Quantifying the Contribution of Obstructions to Anomalous Diffusion in Cell Nuclei
Date:
Authors
Matthew K. Daddysman, Christopher J. Fecko
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. Interestingly, we observed anomalous diffusion of GFP in the chromosomal regions only; GFP diffuses normally in the interchromosomal space of the polytene nuclei. This observation indicates that obstructed diffusion through chromatin is a primary source of anomalous diffusion in cell nuclei. Additionally, we measured the diffusion of GFP in crowded dextran solutions in vitro. At the dextran concentration that results in the same diffusion coefficient as the interchromatin space, GFP also diffuses normally in vitro. These observations indicate that macromolecular crowding is likely not the source of nuclear anomalous diffusion. In order to ensure that the anomalous diffusion observed was not an experimental artifact, we accounted for reversible fluorophore photobleaching in the data analysis.