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: 9195099
• 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/9195099
• 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; Genes; Genetic Screening; Golgi Apparatus; Human Milk; Image; In Vitro; Intervention; Kinetics; Lipids; Malignant Neoplasms; Mediating; Methods; Modeling; Modification; Molecular; Movement; Mutation Analysis; N-terminal; Natural regeneration; Neurons; Organelles; Pathologic; Pathology; Pathway interactions; Photoreceptors; Process; Protein Export Pathway; Protein Family; Proteins; Recruitment Activity; Reporting; Retina; Role; Shapes; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; System; Time; Tissues; Touch sensation; Travel; Variant; Visual system structure; autism spectrum disorder; compound eye; developmental disease; genetic analysis; in vivo; in vivo imaging; milk fat; morphogens; mutant; neural circuit; neuron development; novel; polypeptide; postsynaptic neurons; protein function; protein transport; public health relevance; smoothened signaling pathway; transmission process; vision development

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.

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间通讯途径的缺陷引起的，例如由刺猬蛋白家族介导的那些。最近的研究证明了蛋白质输出机制在塑造 Hedgehog 和其他信号分子在发育和疾病中的活性方面的重要作用。 Hedgehog 可以以大的多聚体或小的单体形式从顶部或基部释放。在释放之前，它可以在长细胞延伸中穿过许多细胞直径，例如细胞线和轴突。 Hedgehog 输出和传播的机制很复杂且尚未解决，但涉及许多细胞机制，这些机制出了问题会产生病理后果。该提案解决了刺猬生物学这一研究较少的领域。我们将讨论 Hedgehog 的输出机制，特别关注一种新的轴突运输机制，该机制将 Hedgehog 运送到称为脂滴的细胞器上的轴突末端。因此，我们将确定控制这条发育、再生和疾病途径的新目标。果蝇视觉系统为阐明刺猬输出和传输的复杂性提供了绝佳的机会。在这个系统中，顶端刺猬分泌物传播小眼发育的时间波，从而产生复眼；光感受器轴突末端的基础靶向、运输和释放触发大脑中突触后神经元的分化。将 Hedgehog 分区以在感光神经元的相对两极释放对于这些神经元的协调发育至关重要，这些神经元组装成精确的神经回路。 Hedgehog 由 N 端和 C 端结构域组成，通过自催化蛋白水解裂解解离。 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 端自裂解产物 HhC 指导。当 HhNp 与 HhC 结合时，HhNp 会并入脂滴中。这些液滴是有针对性的，绕过高尔基体，并通过快速轴突运输到达大脑。一些新生的 Hedgehog 多肽在其 C 末端附近被切割，从而消除了轴突靶向信号。与缩短的 HhC 相关的 HhNp 被分泌到顶部并保留在视网膜中。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动。该提案旨在解决这种新型输出机制的分子细节。我们将定义参与输出途径决定的顺式作用Hedgehog多肽序列。由于我们对脂滴作为轴突运输载体的作用一无所知，因此我们将对潜在分子机器的组成部分进行一般且公正的遗传筛选，并使用生化和细胞生物学方法来研究它们的功能。最后，我们将采用一种新方法对区分感光神经元的轴突运输进行实时成像，以分析 Hedgehog 和脂滴轴突运输的动力学，并表征新识别的输出机械组件。研究这一新途径将发现一组新的干预目标，用于干预涉及刺猬途径、蛋白质运输和轴突运输的发育异常和病理学。