The overall goal of this research is to further develop, test and implement our original experimental technique to study dynamic processes in living cells. Because the cytosol is a densely crowded and dynamically heterogeneous environment, measurements that establish a connection between spatial scale and mobility can provide insight into its organization and mechanisms of cytoskeletal activity. Fourier imaging correlation spectroscopy (FICS) is an attemative to direct imaging methods and is based on selectively detecting fluorescence fluctuations from N labeled sub-cellular species at a specified (and adjustable) spatial scale. Our recent FICS studies of the mitochondriat reticulum in live osteosarcoma cells show that the length-scale-dependence of the organelle's mobility follows a systematic and complex pattern, which likely reflects the relevant length- and time-scales of cytoskeletal motor protein activity. When actin or tubuiin components of the cytoskeleton are selectively removed, the organelle's dynamics become sequentially simplified, in the absence of both cytosketal components, the dynamics of the reticulum become primarily diffusive and may be modelled as a collection of friction points interconnected by elastic springs. The main advantage of FICS is that the signal is enhanced (relative to that from a single particle) by a factor of N and thereby affords superior temporal resolution and signal sensitivity in comparison to fluorescence microscopy. In this proposal, we develop Molecular FICS, a variation of the method that is sensitive to the motions and structural changes of individual proteins and other macromolecules in the celt. The combination of Molecular FICS with the previously established organelle-sensitive version of the method will allow us to study how macromolecular species are transported through their intracellular compartments.
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