Translational Diffusion of Globular Proteins in the Cytoplasm of Cultured Muscle Cells

Abstract

Modulated fringe pattern photobleaching (MFPP) was used to measure the translational diffusion of microinjected fluorescein isothiocyanate (FITC)-labeled proteins of different sizes in the cytoplasm of cultured muscle cells. This technique, which is an extension of the classical fluorescence recovery after photobleaching (FRAP) technique, allows the measurement of the translational diffusion of macromolecules over several microns. Proteins used had molecular masses between 21 and 540 kDa. The results clearly indicated that the diffusivity of the various proteins is a decreasing function of their hydrodynamic radius. This decrease is more rapid with globular proteins than with FITC-labeled dextrans (, Biophys. J. 70:2327-2332), most likely because, unlike globular proteins, dextrans are randomly coiled macromolecules with a flexible structure. These data do not exclude the possibility of a rapid diffusion over a short distance, unobservable with our experimental set-up, which would take place within the first milliseconds after bleaching and would correspond to the diffusion in restricted domains followed by impeded diffusion provoked by the network of microtubules, microfilaments, and intermediate filaments. Thus our results may complement rather than contradict those of Verkman and collaborators (, J. Cell Biol. 138:1-12). The biological consequence of the size-dependent restriction of the mobility of proteins in the cell cytoplasm is that the formation of intracellular complexes with other proteins considerably reduces their mobility.

Diffusion on a curved surface coupled to diffusion in the volume: Application to cell biology

“…The FLIP experimental protocol was then simulated by solving the full model (10) with parameters k off and q, representative of those obtained in the experiments [17], and values of D 2 around 20 μm 2 /s. This is an appropriate range for the diffusion coefficient of a cytosolic protein of the GFP-Rac size (∼50 kDa), according to estimates and recent measurements [26]. The normalized fluorescence acquired from the portion of the bleached region ω 1 (Fig 9), proximal to the unbleached domain, is calculated as where .…”

New insights into diffusion in 3D crowded media by Monte Carlo simulations: effect of size, mobility and spatial distribution of obstacles

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques 13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and from simulations 14,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] . Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) techniques.…”

Diffusion in macromolecular crowded media: Monte Carlo simulation of obstructed diffusion vs. FRAP experiments

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques [7,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and from simulations [10,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) [12-14, 16, 18, 20, 28, 30], fluorescence correlation spectroscopy (FCS) [11-23, 26-27, 29] and single particle tracking (SPT) [15,19,[24][25] techniques.…”