Molecular mechanisms of formation and maintenance of muscle contractile apparatus (myofibrils)

我们主要集中在肌动蛋白结合蛋白Cofilin(CF)和肌球蛋白和连接素结合蛋白C蛋白上，研究了肌原纤维形态发生和维持的分子机制。研究结果概括如下：1)肌原纤维形成中的粘蛋白作用1)肌细胞内Cf过多，导致肌动蛋白细丝断裂和肌原纤维部分解体；2)在基因操作产生的Cf缺乏的小鼠肌细胞中，肌原纤维结构受到严重破坏，并在Cf缺乏的肌细胞集中的区域导致肌肉变性；3)通过增加PIP2在肌细胞中Cf的失活也导致肌原纤维组装不足。根据这些结果，我们认为Cf不仅在肌原纤维的形成中起重要作用，而且在维持正常肌肉结构方面也是重要的。C-蛋白的分子结构和功能1)用基因技术测定了小鼠快骨骼(F)和慢骨骼(S)C-蛋白的分子结构，阐明了它们的表达和组装，并与心脏异构体的分子结构进行了比较；2)在心脏C-蛋白的N-末端确定了肌动蛋白结合结构域；3)发现了一个新的小鼠心脏C-蛋白，它在C-末端的肌球蛋白结合区和连接素结合区略有不同，功能较弱。该变异体在老年心脏中的表达上调。肌球蛋白-肌球蛋白相互作用在肌原纤维形成中的作用我们发现肌球蛋白ATPase的抑制剂BDM(2，3-丁二酮-2-单肟)可以抑制肌原纤维的形成并导致肌原纤维的破坏，从而提示肌动蛋白-肌球蛋白相互作用在肌原纤维形成的早期过程中起着关键作用。

We examined molecular mechanisms of morphogenesis and maintenance of myofibrils by mainly focussing on cofilin (CF), an actin-binding protein, and C-protein, a myosin- and connectin-bindirig protein. The results are summarized as follow :1.Role of cofilin in myofibrillogenesis1) Excess of CF in muscle cells, which was caused by cDNA transfection or injection of recombinant protein led to disruption of actin filaments and partial disorganization of myofibrils ; 2) In CF-deficient muscle cells, which were generated in mice by gene-manipulation, myofibrillar structures were severely damaged and muscle degeneration was induced in the region where CF-deficient muscle cells were concentrated ; 3) Inactivation of CF by increasing PIP2 in muscle cells also led to insufficient myofibril assembly.Based on these results, we conclude that CF is important not only in myofibril formation but also in maintenance of normal muscle structure.2. Molecular structure and function of C-protein1) Molecular structures of mouse fast skeletal (F) and slow skeletal (S) C-proteins were determined by cDNA technology, and their expression and assembly were clarified and compared with that of cardiac isoform ; 2) Actin-binding domain was specified in N-terminal region of cardiac C-protein ; 3) A novel mouse cardiac C-protein was discovered, which slightly differ in the C-terminal myosin-binding and connectin-binding region and functionally weak. The expression of this variant is up-regulated in aged heart.3. Role of actin-myosin interaction in myofibrillogenesisWe found that BDM (2,3-butanedione 2-monoxime), an inhibitor for myosin ATPase, suppresses, myfibrillogenesis and leads to myofibril disruption and thereby suggested that actin-myosin interaction plays a critical role in the early process of myofibrillogenesis.