Probing cytoplasmic viscosity in the confined geometry of tip-growing plant cells via FRAP

Understanding plant growth and development is essential to develop the future technologies necessary to meet the anticipated needs of a growing world population. Because plant growth is a manifestation of cellular growth, it is of prime importance to develop a mechanistic understanding of plant cell growth. Transport of cellular cargo, such as proteins, in growing plant cells is essential as it facilitates growth. Developing a quantitative model of growth requires knowledge of the surrounding medium, i.e. the cytoplasm and its inherent properties. Here, we performed Fluorescence Recovery After Photobleaching (FRAP) in tip-growing Physcomitrella patens cells, to determine the diffusion coefficient of 3xmEGFP, and calculate an effective cytoplasmic viscosity. In order to interpret the experimental measurements correctly and accurately estimate the diffusion coefficient, we developed a three-dimensional comprehensive computational model of the FRAP process, including particle diffusion, the cell boundary effects, and the optical properties of the scanning confocal microscope. To the best of our knowledge, this is the first time such an estimate of the viscosity for particles at this length scale is reported for a plant cell. Our model allows us to determine the degree at which cell boundary and optical effects confound the interpretation of FRAP recovery curves, the bound fraction of fluorescent proteins, and the number of dynamic states of a given fluorescent protein. The presented FRAP model has a wide range of applicability across many cell types including plant, animal, and fungal cells, particularly in the presence of otherwise prohibitive geometries.