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不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。了解如何动员基本的细胞生物学机制来建立特定的背景来调节Notch信号通路，将有助于深入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外周神经系统(PNS)的前体细胞，它们为研究基于Notch活性激活或抑制的二元细胞命运决定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，以利用最先进的成像技术来开发这一系统。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上，即在不对称细胞分裂后，正确的Notch介导的细胞命运分配需要一个特定的保守膜转运调节子子集。这些膜调节剂在组织构型和侧向抑制过程中并不控制Notch信号，而是针对Sanpodo，一种仅在不对称分裂细胞中表达的四遍跨膜蛋白。此前，我们证明了Sanpodo促进Notch活性，从而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，一种Sanpodo蛋白质动力学的活体报告，它忠实地概括了Sanpodo在Notch信号中的功能。此外，通过遗传和生化研究，我们证明了Sanpodo的N-末端区域包含一个以前没有特征的和进化上保守的基序，对Sanpodo的功能至关重要。在活体成像研究中，我们表明，在不对称祖细胞有丝分裂后的几分钟内，Sanpodo将关键的运输调节因子分选到两个离散的膜域是必需的。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域，以促进一个子细胞中的Notch信号，并抑制另一个子细胞中的Notch信号。我们的实验室准备通过结合分子建模、生化分析和活细胞成像完整动物的祖细胞行为的独特能力，剖析在进化上保守的分子、细胞和遗传机制，从而建立不对称的细胞命运决定，并提出以下具体目标：目标1：确定Sanpodo在控制不对称分裂细胞质膜上的Notch信号的机制。目的2：确定囊泡运输的保守调节因子在调控Notch信号中的作用。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效如何导致疾病状态是至关重要的。 公共卫生相关性：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受到调控以及这些机制的失效如何导致疾病状态至关重要。

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.