Probing Short-Range Protein Brownian Motion in the Cytoplasm of Living Cells

Abstract

The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 microsecond, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. In this regard, internal membranes (e.g. the ER sheets, vesicles, Golgi apparatus, etc.) appear to be the more likely candidates as selective disruption of the microtubules network by treatment with Nocodazole did not significantly alter GFP behavior in the cytoplasm. Also, the same measurement in a structurally-different (e.g devoid of membranes) intracellular environment, such as the nucleoplasm, yields a different behavior, in which GFP motion is never coincident with that in a dilute solution. Finally, we believe the present findings coupled with use of genetically-encoded fluorescent markers pave the way to novel studies of biomolecular processes in live cells at the physiologically-relevant spatio-temporal scale. Supported by grants NIH P41-GM103540 and NIH P50-GM076516 (grants to EG), MIUR under FIRB-RBAP11X42L and Fondazione Monte dei Paschi di Siena (grants to FB).