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 thick filaments which interact precisely and specifically with other myofibrillar 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 alpha cardiac myosin heavy chain (MHC) and a truncated heavy meromyosin-like alpha MHC (sHMM) and shown that immunoreactive MHC proteins of the expected sizes were detected in lysates of transfected cells. Surprisingly, the full-length MHC formed large spindle-shaped structures throughout the cytoplasm of transfected cells as determined by immunofluorescence 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 micron to over 20 microns 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 widest point. In addition, the vast majority of transfected MHC did not associate with the endogenous nonmuscle myosin light chains, demonstrating that myosin thick filaments can form in the absence of stoichiometric amounts of myosin light chains.
Vikstrom
K L
KL
Albert Einstein College of Medicine, Bronx, NY 10461-1975.
Rovner
A S
AS
Saez
C G
CG
Bravo-Zehnder
M
M
Straceski
A J
AJ
Leinwand
L A
LA
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001718:Birefringence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism; D008854:Microscopy, Electron; D009218:Myosins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012518:Sarcomeres / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D009218:Myosins