Translational diffusion of globular proteins in the cytoplasm of cultured muscle cells

Modulated fringe pattern photobleaching (MFPP) was used to measure the tran slational diffusion of microinjected fluorescein isothiocyanate (FITC)-labe led proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recover y after photobleaching (FRAP) technique, allows the measurement of the tran slational diffusion of macromolecules over several microns. Proteins used h ad molecular masses between 21 and 540 kDa, The results clearly indicated t hat the diffusivity of the various proteins is a decreasing function of the ir hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (Arrio-Dupont et al., 1996, Biophys. J. 70. 2327-2332), most likely because, unlike globular proteins, dextrans are ran domly coiled macromolecules with a flexible structure. These data do not ex clude the possibility of a rapid diffusion over a short distance, unobserva ble with our experimental set-up, which would take place within the first m illiseconds after bleaching and would correspond to the diffusion in restri cted domains followed by impeded diffusion provoked by the network of micro tubules, microfilaments, and intermediate filaments. Thus our results may c omplement rather than contradict those of Verkman and collaborators (Seksek et al., 1997, J. Cell Biol. 138:1-12), The biological consequence of the s ize-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins consi derably reduces their mobility.