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: 8987570
• 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/8987570
• 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; Health; 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; 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; postsynaptic neurons; protein function; protein transport; 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学病症由保守的细胞-细胞通讯途径的缺陷引起，例如由Hedgehog蛋白家族介导的那些。最近的研究已经证明了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底部释放。在释放之前，它可以在长的细胞延伸物中穿过许多细胞直径，例如细胞丝和轴突。Hedgehog输出和传播的机制是复杂和未解决的，但涉及许多细胞机制，这些机制会导致病理后果。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，特别是集中在轴突运输的一种新的机制，进行刺猬轴突末端的细胞器称为脂滴。因此，我们将确定新的目标，以控制这一途径的发展，再生和疾病。果蝇的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。在该系统中，顶端刺猬分泌传播小眼发育的时间波，产生复眼;从感光轴突末端的基础靶向、运输和释放触发脑中突触后神经元的分化。将Hedgehog分配到光感受器神经元的两极释放，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由N-末端和C-末端结构域组成，其通过自催化的蛋白水解裂解解离。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HhNp输出涉及由C-末端自切割产物HhC指导的输出途径的不寻常选择。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。一些新生的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。这种输出途径的二元选择平衡了发育中的眼睛和大脑之间的Hedgehog活动。该提案旨在解决这种新的输出机制的分子细节。我们将定义参与输出途径决定的顺式作用的Hedgehog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成部分进行一般和无偏见的遗传筛选，并使用生物化学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来区分感光神经元轴突运输的实时成像，以分析刺猬和脂滴轴突运输的动力学，并表征新发现的出口机械组件。研究这种新的途径将发现一组新的干预目标，涉及刺猬途径，蛋白质运输和轴突运输的发育异常和病理。