Vero cells were transfected using Nucleofector by amaxa (Cologne, Germany) according
to the protocol for COS-7 cells (Kit V, program A24). Briefly, 1x106
VERO cells were pelleted and resuspended in 100 µl of solution V, and
electroporated with 1–2.5 µg of DNA. The electroporated cells were
resuspended in 350 µl MEM. Of this solution, 100 µl were seeded on one
18-mm coverslip and incubated over night (15 h) at 37C and 5% CO2;
12–16 h posttransfection cells were imaged live on a temperature-controlled
stage at 37°C.
The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the Endoplasmic Reticulum (ER) was studied using cell biological and computational methods. Fluorescence Recovery After Photobleaching (FRAP) experiments were performed in tissue culture cells expressing GFP-KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER was determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes was simulated using the method of Particle Strength Exchange (PSE). The simulations were compared to experimental FRAP curves of GFP-KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in 2 to 4-fold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Since organelle shape considerably in- fluences diffusive transport it must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. The present novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen.