Mechanisms of Morphogen Secretion in Visual System Development and Disease

项目摘要/摘要 许多发育障碍和病理状况是由保存的细胞缺陷引起的。 通讯途径，例如由Hedgehog蛋白家族介导的那些途径。最近的研究表明 记录了蛋白质输出机制在塑造刺猬和其他动物活动中的重要作用 发育和疾病中的信号分子。刺猬可以在顶部或底部释放，也可以大量释放 多聚体或小单体形式。它可以在长长的细胞延伸中跨越许多细胞直径， 例如细胞丝和轴突，在释放之前。刺猬出口和出口的机制 传递是复杂的和未解决的，但涉及到许多出错的细胞机制 病理性后果。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，并特别关注轴突运输的新机制 这将刺猬带到被称为脂滴的细胞器上的轴突终末。因此，我们将确定新的 在发育、再生和疾病中控制这一途径的目标。 果蝇视觉系统提供了一个很好的机会来解开刺猬出口的复杂性 和变速箱。在这个系统中，心尖刺猬分泌物传播眼小眼的时间波。 产生复眼的发育；基础靶向、运输和从感光细胞释放 轴突终末触发大脑中突触后神经元的分化。为Hedgehog分区 光感受器神经元两极的释放对它们的协调发展至关重要。 神经元，它们组装成一个精确的神经回路。 Hedgehog由N-末端和C-末端结构域组成，它们通过自身催化的蛋白分解而解离 乳沟。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-末端自身- 切割产物HHC。当HhNp与HHC结合时，它被结合到脂滴中。这些水滴 基本是靶向的，绕过高尔基体，通过快速的轴突运输到达大脑。一些人 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。这种二选一的选择 的输出通路平衡了发育中的眼睛和大脑之间的刺猬活动。 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CI- 参与出口途径决定的代理刺猬多肽序列。因为什么都不知道 关于脂滴作为轴突运输载体的作用，我们将进行一般性和不偏不倚的遗传 筛选基础分子机器的组件，并使用生化和细胞生物学 方法对其功能进行研究。最后，我们将使用一种新的方法对轴突进行实时成像 光感受器神经元分化过程中的转运分析Hedgehog和脂滴轴突的动力学 运输和鉴定新确定的出口机械部件。研究这一新的途径 将揭示一套新的目标，用于干预发育异常和病理 Hedgehog途径、蛋白质运输和轴突运输。

PROJECT SUMMARY/ABSTRACT 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.