title: Probing short-range protein Brownian motion in the cytoplasm of living cells.
abstract: The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.
authors.affiliation: 1] Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy [2] NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Carmine
authors.initials: C
authors.lastname: Di Rienzo
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Piazza
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California 92697, USA.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: 1] Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy [2] NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
mesh_terms: D000818:Animals; D016466:CHO Cells; D002477:Cells / Q000737:chemistry / Q000378:metabolism*; D003412:Cricetulus; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D009038:Motion
chemical_list: D049452:Green Fluorescent Proteins