Molecular mechnisms of cell fusion during myogenic differentiation

骨骼肌细胞分化过程中的细胞融合被认为是由特定蛋白与融合肽共同诱导的。该蛋白产生的细胞内信号转导可能调节细胞融合或细胞融合后的一些细胞事件。为了阐明细胞融合的信号转导途径，我们进行了以下三项研究。(1)我们检测了新的膜蛋白ermelin的定位，它包含一个与融合肽同源的序列。结合表位标记和ermelin抗体的免疫荧光显微镜显示该蛋白存在于内质网（ER）中。免疫电镜发现ermelin位于ER膜上。我们正在研究ermelin是否参与内质网膜的融合。(2)据报道，Meltrin α参与肌肉细胞融合，其胞质结构域包含与SH3结构域结合的一致序列。用细菌表达的重组蛋白和牛脑提取物进行的更多夜间实验表明，Src、Yes和Grb2的SH3结构域与meltrin α细胞质结构域结合。因此，meltrin α可能通过与这些蛋白的相互作用参与了肌源性分化的一些信号转导。(3)在研究肌肉细胞融合的信号转导过程中，克隆了新的小GTPases M-Ras和RhoD。我们通过转染cdna和显微注射重组蛋白来检测M-Ras和RhoD的细胞功能。M- Ras引起肌动蛋白细胞骨架的重组，包括微穗的形成和应力纤维的丢失。诱导成纤维细胞的树突状细胞形态和PC12细胞的神经突生长。RhoD诱导应激纤维丢失，抑制细胞运动。它通过阻止细胞分裂产生多核细胞。我们正在研究这些gtpase是否与细胞融合的信号转导有关。少

The cell fusion during skeletal muscle cell differentiation is considered to be induced by certain protein (s) with the fusion peptide. Intracellular signal transduction generated by this protein might regulate the cell fusion or some cellular events following the cell fusion. To elucidate the signal transduction pathway for cell fusion, we addressed the following three studies. (1) We examined the localization of the novel membrane protein ermelin, which contains a sequence homologous to the fusion peptide. Immunofluorescence microscopy in combination with epitope tagging and with an antibody to ermelin exhibited that the protein is present in the endoplasmic reticulum (ER). Immunoelectron microscopy located ermelin on the ER membrane. We are studying whether ermelin is involved in fusion of the ER membrane. (2) Meltrin alpha, which is reported to be participating in muscle cell fusion, contains in its cytoplasmic domain the consensus sequences for binding to the SH3 domain. The bindi … More nig experiments with bacterially expressed recombinant proteins and bovine brain extracts showed that the SH3 domains of Src, Yes, and Grb2 bound to the meltrin alpha cytoplasmic domain. Consequently, meltrin alpha is likely to be involved in some signal transduction for myogenic differentiation through the interaction with these proteins. (3) The novel small GTPases M-Ras and RhoD were cloned during investigation of the signal transduction for muscle cell fusion. We examined the cellularfunctions of M-Ras and RhoD by transfection of the cDNAs and microinjection of the recombinant proteins. M- Ras caused reorganization of the actin cytoskeleton including the microspike fromation and loss of the stress fibers. It induced dendritic cell morphology in fibroblasts and neurite outgrowth in PC12 cells. RhoD induced loss of the stress fibers and inhibited cell motility. It generated multinucleated cells by preventing cytokinesis. We are examining whether these GTPases are implicated in the signal transduction for cell fusion. Less