The mechanism underlying the ATP-dependent permeabilization process will be investigated in 3T6 transformed mouse fibroblasts. Radiolabeled photoaffinity analogs of ATP will be synthesized and used for detailed studies that will correlated binding of ATP to a cell surface receptor with the sequential induction of an ATP- dependent increase in plasma membrane permeability to ions, phosphate esters and finally proteins. Similar experiments with radiolabeled glutathione-maleimide (GS-Mal I) will determine the role of an extracellularly-oriented, disulfide bond-containing protein in the permeabilization process. The effect of extracellular ATP on plasma membrane fluidity will be measured by monitoring changes in fluorescence polarization and intensity of lipid fluidity probes in intact cells. The rapid and transient increase in free cytosolic calcium induced by extracellular ATP under permeabilizing conditions will be evaluated by monitoring ionized calcium levels in 3T6 cells under a variety of experimental conditions using the fluorescent calcium probe, fura-2. Successful elucidation of the molecular mechanism responsible for the ATP-dependent permeabilization process in 3T6 cells will lead to a greater understanding of the documented effects of ATP on physiological processes, relevant to the study of disease states in animals, such as neurotransmission, vascular blood flow and wound healing. The reversibility of the ATP-dependent permeabilization of 3T6 cells will be employed to develop a method to seal soluble proteins of biochemical interest into mammalian cells. A series of studies will examine the kinetics of the increase in pore size of ATP-dependent channels by monitoring the uptake of radio- and fluorescently-labeled dextrans of different molecular weights, and by assaying for the release of (35S)-methionine labeled intracellular proteins. Cell division, DNA, RNA and protein synthesis, morphology, internal structure and long term viability of cells that have been permeabilized to proteins and resealed will also be assayed to determine if this procedure can be used to introduce biomolecules into intact cells without loss of cellular function. Other studies will examine the feasibility of sealing aequorin (M.W. 20,000 Daltons), a photoluminescent calcium- binding protein, into ATP-permeabilized cells in an effort to measure intracellular calcium levels by a method complementary to the fura-2 technique. Successful completion of this objective will result in the development of a general procedure for the selective introduction of high molecular weight biomolecules into viable mammalian cells, a technique that will be invaluable to scientists in all disciplines who are utilizing cell culture to study biochemical, physiological and pathological processes.
Showing the most recent 10 out of 15 publications
