We have developed a laser scanning fluorescence recovery after photobleaching (FRAP) methodology capable of investigating protein diffusion and binding dynamics over several decades in time, from the millisecond through minute timescale.  A several micron diameter bleach spot is established within 800 us, followed by fluorescence monitoring by means of a line scan through the bleach region.  This method of FRAP permits the recovery to be captured with 1.6 ms time resolution, enabling observation of short time diffusion and long time binding kinetics .  This novel implementation is being tested to investigate a fluorescently labeled subunit of RNA Polymerase II (Rpb3-GFP) in the polytene nuclei of Drosophila salivary glands.  This unique chromosomal architecture creates segregated nucleoplasmic and chromatin regions, allowing for discriminating observations of protein dynamics while freely diffusing or in the presence of DNA obstructions and binding sites.  Using the method we have observed three different types of protein diffusion depending on nuclear location.

Modified FRAP methodology with millisecond time resolution for the investigation of heterogeneous RNA polymerase II diffusion dynamics

245th National Meeting (2013)

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