成肌细胞分化过程中KIF5B对CDO信号通路的时空调控机制研究

发布时间：2018-04-29 01:42

本文选题：Kinesin + BNIP-2　；参考：《浙江大学》2016年博士论文

【摘要】：成肌细胞是未分化的肌肉前体细胞,可以分化融合形成肌管(Myotube),在肌肉生成中扮演着重要的角色。Cdo-p38MAPK信号通路是调控骨骼肌生成过程的主要信号通路之一,在成肌细胞分化过程中细胞表面受体蛋白Cdo可通过与支架蛋白BNIP-2和JLP相结合激活p38MAPK进而促进成肌细胞的分化和肌管的形成,但是其中的时空调控机制尚不清楚。本研究中我们发现KIF5B蛋白(又被称为Kinesin-1的分子马达蛋白)可与支架蛋白BNIP-2相互作用并参与其在成肌细胞C2C12内的转运,调控BNIP-2促进成肌细胞分化的作用。首先我们通过蛋白GST-Pull down实验、蛋白质谱分析、蛋白免疫共沉淀实验发现和确认KIF5B蛋白通过其motor和tail结构域与BNIP-2蛋白的BCH结构域相互结合。其次,通过免疫荧光和活细胞成像实验我们发现在成肌细胞中BNIP-2蛋白定位于细胞早期内涵体,并且BNIP-2蛋白沿细胞微管顺行性运动。同时我们还发现在细胞中过量表达KIF5B蛋白的motor或tail结构域均可阻碍BNIP-2蛋白的这种顺行性运动,并且通过siRNA干扰降低KIF5B蛋白的表达也可以抑制BNIP-2蛋白在成肌细胞C2C12中的顺行性运动。由此可以确认KIF5B蛋白对BNIP-2蛋白在细胞中的顺行性运动起着致关重要的作用。然后,我们在成肌细胞分化过程中通过稳定过量表达和siRNA干扰实验发现蛋白KIF5B可以通过激活p38MAPK的活性进而促进成肌细胞分化,更重要的是我们研究中还发现成肌细胞分化过程中BNIP-2蛋白依赖于KIF5B蛋白的顺行性运动是其发挥促进成肌细胞分化作用所必须的。综上所述,本实验研究发现了KIF5B蛋白在成肌细胞分化过程中对Cdo-BNIP-2-p38MAPK信号通路的时空调控机制,同时也表明了细胞内的物质转运系统在成肌细胞分化过程中起着重要作用。
[Abstract]:Myoblasts are undifferentiated muscle progenitor cells, which can differentiate and fuse to form myotube.Cdo-p38 MAPK signaling pathway is one of the major signaling pathways regulating skeletal muscle formation. During the process of myoblast differentiation, cell surface receptor protein Cdo can activate p38MAPK by combining with scaffold protein BNIP-2 and JLP to promote myoblast differentiation and myotube formation. In this study, we found that KIF5B protein (also known as Kinesin-1 molecular motor protein) can interact with scaffold protein BNIP-2 and participate in its transport in myoblast C2C12, and regulate the role of BNIP-2 in promoting myoblast differentiation. Firstly, we found and confirmed that KIF5B protein binds to BCH domain of BNIP-2 protein through its motor and tail domains through protein GST-Pull down experiment, protein spectrum analysis and protein immunoprecipitation assay. Secondly, by immunofluorescence and living cell imaging, we found that BNIP-2 protein was located in the early intension of cells in myoblasts, and BNIP-2 protein moved along the microtubule of cells. At the same time, we also found that motor or tail domains, which overexpressed KIF5B protein in cells, could block the anterograde movement of BNIP-2 protein. Furthermore, decreasing the expression of KIF5B protein by siRNA interference could also inhibit the anterograde movement of BNIP-2 protein in myoblast C2C12. It can be concluded that KIF5B protein plays an important role in the direct movement of BNIP-2 protein in cells. Then, we found that protein KIF5B can promote myoblast differentiation by activating the activity of p38MAPK through stable overexpression and siRNA interference during myoblast differentiation. More importantly, we also found that BNIP-2 protein is dependent on the anterograde movement of KIF5B protein in the process of myoblast differentiation, which is necessary to play a role in promoting myoblast differentiation. To sum up, we found that KIF5B protein plays an important role in the process of myoblast differentiation by regulating the Cdo-BNIP-2-p38MAPK signaling pathway in time and space. At the same time, the material transport system in cells plays an important role in the process of myoblast differentiation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q254

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