Functions of membrane deforming-proteins during neuronal development

描述（申请人提供）：在大脑发育过程中，适当的神经元迁移和形态发生对功能回路的建立至关重要。神经元迁移以及轴突和树突分化都需要广泛的膜重塑和细胞骨架动力学。直到最近，该领域的大多数研究都集中在直接调节微管和肌动蛋白细胞骨架动力学的蛋白质上。然而，最近的证据表明，一类直接控制膜变形和动力学的新分子（BAR-样超家族细分为BAR / N-BAR， F-BAR和I-BAR结构域）调节从膜内陷（内吞作用）到膜突出（丝状足形成）的重要细胞生物学过程。最近发现的F-BAR亚家族，主要是在细胞系或更还原的体外系统中研究的，该亚家族的23个人类基因在体内的功能特征不明显。最近，一种名为srGAP3或MEGAP的基因的大量缺失被证明会导致一种称为3p-综合征的家族性严重智力迟钝，这表明一些含有F-BAR的蛋白质可能在大脑发育过程中发挥重要作用。我们已经积累的证据表明，含有F-BAR的蛋白质srGAP2是神经元迁移和形态的一种新的负调节因子。该功能需要其n端F-BAR结构域，令人惊讶的是，与该家族先前表征的结构域不同，我们发现srGAP2的F-BAR结构域在体外和体内细胞系和皮质神经元中诱导丝状足样膜突起，类似于I-BAR结构域诱导的膜突起。先前的研究表明，在非神经元细胞系中，丝状足的诱导降低了细胞迁移的速度和前缘突起的持久性。我们发现srGAP2表达的下调减少了前导过程的形态，增加了神经元在体内的迁移速度。srGAP2或其F-BAR结构域的过表达则会产生相反的效果，增加主导过程的动态并阻断迁移。重要的是，F-BAR结构域c端49aa截断，定位于膜，但不能引起丝状足样膜突起，不抑制神经元迁移。最后，我们发现srGAP2的另外两个功能域（一个rac1特异性的GAP结构域和一个SH3结构域）也参与了srGAP2在神经元迁移中的功能。这些结果使我们提出了f - bar样蛋白对膜变形的直接调节在调节神经元迁移和形态发生中起关键作用的假设。我们将使用分为三个特定目标的多种方法来验证这一假设：在Aim1中，我们将通过使用生化和细胞生物学方法的组合来确定调节srGAP2在皮质神经元中的功能的分子机制，以确定和表征其SH3结构域的结合伙伴，这对神经元迁移和形态发生过程中srGAP2活性的调节至关重要。在Aim 2中，我们将利用我们最近获得的srGAP2基因诱捕小鼠系，利用遗传功能缺失方法探索srGAP2在体内神经元迁移和形态发生中的功能。在Aim 3中，我们将使用体外和体内结合的方法测试另外三种未表征的脑特异性含F-BAR蛋白（srGAP3/MEGAP， Gas7和FCHo1）在神经元迁移和形态发生中的功能。公共卫生相关性：在大脑发育过程中，适当的神经元迁移和形态发生对于功能性神经元回路的建立至关重要。在这里，我们建议研究一个新的调节膜变形的蛋白家族（F-BAR蛋白）在神经元迁移和形态发生中的功能。这项工作将为包括严重智力迟钝（3p-综合征）在内的一系列疾病的发展机制提供重要的新见解。

DESCRIPTION (provided by applicant): During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional circuits. Both neuronal migration as well as axon and dendrite differentiation requires extensive membrane remodeling and cytoskeleton dynamics. Until recently, most studies in this field have focused on proteins directly regulating microtubules and actin cytoskeletal dynamics. However, recent evidence suggests that a new class of molecules directly controlling membrane deformation and dynamics (BAR-like superfamily subdivided into BAR / N-BAR, F-BAR, and I-BAR domains) regulate important cell biological processes ranging from membrane invagination (endocytosis) to membrane protrusion (filopodia formation). The most recently identified, the F-BAR subfamily, has mostly been studied in cell lines or more reductionist in vitro systems and the 23 human genes of this sub-family have poorly characterized functions in vivo. Recently, a large deletion in one of these genes called srGAP3 or MEGAP was shown to cause a familial form of severe mental retardation called 3p- syndrome suggesting that some F-BAR containing proteins might play important functions during brain development. We have accumulated evidence demonstrating that a F-BAR containing protein called srGAP2 is a novel negative regulator of neuronal migration and morphology. This function requires its N-terminal F-BAR domain and surprisingly, unlike previously characterized domains of this family, we found that the F-BAR domain of srGAP2 induces filopodia-like membrane protrusions resembling those induced by I-BAR domains in cell lines and in cortical neurons in vitro and in vivo. Previous work has demonstrated that in non-neuronal cell lines, induction of filopodia decreases the rate of cell migration and the persistence of leading edge protrusions. We found that knockdown of srGAP2 expression reduces leading process morphology and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain have the opposite effects, increasing leading process dynamics and blocking migration. Importantly, expression of the F-BAR domain with a 49aa truncation in its C-terminal domain which localizes to the membrane but fails to elicit filopodia-like membrane protrusions, does not inhibit neuronal migration. Finally, we found that the two other functional domains of srGAP2 (a Rac1-specific GAP domain and a SH3 domain) also participate to srGAP2 function in neuronal migration. These results led us to formulate the hypothesis that direct regulation of membrane deformation by F-BAR-like proteins plays critical roles in regulating neuronal migration and morphogenesis. We will test this hypothesis using multiple approaches divided in three specific aims: in Aim1, we will identify the molecular mechanisms regulating the function of srGAP2 in cortical neurons by using combinations of biochemical and cell biological approaches in order to identify and characterize the binding partners of its SH3 domain which is critical for the regulation of srGAP2 activity during neuronal migration and morphogenesis. In Aim 2, we will explore the function of srGAP2 in neuronal migration and morphogenesis in vivo using a genetic loss-of- function approach taking advantage of a srGAP2 gene trap mouse line that we recently acquired. In Aim 3, we will test the function of three other uncharacterized, brain-specific F-BAR containing proteins (srGAP3/MEGAP, Gas7, and FCHo1) in neuronal migration and morphogenesis using combinations of in vitro and in vivo approaches. PUBLIC HEALTH RELEVANCE: During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neuronal circuits. Here we propose to study the function of a novel family of proteins regulating membrane deformation (F-BAR proteins) in neuronal migration and morphogenesis. This work will provide important new insights into the developmental mechanisms leading to a wide range of pathologies including severe mental retardation (3p- syndrome).