The mechanism by which nonmuscle cells change their shape is an area of interest to many fields of biology and medicine. Numerous protozoan and fungal systems have been used as model systems for higher cell types, in part because these cells can be easily cultured and studied. This proposal recommends two pilot studies on one such protozoan system. These studies should indicate the feasibiity of using this system to answer the long range question, do cell shape changes in Euglena involve an actomyosin-based contractile system similar to smooth muscle cells? Initial studies on this system have used indirect immunofluorescence directed against two proteins thought to be involved, actin and myosin. The antibodies were obtained commercially and were not made against Euglena antigen. Although immunofluorescent controls indicate that the antibodies specifically cross react with actin and myosin, these two proteins have not been demonstrated beyond a doubt to be Euglena proteins although other investigators have reported them. It is proposed in the first pilot study that the immunoblotting technique be used to verify the cross reactivity of the antibodies to the Euglena antigen. Two cellular fractions, each reported to contain actin and myosin, will be used for extraction with subsequent separation of proteins by electrophoresis. If the antibodies are shown to specifically cross react, then they will be used in a second pilot study intended to localize these proteins by electron microscopy. The technique of gold-labeled antibody localization will be used for several proteins including actin, myosin and calmodulin. The technique will use nonembedded thin sections (sectioning in diethylene glycol distearate, followed by removal of the wax) which should allow for maximal exposure of the cellular proteins to the antibodies. This technique will be used to answer numerous questions which collectively will indicate if the system can be used to answer the primary question "can an actomyosin contractile system be implicated as a cause of cell shape changes?"
NIH ExPorter
NIH ExPorter