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状况是由保守的细胞-细胞通讯途径的缺陷引起的，例如由刺猬蛋白家族介导的细胞-细胞通讯途径。最近的研究已经证明了蛋白质输出机制在发育和疾病中塑造Hedgehog和其他信号分子的活性中的重要作用。Hedgehog基因可以顶端或基部释放，以大的多聚体或小的单体形式释放。在释放之前，它可以在很长的细胞延伸中传播许多细胞直径，例如细胞素和轴突。Hedgehog基因输出和传递的机制是复杂且未解决的，但涉及许多导致病理后果的细胞机制。这一建议解决了刺猬生物学中这一研究较少的领域。我们将讨论Hedgehog基因的输出机制，并特别关注轴突运输的一种新机制，该机制将Hedgehog基因运送到被称为脂滴的细胞器上的轴突末端。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。果蝇的视觉系统为揭示Hedgehog基因输出和传播的复杂性提供了一个极好的机会。在这个系统中，根尖的Hedgehog基因分泌传播孔眼发育的时间波，从而产生复眼；光感受器轴突末端的基底靶向、运输和释放触发脑突触后神经元的分化。将Hedgehog基因分配到感光神经元的两极释放对于这些神经元的协调发育至关重要，这些神经元组装成一个精确的神经回路。Hedgehog基因由n端和c端结构域组成，它们通过自催化的蛋白水解裂解分离。n端产物HhNp包含所有已知的信号活动。我们最近发现极化HhNp输出涉及到一种不寻常的输出路径选择，该路径是由c端自裂解产物HhC引导的。当HhNp与HhC结合时，HhNp被纳入脂滴。这些液滴是基本目标，绕过高尔基体，并通过快速轴突运输到大脑。一些新生的刺猬多肽在其c端附近被切割，从而去除轴突靶向信号。与这种缩短的HhC相关的HhNp在根尖分泌，并保留在视网膜中。这种输出途径的二元选择平衡了发育中的眼睛和大脑之间的刺猬活性。本提案旨在解决这种新型出口机制的分子细节。我们将定义参与输出通路决策的顺式作用的刺猬多肽序列。由于脂滴作为轴突运输载体的作用尚不清楚，我们将对潜在分子机制的组成部分进行一般和公正的遗传筛选，并使用生化和细胞生物学方法来研究它们的功能。最后，我们将采用一种新的方法，对不同感光神经元的轴突运输进行实时成像，分析刺猬蛋白和脂滴轴突运输的动力学，并表征新发现的出口机械部件。研究这一新的通路将发现一组新的目标，用于干预涉及Hedgehog通路、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.