This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project seeks to characterise the real time diffusion dynamics of fluoresently labelled actin molecules within the nuclei of living cultured mammalian cells.
We aim to determine if nuclear Actin molecules exhibit evidence of self binding (polymerization) and if they show binding or barriers to diffusion in relation to nuclear components (chromatin and transcription factors). The major experiments for this work include; 1) cell culture of L6 Myoblast adherent cells using standard culture reagents and techniques. 2) Transfection of the cultured cells with fluorescently labelled actin plasmid DNA. 3) Confocal imaging and spectroscopy of cells expressing fluorescently labelled actin molecules. Spectroscopy tecniques used will include scanning fluorescence correlation spectroscopy, rast image correlation spectroscopy, number and brightness analysis, and photon counting histogram.
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