Understanding boundary effects and confocal optics enables quantitative FRAP analysis in the confined geometries of animal, plant, and fungal cells

Abstract

Fluorescence Recovery After Photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus or cell membrane. While many FRAP models have been developed over the past decades, the influence of the complex boundaries of three-dimensional cellular geometries on the recovery curves, in conjunction with ROI and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied. Here, we developed a three-dimensional computational model of the FRAP process that incorporates particle diffusion, cell boundary effects, and the optical properties of the scanning confocal microscope, and validated this model using the tip-growing cells of Physcomitrella patens. We then show how these cell boundary and optical effects confound the interpretation of FRAP recovery curves, including the number of dynamic states of a given fluorescent protein, in a wide range of cellular geometries-both in two and three dimensions-namely nuclei, filopodia, and lamellipodia of mammalian cells, and in cell types such as the budding yeast, S. pombe, and tip-growing plant cells. We explored the performance of existing analytical and algorithmic FRAP models in these various cellular geometries, and determined that the VCell VirtualFRAP tool provides the best accuracy to measure diffusion coefficients. Our computational model is not limited only to these cells types, but can easily be extended to other cellular geometries via the graphical Java-based application we also provide. This particle-based simulation-called the Digital Confocal Microscopy Suite, DCMS-can also perform fluorescence dynamics assays, such as Number and Brightness (N&B), Fluorescence Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy (RICS), and could help shape the way these techniques are interpreted.

F-Actin Mediated Focusing of Vesicles at the Cell Tip Is Essential for Polarized Growth

“…In addition, fluorescence labeling of the endoplasmic reticulum and Golgi (Furt et al, 2012) did not match the fluorescence intensity distribution found with VAMP72. Furthermore, the vesicle diffusion coefficient we measured is in agreement with previously measured pollen tube vesicle diffusion coefficients (Bove et al, 2008;Kroeger et al, 2009) and measurements of the viscosity of the moss cytoplasm (Kingsley et al, 2017).…”

“…This is not the case at the cell shank, where fluorescence recovery can happen from all directions. To accurately determine the diffusion coefficient, we used a particle-based computational model of FRAP that incorporates the properties of our confocal imaging system, the three-dimensional moss cell shape, and the thermal fluctuations of the fluorophore (Kingsley et al, 2017).…”

“…Moss samples were cultured using the protocol described previously (Kingsley et al, 2017). Samples were prepared in QR-43C chambers (Warner Instruments) and perfused with 5 mM latrunculin B.…”

“…To probe the utility of actin-mediated active transport, we used the small molecule inhibitor latrunculin B to depolymerize actin and uncouple vesicle diffusion from active transport. With FRAP as well as number and brightness analyses Lorén et al, 2015;Kingsley et al, 2017), we measured vesicle diffusion rates and concentrations, respectively. We then developed a diffusion-limited analytical model and numerically solved it using these parameters as well as previously measured cellular growth rates (Furt et al, 2013).…”

“…Diffusion coefficient, D, was measured as described in the previous section with FRAP analysis (Kingsley et al, 2017). To determine c L , we used our confocal particlebased simulation (Kingsley et al, 2017) to produce a range of potential concentrations and found that resolving individual particles becomes impossible at concentrations above 10 vesicles mm 23 (Fig. 4C).…”