ASPECTS OF CARDIAC, SKELETAL, AND SMOOTH MUSCLE MYOGENESIS

最近的许多工作都集中在编码各种蛋白质的基因上。 收缩蛋白 也有大量的文献集中在 单个纯化的收缩蛋白在无细胞环境中的行为 系统. 肌球蛋白和肌动蛋白的体外自组装研究 和肌动蛋白结合蛋白继续产生新的信息。 然而，在这方面， 在活细胞中，这些蛋白质中的大约20多种是如何被 在空间上被分类，使得肌原纤维在肌原细胞中组装， 应力纤维在非肌肉细胞中组装。 为了研究横纹肌原纤维或肌纤维在体内的组装规律， 我们选择性地干扰一种肌肉特异性蛋白质， 评估对剩余收缩结构的影响。 为 例如，有相当多的文献声称结蛋白IFs起关键作用， 在组装Z带和连接肌原纤维到 肌膜 为了验证这一点，我们最近用一种 截短的结蛋白cDNA。 将结蛋白聚合成长IF， 在转染细胞中完全阻断。 然而，这些细胞 组装的正常收缩的横纹肌原纤维。 显然， 必须重新考虑结蛋白IFs在肌原纤维发生中的作用。 我们将应用这种新的缺失诱变技术， 转染，首先干扰单个收缩蛋白， 第二，跟踪这种扰动对组装的影响， 肌原纤维和应力纤维。 具体而言，我们将重点关注 （a）肌节α-辅肌动蛋白Z-带和非肌节α-辅肌动蛋白Z-带的组装 α-辅肌动蛋白致密体，（B）A-肌节MHC粗丝， 带和应力纤维中的非肌节MHC细丝，和9 c） I带中的α-肌动蛋白细丝和I带中的β-和γ-肌动蛋白细丝。 应力纤维 原则上，这应该允许我们逐个测试， 每种收缩蛋白在肌原纤维组装中的作用 或应力纤维。

Much recent work has focused on the genes that encode the various contractile proteins. There is also an extensive literature focused on the behavior of individual purified contractile proteins in cell-free systems. Studies of the in vitro self-assembly of myosin, and of actin and actin-binding proteins continue to yield new information. However, little is known of how in living cells some 2 dozen of these proteins are sorted out spatially so that myofibrils are assembled in myogenic cells and stress fibers are assembled in non-muscle cells. To study the rules for the in vivo assembly of striated myofibrils or of stress fibers we selectively perturb one muscle-specific protein and then assess the consequences for the remaining contractile structures. For example, there is a sizeable literature claiming that desmin IFs play key roles in the assembly of Z-bands and in linking myofibrils to the sarcolemma. To test this we recently transfected myogenic cells with a truncated desmin cDNA. The polymerization of desmin into long IFs was totally blocked in the transfected cells. Nevertheless, these cells assembled normal, contracting striated myofibrils. Clearly the putative roles of desmin IFs during myofibrillogenesis have to be re-considered. We shall apply this novel technique of deletion mutagenesis and transfection, first to perturb individual contractile proteins and second, to follow the effects of such perturbations on the assembly of both myofibrils and stress fibers. Specifically we shall focus on the assembly of (a) sarcomeric alpha-actinin Z-bands and of non-sarcomeric alpha-actinin dense bodies, (b) sarcomeric MHC thick filaments in a A- bands and of non-sarcomeric MHC filaments in stress fibers, and 9c) alpha-actin thin filaments in I-bands and beta- and gamma- actin in stress fibers. In principle this should allow us to test, one-by-one, the role of each contractile protein in the assembly of either myofibrils or stress fibers.