The proposed research is aimed at comparing the molecular events involved in muscle contraction to the interactions between cytoplasmic actins and myosins that produce streaming in amoeboid cells. First, the changes in birefringence, linear dichroism, difluorescence, and circular dichroism, (phase modulation microspectrophotometry), will be measured in the rigor-relax-contraction transition of rabbit psoas muscle fibers. These experiments will relate the polarized light measurements to structural changes that have been characterized by x-ray diffraction, electron microscopy and solution spectroscopy. Then, pooled cytoplasm from Amoeba proteus will be fractionated in order to prepare a calcium sensitive amoeba actin-myosin system. Unidirectional contractions or cytoplasmic streaming will be ellicited in this model system by controlling the gradient of calcium and magnesium-ATP. The polarization properties of contracting and streaming amoeba actin-myosin can then be investigated and compared to the molecular events during muscle contraction. The possible role of filamentagenesis in cytoplasmic streaming of amoeba extracts will be tested with combinations of light and electron microscopy. In addition, intact amoebas will be studied in order to relate the dynamics of calcium controlled contractions to cytoplasmic streaming and amoeboid movement. The sites of calcium release and calcium sequestering will be identified and related to the contractile events. Finally, membrane formation and dynamics will be characterized and its role in controlling movements determined.
NIH ExPorter
NIH ExPorter