Final Activity Report Summary - IMAGING SINGLE LCA S (Single-molecule interaction between calmodulin and the L-type calcium channel)
Invidual molecules of Calmodulin (CaM), a small Ca2+-sensing protein that binds four Ca2+ ions and regulates cellular signals by binding to enzymes, were tracked in living cells. The CaM were observed by attaching a fluorophore, a tiny "light bulb", to them so that they would shine brightly but would not lose their Ca2+ or enzyme binding properties in living biological cells in concentrations so low that they would not disrupt the normal physiological activities of the cells. The single CaM could be detected with a Single-Molecule fluorescence microscope: an optical microscope that takes fast and consecutive "snapshots and movies" with a synchronised laser to make the fluorophores shine, and with the highest possible sensitivity digital cameras, resolution and throughput optics. With this microscope and method, we were able to "spy on" individual CaMs.
Why would one want to spy on single CaM in cells? In the past, different spying methods have been used to observe CaM in living cells: by biochemical techniques -purifying large amounts of the protein and observing its Ca2+ (by titration) and protein (by co-immunoprecipitation) binding properties and by microscopic techniques - by labelling large quantities of CaM in cells at un-natural levels and observing the cell property. We know that CaM responds to Ca2+ levels in cells, so at high calcium, CaM will fold up and bind a number of protein targets, including Calcium Channels (LCa), Calcium Pumps (PMCA), and Nitric Oxide Synthase (eNOS). However, we had previously not been good enough spies to see how fast CaM runs around in the cell, or whether CaM binds or "hides" in one place in the cell, or "sneaks" from one place to another through the cell. Such information is of importance to us because Ca2+ through CaM sends some vital messages to proteins involved in "excitation-contraction coupling" or the essence of the heart beat. Thus, CaM is involved in heart related diseases, the leading cause of death in industrialised nations in the world. From our reconnaissance, we obtain information potentially useful for drug screening and discovery.
By observing the single CaM, we unravelled some new information because the CaM was distinguishable and non-perturbing. First, the individual CaMs displayed a wide-range of motion: from extremely fast, free cytosolic motion bound to a static protein to directed motion along the cytoskeletal tracks of cells. Second, the membrane binding and motion was vastly changed upon cellular Ca2+ level changes. Third, individual CaMs were counted in co-localisation experiments to potential membrane protein binding partners and have high binding to eNOS, moderate binding to PMCA, and relatively less binding to LCa.
Why would one want to spy on single CaM in cells? In the past, different spying methods have been used to observe CaM in living cells: by biochemical techniques -purifying large amounts of the protein and observing its Ca2+ (by titration) and protein (by co-immunoprecipitation) binding properties and by microscopic techniques - by labelling large quantities of CaM in cells at un-natural levels and observing the cell property. We know that CaM responds to Ca2+ levels in cells, so at high calcium, CaM will fold up and bind a number of protein targets, including Calcium Channels (LCa), Calcium Pumps (PMCA), and Nitric Oxide Synthase (eNOS). However, we had previously not been good enough spies to see how fast CaM runs around in the cell, or whether CaM binds or "hides" in one place in the cell, or "sneaks" from one place to another through the cell. Such information is of importance to us because Ca2+ through CaM sends some vital messages to proteins involved in "excitation-contraction coupling" or the essence of the heart beat. Thus, CaM is involved in heart related diseases, the leading cause of death in industrialised nations in the world. From our reconnaissance, we obtain information potentially useful for drug screening and discovery.
By observing the single CaM, we unravelled some new information because the CaM was distinguishable and non-perturbing. First, the individual CaMs displayed a wide-range of motion: from extremely fast, free cytosolic motion bound to a static protein to directed motion along the cytoskeletal tracks of cells. Second, the membrane binding and motion was vastly changed upon cellular Ca2+ level changes. Third, individual CaMs were counted in co-localisation experiments to potential membrane protein binding partners and have high binding to eNOS, moderate binding to PMCA, and relatively less binding to LCa.