The overall research goals of the investigators requesting the cryofacility described here is to define the structural organization, relationships, alterations and mechanisms operating during physiological activity, in elements of both the contractile apparatus and the cytoscaffold of muscle and non-muscle cells. We wish to examine these parameters under physiological conditions, or those that mimic physiological states (in the case of isolated macromolecular assemblies and molecules) more closely than is possible using the techniques presently available to us that require fixation and/or staining with heavy metals and prevent determination of the structural parameters we seek in the """"""""native"""""""" specimens. Cryo-preparation: freeze-etching and examination of some specimens as frozen-hydrated samples will enable us to """"""""catch"""""""" our materials in a state close, if not identical, to the native one, and thus to achieve our individual research goals much more directly and expeditiously. Currently we are singly and jointly funded: to examine the arrangement of crossbridges on the surfaces of thick filaments in skeletal and cardiac muscles of invertebrates (RL) and vertebrates (RK), to determine the influence of physiological, environmental alterations on the orientation of the crossbridges and thick filament organization (RL), to identify sites on thick filaments and isolated myosin molecules that are involved in cooperative and other interactions that may occur during activation in myosin-regulated invertebrate thick filaments (PC, RCL), to identify sites along thick filaments and myosin molecules that bind accessory proteins (C, M-band, H, F & Xproteins -JW) and to determine structural and biochemical states of the elements of the cytoscaffold and contractile apparatus involved in neurite elongation (or the inhibition of this process) in culture of dorsal root ganglion (PC). The state-of-the-art cryofacility requested here: Balzers propane jet quick-freezing and freeze-etching devices (with accessories), plus the Gatan specimen cryotransfer system and a low-dose package as a modification for our Jeolco 100CX electron microscope will greatly enhance our research productivity and significantly aid our interpretation of structural correlates of physiological events during cell and muscle motility.
