This project studies the organization of structural and diffusible components of nerve, muscle and glia, particularly with respect to the function of specialized membrane regions such as synapses. A new and unique instrument -- the low-temperature, high-resolution, field-emission scanning transmission electron microscope (STEM) -- has been developed and shown to reach new levels of sensitivity and resolution for molecular weight mapping and chemical analysis of native proteins. This STEM has now been used to determine the tertiary structure and molecular weight distribution of individual, native molecules of kinesin, as described in Project ZO1-NS-02700-05 LN. This instrument will also be essential for extending analytical microscopy studies on calcium regulation in dendritic spines and shafts, which have been advanced by the development of suitable organotypic cultures of hippocampal slices. In parallel, uptake and release of calcium from the endoplasmic reticulum (ER) of a model for calciumregulated neurosecretion, the sea urchin egg cortex, has been studied using calcium indicator dyes. Light and electron microscopy immunocytochemistry has indicated that transport on microtubules is also the basis of anterograde and retrograde vesicular transport of myelin-specific proteins in oligodendrocytes. Thus, the depolymerization of microtubule bundles, the distribution and polarity of which support a role in organelle translocation, reversibly block transport of myelin proteins from the Golgi apparatus. Microtubule depolymerization also causes reorganization of organelles involved in protein synthesis in cultured cells in living cells, the three-dimensional organization of the ER in relation to microtubules is being revealed by confocal microscopy. Immunocychemistry has been used to localize the alpha-1 and alpha-2 subunits of the dihydropyridine receptor to the t-tubule system of skeletal muscle, thereby indicating the association of these polypeptides in the excitation-contraction (EC) coupling couple. This contrasts with the distribution of these receptors in dysgenic muscle (mdg/mdg) where the absence of the alpha-1 subunit is associated with disorganization of the remaining alpha-2 protein. During myogenesis in normal muscle, the development of the E/C coupling apparatus is characterized by the formation of t-tubule membranes prior to connection with the plasma membrane.
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