Modified FRAP Methodology with Millisecond Time Resolution for the Investigation of Heterogeneous RNA Polymerase II Diffusion Dynamics

Abstract

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 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. The rapid time resolution, precisely defined bleach geometry, and well-characterized bleach depth enable this method to be made highly quantitative through the application of a diffusion-anomalous subdiffusion model. This modeling can provide insight into the local environment through which biological molecules move. We have applied this novel implementation 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. This has allowed for discriminating observations of protein dynamics while diffusing in the presence or absence of DNA obstructions and binding sites, permitting observation of three distinct regimes of protein diffusion - nucleoplasmic (absence of binding/obstruction), intrachromatin (binding/obstruction), and active transcription regions. We have observed that Rpb3-GFP diffuses anomalously in the nucleoplasm, in contrast to our studies of GFP expressed in polytene cells, which moves by Brownian diffusion through the nucleoplasm. Given the comparable size between the proteins and absence of possible chromatin binding sites, this indicates that Rpb3-GFP is engaged with other RNAP II subunits, giving rise to a distribution of diffusion coefficients, manifest as anomalous subdiffusion. We also see highly anomalous behavior for Rbp3-GFP moving through chromatin regions coupled with reduced diffusion coefficients.