Quantifying the Contribution of Obstructions to Anomalous Diffusion in Cell Nuclei

Abstract

Most proteins exhibit anomalous subdiffusion in cell nuclei. The source of this behavior is not well understood but is likely due to a combination of crowding and binding of proteins. In order to separate the contributions of these two effects, we measured the diffusion of unconjugated GFP in HeLa cells and the polytene cells of Drosophila salivary glands using two-photon excited fluorescence recovery after photobleaching (FRAP). Our experiments use a diffraction-limited bleaching and observation volume to quantitatively characterize diffusion in specific nuclear substructures. In polytene cells unlike HeLa cells, we can resolve GFP diffusion behavior in nuclear regions containing chromosomes from regions devoid of chromosomes (Figure). Interestingly, we observed anomalous diffusion of GFP in the chromosomal regions only; GFP diffuses normally in the interchromosomal space of the polytene nuclei (Figure). This observation indicates that obstructed diffusion through chromatin is a primary source of anomalous diffusion in cell nuclei. Our presentation will focus on technical challenges of applying quantitative point FRAP, including the need to account for reversible fluorophore photobleaching in data analysis, as well as results and interpretation of anomalous diffusion measurements of unconjugated GFP in cells.View Large Image | View Hi-Res Image | Download PowerPoint Slide