A 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结合。MACF具有复杂的 域结构MACF的N-末端部分是由一种钙调蛋白型的 肌动蛋白结合结构域和斑蛋白样结构域。此域对于 已显示连接细胞骨架元件的斑蛋白家族 以及质膜上的连接复合物。的 斑蛋白样结构域之后是由血影蛋白组成的中心杆结构域 重复和钙调素样EF手基序，类似于血影蛋白的成员 超家族最后，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 BPAG1的神经元亚型，小鼠的突变基因产物 变种人肌张力障碍（DT）。MACF的杆状结构域和EF手型 与抗肌萎缩蛋白高度同源MACF广泛表达于 小鼠胚胎在神经系统中具有高表达水平， 在肌肉中的表达水平。鸮鹦鹉，也被称为短停，是 MACF的果蝇同源物，对神经元生长和粘附至关重要 在细胞层之间和细胞层内。Kakapo/ShortStop基因突变 果蝇导致肌肉肌腱细胞分化缺陷，局部 神经元突起和轴突生长的发育。的性质 鸮鹦鹉/短停使MACF成为轴突生长的潜在关键因素， 因此，建议研究MACF的功能和互动伙伴， 更多细节在本提案中，我们将确定表达模式， MACF的定位：将产生抗MACF的抗体并用于研究 MACF的表达模式。我们将研究功能丧失表型 in C.使用双链RNA的具有类型特异性GFP标记的神经元的线虫 干扰为C。我们将进行一系列的 对培养的dt和野生型的挽救实验和显性阴性实验 型神经元。我们将定义MACF的关联合作伙伴： 将在体内解剖MACF C末端的微管结合结构域 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的新剪接变体。

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.