Mechanisms of Morphogen Secretion in Visual System Development and Disease

视觉系统发育和疾病中形态原分泌的机制

• 批准号: 8621495
• 负责人: Samuel M Kunes
• 金额: $ 42.25万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621495
• 关键词: Address; Adult; Animals; Apical; Area; Axon; Biochemical; Biochemical Pathway; Biogenesis; Biological; Biology; Brain; Bypass; C-terminal; Caliber; Cell Communication; Cells; Child; Cholesterol; Cleaved cell; Complex; Defect; Development; Disease; Drosophila genus; Environment; Equilibrium; Erinaceidae; Eye; Fatty acid glycerol esters; Genes; Genetic Screening; Golgi Apparatus; Human Milk; Image; In Vitro; Intervention; Kinetics; Life; Lipids; Malignant Neoplasms; Mediating; Methods; Modeling; Modification; Molecular; Movement; N-terminal; Natural regeneration; Neurons; Organelles; Pathology; Pathway interactions; Photoreceptors; Process; Protein Export Pathway; Protein Family; Proteins; Reporting; Retina; Role; Shapes; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Synapses; System; Time; Tissues; Touch sensation; Travel; Variant; Visual system structure; autism spectrum disorder; compound eye; developmental disease; genetic analysis; in vivo; in vivo imaging; morphogens; mutant; neural circuit; neuron development; novel; polypeptide; protein function; protein transport; public health relevance; smoothened signaling pathway; transmission process; vision development

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.

项目总结/摘要 许多发育障碍和病理状况是由保守的细胞-细胞间相互作用的缺陷引起的。 通信途径，例如由Hedgehog蛋白家族介导的那些。最近的研究 记录了蛋白质输出机制在塑造Hedgehog和其他蛋白质的活性中的重要作用。 信号分子在发育和疾病中的作用刺猬可以释放顶部或底部，在大 多聚体或小单体形式。它可以在长的细胞延伸中穿过许多细胞直径， 例如细胞丝和轴突。Hedgehog出口的机制， 传播是复杂的和未解决的，但触及许多细胞机制， 病理后果。这项提议解决了刺猬生物学这一研究较少的领域。我们 将解决刺猬的出口机制，特别是集中在一个新的机制轴突运输 将Hedgehog运送到轴突末端的细胞器称为脂滴。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。 果蝇的视觉系统提供了一个极好的机会来解开刺猬出口的复杂性 和传输。在这个系统中，顶端Hedgehog分泌传播小眼的颞波 产生复眼的发育;基础靶向、运输和从感光细胞释放 轴突末端触发脑中突触后神经元的分化。分区刺猬为 在光感受器神经元的相反两极的释放对于这些细胞的协调发展是至关重要的。 神经元，它们组装成一个精确的神经回路。 Hedgehog由N端和C端结构域组成，它们通过自催化蛋白水解而解离。 乳沟N-末端产物HhNp具有所有已知的信号传导活性。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，该途径由C末端自身介导， 裂解产物HhC。当HhNp与HhC结合时，其被掺入脂滴中。这些液滴 是基底靶向的，绕过高尔基体，并通过快速轴突运输到达大脑。一些 新生Hedgehog多肽在其C末端附近被切割，这去除了轴突靶向信号。 与这种缩短的HhC相关的HhNp在顶部分泌并保留在视网膜中。这种二元选择 输出途径的平衡在发育中的眼睛和大脑之间的刺猬活动。 该提案旨在解决这种新的输出机制的分子细节。我们将定义顺- 参与输出途径决定的作用Hedgehog多肽序列。既然我们一无所知 关于脂滴作为轴突运输载体的作用，我们将进行一个普遍的和无偏见的遗传学研究。 筛选潜在的分子机制的组成部分，并使用生物化学和细胞生物学 方法研究其功能。最后，我们将采用一种新的方法来进行轴突的实时成像 光感受器神经元分化过程中的转运，以分析Hedgehog和脂滴轴突的动力学 运输和表征新确定的出口机械部件。研究这种新途径 将发现一组新的目标，用于干预发育异常和病理， Hedgehog途径、蛋白质运输和轴突运输。