Cytoskeletal Linker Protein Involved in Axon Outgrowth

描述(由申请人提供)： MACF(微管肌动蛋白交联因子)是一种大蛋白(608 KDa)。 可以与肌动蛋白和微管网络相联系。MACF有一个复杂的 域结构。MACF的N-末端部分由一种钙蛋白组成 肌动蛋白结合域和类粘连蛋白结构域。此域是 Plakin蛋白家族，已被证明连接细胞骨架元素 相互之间以及质膜上的连接复合体。这个 Plakin样结构域之后是由血影蛋白组成的中心杆状结构域 重复和钙调素样的EF手型，类似于幽灵蛋白的成员 超级大家庭。最后，C-末端结构域包含一个新的 微管结合域。N-末端结构域显示出惊人的同源性 小鼠突变基因产物BPAG1的神经元亚型 变种人。肌张力障碍(DT)。MACF的杆状结构域和EF手形 与抗肌营养不良蛋白高度同源。MACF普遍表达在 小鼠胚胎在神经系统中高表达，中等水平表达 肌肉中的表达水平。Kakapo，也被称为短停，是 果蝇MACF的同源物，对神经元生长和黏附是必不可少的 在细胞层之间和内部。鹦鹉/短停基因突变的研究 果蝇导致肌肉-肌腱细胞分化缺陷，局部 神经元突起的发育和轴突的生长。的属性 Kakapo/Short Stop使MACF成为轴突生长的潜在关键参与者，我们 因此，建议研究MACF的功能和互动伙伴在 更多细节。在这个提案中，我们将确定表达模式和 MACF的定位：将提高抗MACF的抗体并用于研究 MACF的表达模式。我们将研究功能丧失的表型 在线虫中使用双链RNA进行类型特异性GFP标记的神经元 干扰线虫MACF同源物，我们将进行一系列 养殖DT和野生DT的抢救试验和显性阴性试验 类型神经元。我们将定义MACF的协会合作伙伴： MACF C末端微管结合区将在体内被解剖 通过转染研究和体外微管结合试验。其他 MACF不同域的关联伙伴将通过使用 酵母双杂交系统和免疫共沉淀实验。最后，我们 将表征MACF的一种新的剪接变体。

DESCRIPTION (provided by applicant): MACF (Microtubule actin crosslinking factor) is a large protein (608 kDa) that can associate with the actin and microtubule networks. MACF has a complex domain structure. The N-terminal part of MACF is made up of a calponin-type actin-binding domain and a plakin-like domain. This domain is common to the plakin family of proteins that has been shown to connect cytoskeletal elements to each other and to the junctional complexes at the plasma membrane. The plakin-like domain is followed by a central rod domain composed of spectrin repeats and calmodulinlike EF hand motifs, similar to members of the spectrin superfamily. Finally, the C-terminal domain contains a novel microtubule-binding domain. The N-terminal domain exhibits a striking homology to a proposed neuronal isoform of BPAG1, the mutated gene product of the mouse mutant. dystonia musculorum (dt). The rod domain and the EF hand motifs of MACF are highly homologous to dystrophin. MACF is ubiquitously expressed in the mouse embryo with high expression levels in the nervous system and moderate expression levels in muscles. Kakapo, also known as short stop, is the Drosophila homologue of MACF and is essential for neuronal growth and adhesion between and within cell layers. Mutations in the kakapo/short stop gene in Drosophila cause defects in muscle-tendon cell differentiation, local development of neuronal processes and axonal outgrowth. The properties of kakapo/short stop make MACF a potential key player in axonal outgrowth and we therefore propose to study the function and interaction partners of MACF in more detail. In this proposal, we will determine the expression pattern and localization of MACF: antibodies against MACF will be raised and used to study the expression pattern of MACF. We will investigate loss-of-function phenotypes in C. elegans with type-specific GFP-labeled neurons using double-stranded RNA interference to a C. elegans MACF homologue and we will carry out a series of rescue experiments and dominant negative experiments on cultured dt and wild type neurons. We will define the association partners of MACF: the microtubule-binding domain at the C-terminus of MACF will be dissected in vivo by transfection studies and in vitro by microtubule-binding assays. Other association partners to different domains of MACF will be isolated by using the yeast two-hybrid system and by co-immunoprecipitation experiments. Finally, we will characterize a novel splice variant of MACF.