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

基本信息

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
项目类别：
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

项目摘要

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通信途径中的缺陷引起的，例如由刺猬蛋白质介导的缺陷。最近的研究记录了蛋白质出口机制在塑造刺猬和其他信号分子在发育和疾病中的活性中的重要作用。刺猬可以在大型多聚体或小单体形式中以顶端或基础释放。在释放之前，它可以在长细胞延伸（例如细胞质和轴突）中以许多细胞直径的速度传播。刺猬出口和传播的基础机制是复杂且未解决的，但却触及了许多细胞机制，这些机制与病理后果有关。该提案探讨了刺猬生物学的这个研究不善的领域。我们将解决刺猬的出口机制，尤其是专注于一种新型的轴突运输机制，该机制将刺猬带到轴突末端，称为脂质液滴。因此，我们将确定控制这一发展，再生和疾病的新目标。果蝇视觉系统提供了一个绝佳的机会，可以揭示刺猬出口和传输的复杂性。在该系统中，根尖刺猬的分泌传播了产生复合眼的典型发育的时间波。从光感受器轴突末端的基础靶向，运输和释放会触发大脑中突触后神经元的分化。在光感受器神经元的相对极点释放的刺猬对这些神经元的协调发展至关重要，这些神经元聚集成精确的神经回路。刺猬由通过自催化的蛋白水解裂解解离的N末端和C末端结构域组成。 N末端产品HHNP拥有所有已知的信号活动。我们最近发现，极化HHNP导出涉及由C末端自裂解产品HHC指导的异常选择的出口途径。当与HHC相关联时，将HHNP掺入脂质液滴中。这些液滴基在基本上是靶向的，绕过高尔基体，并通过快速轴突传输到大脑。一些新生的刺猬多肽在其C末端裂解，从而去除轴突靶向信号。与此缩短的HHC相关的HHNP是顶端分泌的，并且仍留在视网膜中。这种出口途径的二元选择可以平衡发育中的眼睛和大脑之间的刺猬活动。该建议旨在解决这种新型出口机制的分子细节。我们将定义与出口途径决策有关的刺猬多肽序列。由于对脂质液滴作为轴突传输载体的作用一无所知，因此我们将对基础分子机器的组成部分进行一般且无偏的遗传筛选，并使用生化和细胞生物学方法研究其功能。最后，我们将采用一种新方法来分化光感受器神经元的轴突传输成像，以分析刺猬和脂质液滴轴突传输的动力学，并表征新鉴定的出口机械组件。研究这种新颖的途径将发现一组新的目标，以干预涉及刺猬途径，蛋白质运输和轴突运输的发育异常和病理。