Abstract
We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.
Highlights
Living cells are multifunctional organisms that exhibit remarkable dynamic phenomena including, e.g., cell motility, and vesicular, cytoplasmic and nuclear transport
We introduce the new methods by determining the diffusion coefficient of a freely-diffusing model protein, enhanced yellow fluorescent protein (EYFP), in two continuous cell lines, fibroblastlike Norden Laboratory Feline Kidney (NLFK) [25] and cervical carcinoma HeLa [26]
When the measured recovery data were analyzed by the Soumpasis method, we found a cytoplasm diffusion coefficient of D~0:75+0:3 mm2/s (n = 8) for the NLFK cells and D~1:83+0:28 mm2/s (n = 13) for the HeLa cells
Summary
Living cells are multifunctional organisms that exhibit remarkable dynamic phenomena including, e.g., cell motility, and vesicular, cytoplasmic and nuclear transport. The cytoplasm consists of a viscous liquid phase (the cytosol) and a non-liquid phase that will be called here the solid phase. The protein concentration in the cytoplasm has been estimated to be 100 mg/ ml [1], and its total macromolecular concentration (proteins, lipids, nucleic acids, and sugars) can be as high as 400 mg/ml [2]. The cytoplasm can be described as a ‘molecularly crowded’ environment, where macromolecules can occupy 20–30% of its volume [3]. Macromolecular diffusion in the cytoplasm can be severely restricted in such an environment [6]. The same applies to the nucleus [7,8,9] that is composed of a liquid phase, the nucleosol, and a solid phase comprising, e.g., chromatin and proteinaceous nuclear bodies
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