SARCOMERIC MYOSIN HEAVY-CHAIN EXPRESSED IN NONMUSCLE CELLS FORMS THICK FILAMENTS IN THE PRESENCE OF SUBSTOICHIOMETRIC AMOUNTS OF LIGHT-CHAINS

Central to the function of myosin is its ability to assemble into thic k filaments which interact precisely and specifically with other myofi brillar proteins. We have established a novel experimental system for studying myofibrillogenesis using transient transfections of COS cells , a monkey kidney cell line. We have expressed both full-length rat a cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin-like alpha MHC (sHMM) and shown that immunoreactive MHC proteins of the ex pected sizes were detected in lysates of transfected cells. Surprising ly, the full-length MHC formed large spindle-shaped structures through out the cytoplasm of transfected cells as determined by immunofluoresc ence microscopy. The structures were not found in cells expressing the sHMM construct, indicating that their formation required an MHC rod. The spindle-shaped structures ranged in length from approximately 1 mu m to over 20 mum in length and were birefringent suggesting that they are ordered arrays of thick filaments. This was confirmed by electron microscopic analysis of the transfected cells which revealed arrays of filamentous structures approximately 12 nm in diameter at their wides t point. In addition, the vast majority of transfected MHC did not ass ociate with the endogenous nonmuscle myosin light chains, demonstratin g that myosin thick filaments can form in the absence of stoichiometri c amounts of myosin light chains. (C) 1993 Wiley-Liss, Inc.