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。神经元的迁移以及轴突和树突的分化都需要广泛的膜重建和细胞骨架动力学。直到最近，这一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，一类新的直接控制膜变形和动力学的分子(棒状超家族细分为bar/N-bar、F-bar和I-bar结构域)调节从膜内陷(内吞)到膜突起(丝足形成)等重要的细胞生物学过程。最近发现的F-bar亚家族，大多是在体外细胞系或更多的还原剂系统中进行研究的，这个亚家族中的23个人类基因在体内的功能很差。最近，其中一种名为srGAP3或MEGAP的基因的大量缺失被证明会导致一种名为3P综合征的家族性严重智力低下，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。我们积累的证据表明，一种名为SRGAP2的含有F-bar的蛋白是一种新的神经元迁移和形态的负调节因子。这一功能需要其N-末端的F-bar结构域，令人惊讶的是，与该家族先前描述的结构域不同，我们发现SRGAP2的F-bar结构域在细胞系和体外和体内的皮质神经元中诱导出类似于I-bar结构域的丝状足样膜突起。先前的工作已经证明，在非神经细胞系中，诱导丝状足可以降低细胞的迁移速度和前沿突起的持久性。我们发现，在活体中，下调SRGAP2的表达减少了前导突起的形态，增加了神经元的迁移速度。SRGAP2或其F-bar结构域的过表达具有相反的作用，增加了领先过程的动力学并阻止了迁移。重要的是，F-bar结构域在其C-末端结构域中的表达具有49aa的截短，该结构域定位于膜上，但不能引起丝状足样膜突起，并不抑制神经元的迁移。最后，我们发现SRGAP2的另外两个功能结构域(一个是rac1特异的GAP结构域，一个是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一种假设，即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方面发挥着关键作用。我们将使用分为三个特定目标的多种方法来验证这一假说：在Aim1中，我们将结合生化和细胞生物学方法确定SRGAP2在皮质神经元中功能的分子机制，以确定和表征其SH3结构域的结合伙伴，该结合伙伴在神经元迁移和形态发生过程中对SRGAP2活性的调节至关重要。在目标2中，我们将利用我们最近获得的SRGAP2基因陷阱小鼠系，利用遗传功能丧失的方法来探索SRGAP2在体内神经元迁移和形态发生中的作用。在目标3中，我们将采用体外和体内相结合的方法，测试另外三个未鉴定的、大脑特异的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建立功能神经元回路至关重要。在这里，我们建议研究一类新的调控膜变形的蛋白质家族(F-bar蛋白)在神经元迁移和形态发生中的作用。这项工作将对导致包括严重精神发育迟滞(3P综合征)在内的广泛病理的发育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).