端产物HhNp包含所有已知的信号活动。我们最近发现，极化的HhNp输出涉及一种不寻常的输出途径选择，这是由C端自切割产物HHC引导的。当HhNp与HHC结合时，它被结合到脂滴中。这些液滴具有基本的靶向性，绕过高尔基体，通过快速的轴突运输进入大脑。一些新生的Hedgehog多肽在其C末端附近被切割，从而移除轴突靶向信号。与这种缩短的HHC相关的HhNp在视网膜顶端分泌并保留在视网膜中。这种输出路径的二元选择平衡了刺猬发育中的眼睛和大脑之间的活动。这项提议旨在解决这一新的出口机制的分子细节。我们将定义出口途径决定中涉及的顺式作用刺猬多肽序列。由于对脂滴作为轴突运输载体的作用一无所知，我们将对潜在分子机制的组件进行全面和公正的遗传筛选，并使用生化和细胞生物学方法来研究它们的功能。最后，我们将使用一种新的用于区分光感受器神经元的轴突运输的实时成像方法来分析Hedgehog和脂滴轴突运输的动力学，并表征新发现的出口机械部件。对这一新途径的研究将揭示一套新的靶点，用于干预涉及Hedgehog途径、蛋白质运输和轴突运输的发育异常和病理。

DESCRIPTION (provided by applicant): A number of developmental disorders and pathological conditions arise from defects in conserved cell-cell communication pathways, such as those mediated by the Hedgehog family of proteins. Recent studies have documented the important role of protein export mechanisms in shaping the activity of Hedgehog and other signaling molecules in development and disease. Hedgehog can be released apically or basally, in large multimeric or small monomeric forms. It can travel over many cell diameters in long cellular extensions, such as cytonemes and axons, prior to release. The mechanisms underlying Hedgehog export and transmission are complex and unresolved, and yet touch on many cellular mechanisms that go awry with pathological consequences. This proposal addresses this less well-studied area of Hedgehog biology. We will address Hedgehog's export mechanisms, and especially focus on a novel mechanism for axon transport that carries Hedgehog to axon termini on organelles known as lipid droplets. We will thus identify new targets for controlling this pathway in development, regeneration and disease. The Drosophila visual system offers an excellent opportunity to unravel the complexity of Hedgehog export and transmission. In this system, apical Hedgehog secretion propagates the temporal wave of ommatidial development that gives rise to the compound eye; basal targeting, transport and release from photoreceptor axon termini triggers the differentiation of post-synaptic neurons in the brain. Partitioning Hedgehog for release at the opposite poles of a photoreceptor neuron is critical to the coordinated development of these neurons, which assemble into a precise neural circuit. Hedgehog is composed of N-terminal and C-terminal domains that dissociate by self-catalyzed proteolytic cleavage. The N-terminal product, HhNp, harbors all known signaling activities. We recently discovered that polarized HhNp export involves an unusual choice of export pathway that is directed by the C-terminal self- cleavage product HhC. HhNp, when associated with HhC, is incorporated into lipid droplets. These droplets are basally targeted, bypass the Golgi apparatus, and travel by fast axon transport to the brain. Some nascent Hedgehog polypeptide is cleaved near its C-terminus, which removes an axonal targeting signal. HhNp associated with this shortened HhC is secreted apically and remains in the retina. This binary choice of export pathways balances Hedgehog activity between the developing eye and brain. This proposal aims to resolve the molecular details of this novel export mechanism. We will define the cis- acting Hedgehog polypeptide sequences involved in export pathway decisions. Since nothing is known about a role of lipid droplets as axon transport carriers, we will conduct a general and unbiased genetic screen for components of the underlying molecular machineries, and use biochemical and cell biological methods to investigate their functions. Finally, we will employ a new method for live imaging of axon transport in differentiating photoreceptor neurons to analyze the kinetics of Hedgehog and lipid droplet axon transport and characterize newly identified export machinery components. Investigating this novel pathway will uncover a new set of targets for interventions into developmental anomalies and pathologies involving the Hedgehog pathway, protein trafficking and axon transport.