In continuing studies of myosin function in vitro, we will explore the myosin step length as measured by the nanometer analyses of movements of particles attached either to the myosin or to actin filaments. We will compare the behavior of myosins which differ in movement velocities and structure to determine if step distance is constant for the different velocities and structures. The mechanisms whereby many intracellular organelles actually move through cytoplasm or particles move on cell surfaces are not well understood. With the ability to analyze the movements at the nanometer level we can determine whether or not particles are actively moving on a stable filament or are moving by another mechanism. Saltatory organelle movements are characterized by frequent reversals in direction which may be recoil events or active movements. Capping of cell surface glycoproteins may be caused by the biased diffusion of protein aggregates in a lipid flow or by the active movement of those aggregates on cytoskeletal filaments. We will now analyze in vivo movements to determine whether saltatory organelle movements and extracellular particle capping involve active movements on filaments. A major emphasis of this grant is to extend our in vitro studies of tubulovesicular networks to understand endoplasmic reticulum (ER) structure, dynamics, and function. Recently, we have developed a method to prepare a two-dimensional network from isolated components of the ER (microsomes) which involves the active movement of membrane strands on microtubules driven by microtubule-dependent motors. The morphology and dynamics of the in vitro networks mimic those of the in vivo ER networks. We will now determine whether Golgi, lysosomes, rough microsomes, and smooth microsomes can form or fuse with the in vitro networks. Membrane fusion may be a major factor involved in network formation and dynamics. Using fluorescent probes we will examine the rates of membrane fusion into and budding from the networks in vitro and the dependence of the fusion and budding processes on ATP, GTP, microtubule motors and microtubules. Because the networks may facilitate diffusion of some components while restricting others, fluorescent probes and photobleaching experiments will be used to measure the diffusion coefficients of membrane and luminal components through the networks. ER functions such as protein synthesis, processing and glycosylation may be greatly facilitated by network formation, and tests of those possibilities will be performed.
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