Endocytic Control of Notch-Mediated Cell Fate Decisions in Neurogenesis

描述（由申请人提供）：本提案的目标是确定非对称细胞分裂过程中的膜运输如何控制Notch信号通路介导的细胞命运开关。了解基本的细胞生物学机制是如何被动员起来，以建立一个特定的环境来调节Notch信号通路，将有助于了解这些机制在干细胞和祖细胞中的失调如何导致人类疾病，包括癌症。一个这样的背景是果蝇成年周围神经系统（PNS）的祖细胞，它为基于Notch活性激活或抑制的二元细胞命运决定的研究提供了一个强大的模型。我们已经产生了令人兴奋的新的体内试剂，利用最先进的成像技术来开发这个系统。我们将建立在遗传框架的基础上，我们的实验室和其他人已经建立了一个特定的保守膜运输调节子子集，在不对称细胞分裂后，Notch介导的细胞命运分配是正确的。这些膜调节因子在组织模式和侧抑制过程中不控制Notch信号，相反，它们似乎靶向Sanpodo，这是一种仅在非对称分裂细胞中表达的四通跨膜蛋白。先前，我们证明了Sanpodo促进Notch活性，在成人周围神经系统细胞不对称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，这是一个Sanpodo蛋白动态的体内报告者，它忠实地概括了Sanpodo在Notch信号传导中的功能。此外，通过遗传和生化研究，我们证明了Sanpodo的n端区域包含一个以前未被表征和进化保守的基序，对Sanpodo的功能至关重要。在实时成像研究中，我们发现在不对称祖细胞有丝分裂后的几分钟内，Sanpodo分选到两个离散的膜结构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功能是建立这些膜结构域，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。我们的实验室准备剖析进化上保守的分子、细胞和遗传机制，通过我们独特的能力结合分子建模、生化分析和完整动物祖细胞行为的活细胞成像，并提出以下具体目标：目的1：确定Sanpodo功能在不对称分裂细胞中控制质膜Notch信号传导的机制。目的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的时空控制机制对于我们理解脊椎动物神经发生是如何调控的以及这些机制的失败是如何导致疾病状态的至关重要。

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.