Sussman M A, Sakhi S, Barrientos P, Ito M, Kedes L
Institute for Genetic Medicine, University of Southern California, School of Medicine, Los Angeles 90033.
Circ Res. 1994 Aug;75(2):221-32. doi: 10.1161/01.res.75.2.221.
Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43,000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with alpha-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinuclear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.
原肌球蛋白是一种40.6-kD的蛋白质，在骨骼肌中与肌动蛋白丝的尖端共定位。我们报告了大鼠心脏原肌球蛋白的两个部分长度互补DNA（cDNA）克隆的序列，它们覆盖了90%的编码区域。该cDNA序列在人和大鼠之间有90%的保守性，预测的氨基酸序列相似性更高，为95%。在大鼠心肌的蛋白质印迹分析中，抗原肌球蛋白抗体标记出一条表观迁移率为43,000的单一多肽。使用这种抗原肌球蛋白抗体进行的免疫荧光实验结果显示，标记与细肌丝末端一致，这通过与α-辅肌动蛋白抗体的双重定位得以证明。原肌球蛋白蛋白在大鼠心肌细胞中最早出现肌原纤维时就组织成肌节染色模式。原肌球蛋白蛋白在细肌丝末端或其附近的定位促使我们研究体外肌原纤维发育过程中原肌球蛋白信使RNA（mRNA）的分布。在培养3至5天的心肌细胞中使用原肌球蛋白cDNA探针进行的荧光原位杂交实验显示出大的mRNA斑块分布。此时原肌球蛋白mRNA的细胞质位置与已发育的肌原纤维无关，这表明原肌球蛋白蛋白是靶向细肌丝末端的，而不是使用局部翻译机制。然而，在接下来的2周内，培养的心肌细胞中原肌球蛋白mRNA的分布从大的核周斑块变为沿整个细胞的肌原纤维排列的小浓度。整个心肌细胞中原肌球蛋白mRNA的重新组织似乎与同一时期内甘油醛-3-磷酸脱氢酶mRNA的模式不同。发育中的心肌细胞内肌原纤维细胞内密度的增加可能导致选定的mRNA重新分布以进行局部翻译。
