Endocytic Control of Notch-Mediated Cell Fate Decisions in Neurogenesis

神经发生中Notch介导的细胞命运决定的内吞控制

• 批准号: 7624703
• 负责人: Fabrice J. Roegiers
• 金额: $ 34.35万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7624703
• 关键词: Adult; Alleles; Animals; Arts; Biochemical; Biological; Cell Fate Control; Cell Separation; Cell division; Cell membrane; Cells; Collection; Complex; Coupled; Development; Disease; Dominant-Negative Mutation; Drosophila genus; Early Endosome; Failure; Future; Genetic; Goals; Golgi Apparatus; Image; Imaging Techniques; Integral Membrane Protein; Lateral; Life; Malignant Neoplasms; Mediating; Membrane; Membrane Protein Traffic; Mitosis; Modification; Molecular; Molecular Models; Monitor; N-terminal; Nervous system structure; Notch Signaling Pathway; Pattern; Peripheral Nervous System; Phosphorylation; Protein Dynamics; Proteins; Pupa; Reagent; Reporter; Role; Screening procedure; Signal Transduction; Sorting - Cell Movement; Stem cells; Study models; System; Time; Tissues; To specify; Transgenes; Ubiquitin; Vesicle; base; cell behavior; cell fate specification; cellular imaging; daughter cell; fly; genetic analysis; human disease; in vivo; in vivo Model; insight; molecular modeling; mutant; neurogenesis; notch protein; public health relevance; rab GTP-Binding Proteins; research study; stem; trafficking

DESCRIPTION (provided by applicant): The goal of this proposal is to determine how membrane trafficking during asymmetric cell division controls a Notch signaling pathway-mediated cell fate switch. Understanding how fundamental cell biological mechanisms are mobilized to establish a specific context to regulate the Notch signaling pathway will provide insights into how dysregulation of these mechanisms in stem and progenitor cells contributes to human diseases, including cancer. One such context is the progenitor cells of the Drosophila adult peripheral nervous system (PNS), which provide a powerful model for the study of binary cell fate decisions based on the activation or inhibition of Notch activity. We have generated exciting new in vivo reagents to exploit this system using state of the art imaging techniques. We will build on the genetic framework our lab and others have established that a specific subset of conserved membrane trafficking regulators are required for correct Notch- mediated cell fate assignments after asymmetric cell division. These membrane regulators do not control Notch signaling during tissue patterning and lateral inhibition, rather they appear to target Sanpodo, a four pass transmembrane protein expressed exclusively in asymmetrically dividing cells. Previously, we demonstrated that Sanpodo promotes Notch activity to confer correct cell fates after asymmetric cell division in the adult peripheral nervous system. In preliminary studies, we developed Sanpodo-GFP, an in vivo reporter of Sanpodo protein dynamics, which faithfully recapitulates Sanpodo function in Notch signaling. Furthermore, through genetic and biochemical studies, we demonstrate that the N-terminal region of Sanpodo, which contains a previously uncharacterized and evolutionarily conserved motif, is critical for Sanpodo's function. In live imaging studies we show that key trafficking regulators are required for Sanpodo sorting to two discrete membrane domains within minutes after asymmetric progenitor cell mitosis. We hypothesize that evolutionarily conserved vesicle trafficking regulators function to establish these membrane domains to promote Notch signaling in one daughter cell, and to inhibit Notch signaling in the other daughter cell. Our lab is poised to dissect the evolutionarily conserved molecular, cellular, and genetic mechanisms underlying the establishment asymmetric cell fate decisions by our unique ability to combine molecular modeling, biochemical analysis, and live cell imaging of progenitor cell behavior in the intact animal, and propose the following specific aims: Aim 1: To determine the mechanism of Sanpodo function in controlling Notch signaling at the plasma membrane in asymmetrically dividing cells. Aim 2: To determine the roles of conserved regulators of vesicle trafficking in regulating Notch signaling. Elucidating the mechanisms of spatial-temporal control of signaling in cell fate decisions is critical to our understanding of how vertebrate neurogenesis is regulated and how failure of these mechanisms leads to disease states. PUBLIC HEALTH RELEVANCE: Elucidating the mechanisms of spatial-temporal control of signaling in cell fate decisions is critical to our understanding of how vertebrate neurogenesis is regulated and how failure of these mechanisms leads to disease states.

描述（由申请人提供）：该提案的目标是确定不对称细胞分裂期间的膜运输如何控制Notch信号通路介导的细胞命运转换。了解如何调动基本细胞生物学机制来建立特定环境来调节 Notch 信号通路，将有助于了解干细胞和祖细胞中这些机制的失调如何导致包括癌症在内的人类疾病。其中一个背景是果蝇成体周围神经系统 (PNS) 的祖细胞，它为研究基于 Notch 活性的激活或抑制的二元细胞命运决策提供了强大的模型。我们已经产生了令人兴奋的新体内试剂，以利用最先进的成像技术来开发该系统。我们将建立在我们的实验室和其他人已经建立的遗传框架的基础上，即不对称细胞分裂后正确的Notch介导的细胞命运分配需要保守的膜运输调节因子的特定子集。这些膜调节剂在组织模式化和侧向抑制过程中不控制Notch信号传导，而是以Sanpodo为目标，Sanpodo是一种仅在不对称分裂细胞中表达的四次跨膜蛋白。之前，我们证明了 Sanpodo 可以促进 Notch 活性，从而在成人周围神经系统中的不对称细胞分裂后赋予正确的细胞命运。在初步研究中，我们开发了Sanpodo-GFP，一种Sanpodo蛋白动力学的体内报告基因，它忠实地再现了Sanpodo在Notch信号传导中的功能。此外，通过遗传和生化研究，我们证明 Sanpodo 的 N 末端区域包含一个以前未表征且进化上保守的基序，对于 Sanpodo 的功能至关重要。在实时成像研究中，我们表明，在不对称祖细胞有丝分裂后几分钟内，Sanpodo 分选到两个离散的膜域需要关键的运输调节因子。我们假设进化上保守的囊泡运输调节因子的作用是建立这些膜域，以促进一个子细胞中的 Notch 信号传导，并抑制另一个子细胞中的 Notch 信号传导。我们的实验室准备通过我们将分子建模、生化分析和完整动物祖细胞行为的活细胞成像相结合的独特能力，剖析建立不对称细胞命运决定背后的进化保守的分子、细胞和遗传机制，并提出以下具体目标： 目标 1：确定 Sanpodo 功能在控制细胞质膜上的 Notch 信号传导方面的机制。 细胞分裂不对称。目标 2：确定囊泡运输的保守调节因子在调节 Notch 信号传导中的作用。阐明细胞命运决定中信号传导的时空控制机制对于我们理解脊椎动物神经发生如何调节以及这些机制的失败如何导致疾病状态至关重要。 公共卫生相关性：阐明细胞命运决定中信号传导的时空控制机制对于我们理解脊椎动物神经发生如何调节以及这些机制的失败如何导致疾病状态至关重要。