Abstract
We describe a general, versatile and non-invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in C. elegans embryos. We exploit tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single particle tracking (SPT) analysis. We also introduce and validate a new method called smPReSS that infers exchange rates from quantitative analysis of single molecule photobleaching kinetics, without using SPT. Combining SPT and smPReSS allows spatially and temporally resolved measurements of protein mobility and exchange kinetics. We use these methods (a) to resolve distinct mobility states and spatial variation in exchange rates of the polarity protein Par-6 and (b) to measure spatiotemporal modulation of actin filament assembly and disassembly. The introduction of these methods in a powerful model system offers a promising new avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.
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