QUICK-FREEZE DEEP-ETCH ELECTRON-MICROSCOPY OF THE ACTIN-HEAVY MEROMYOSIN COMPLEX DURING THE IN-VITRO MOTILITY ASSAY

Since mica is a substitute for glass in the in vitro actin motility as say, I examined the structure of heavy meromyosin (HMM) crossbridges s upporting actin filaments by quick-freeze deep-etch replica electron m icroscopy. This method Mras capable of resolving the inter-domain clef t of the monomeric actin molecule. HMM heads that are not bound to act in, when observed by this technique, were straight and elongated in th e absence of ATP but strongly kinked upon addition of ATP or ADP.inorg anic vanadate to produce the putative long-lived analog of HMM-ADP.ino rganic phosphate. The low-magnification image of the Am-containing act a-HMM preparation showed features characteristic of sliding actin fila ments on glass coverslips. At high magnification, all the HMM molecule s were found attached to actin by one head with the majority projectin g perpendicular to the filament axis, whereas in the absence of ATP, H MM exhibited two-head binding with a preponderance of molecules tilted at 45 degrees. Detailed examination of the shape of HMM heads involve d in sliding showed a rounded, and flat appearance of the tip and comp aratively thin neck portion as if the heads grasp actin filament, in c ontrast to rigor crossbridges which have a pear-shaped configuration w ith more gradual taper. Such configurations of HMM heads were essentia lly the same as I observed previously on acto-myosin subfragment-l (S1 ) by the same technique, except for the presence of an additional neck portion of HMM which makes interpretaion of the images easier. Intere stingly, under actively sliding conditions, very few heads were tilted in the rigor configuration. At first glance, the addition of ADP to t he rigor-complex gave images rather like those obtained with ATP, but they turned out to be different. The contribution of the structural ch ange of crossbridges to the force development is discussed. (C) 1998 A cademic Press Limited.