In addition to FRAP studies, confocal microscopy can also be used to assess the mobility of proteins in cells by homotransfer AFRET (anisotropy-based Förster Resonance Energy Transfer) . Molecules tagged with a fluorescent probe can tumble at different rates, depending on their mobility. Highly mobile proteins tumble rapidly and when visualized with polarized optics show little to no anisotropy. Immobile proteins tumble slowly and thus are more anisotropic. When two molecules of the same type interact, or if they interact with another (untagged) molecule, they tumble more slowly and FRET between identical fluorophores can be detected if they are near one another. This is called homotransfer FRET. With the collaboration of Dr. M. Rizzo (Dept. of Physiology, University of Maryland School of Medicine), who developed these methods, we have been using homotransfer AFRET to assess the state of dysferlin (tagged with Venus) in the transverse tubules of muscle cells and the ability of sAnk1 (tagged with GFP) to associate with SERCA (tagged with Cerulean) when both proteins are overexpressed in COS7 cells. The figure below shows that, although the complex formed by SERCA and sAnk1 is not as anisotropic as that formed by SERCA and SLN, it is more anisotropic than either protein expressed with an irrelevant (i.e., non-binding) partner (Dysf; the y-axis is a measure of anisotropy). The evidence indicates that sAnk1 and SERCA associate directly.
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