Abstract
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in Caenorhabditis elegans embryos. We exploited 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). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.
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Acknowledgements
This research was supported by the US National Institutes of Health R01 grant GM098441 (to E.M.M.) and by the University of Chicago Materials Research Science & Engineering Center. We thank G. Seydoux (Johns Hopkins University), J. Nance (NYU School of Medicine), K. Kemphues (Cornell University), M. Glotzer and K. Longhini (University of Chicago) for strains; A. Sailer for technical support; N. Bartley, X. Zhang and W. Dong for their contributions to the initial stages of this project; and the Glotzer lab for sharing strains and reagents.
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Contributions
E.M.M. conceived the imaging approach and provided overall guidance. F.B.R. conceived and developed the smPReSS approach and, with E.M.M., developed the tracking-based turnover analysis. F.B.R. and W.M.M. acquired the data for actin; B.Y. and F.B.R. acquired the data for PAR-6; W.M.M., F.B.R. and B.Y. performed the nonlinear fitting; E.M.M., F.B.R., B.Y. and W.M.M. developed and performed the mobility analysis. F.B.R. performed the tracking-based turnover analysis, and F.B.R., W.M.M., B.Y. and E.M.M. made the figures. E.M.M. and F.B.R. wrote the text.
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Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Notes 1 and 2 (PDF 2656 kb)
Single molecule movie of PAR-6::GFP
A one cell embryo during maintenance phase expressing PAR-6::GFP at single molecule levels and imaged for 20 s at 30 frames per second, at 100% laser power. Replay speed is 1×. Image size = 331 × 231 pixels, pixel size = 107 nm. Anterior is to the left. Note the range of particle behaviors and mobilities are readily apparent (see main text, and Supplementary Video 3,4). (MOV 13657 kb)
Single molecule movie of GFP::Actin.
A one-cell embryo expressing GFP::Actin at single molecule levels and depleted of NMY-2 by RNAi. The embryo was imaged for 500s at 10 frames per second, at 30% laser power, beginning in maintenance phase and proceeding through anaphase and into telophase. Replay speed is 12×. Image size = 424 × 267 pixels, pixel size = 107 nm. (MOV 22442 kb)
Single molecule movie of GFP::Actin.
Same embryo as in Video 2, showing the entire acquisition sequence. Replay speed is 48×. Image size = 424 × 267 pixels, pixel size = 107 nm. Note that actin is initially evenly distributed along the anterior posterior axis, but during anaphase and telophase, it accumulates at the equator and is depleted from the poles. (MOV 21895 kb)
A single PAR-6::GFP molecule displaying simple diffusive behavior.
Original image size: 45 × 45 pixels, pixel size: 107 nm. Imaging conditions as in Supplementary Video 1. Transient failures to detect the particle in a given frame due to motion blur are indicated by the absence of a red circle. (MOV 2140 kb)
A single PAR-6::GFP molecule displaying sub-diffusive behavior.
Original image size: 45 × 45 pixels, pixel size: 107 nm. Imaging conditions as in Supplementary Video 1. For this slower moving particle, there are no detection failures. (MOV 1577 kb)
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Robin, F., McFadden, W., Yao, B. et al. Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos. Nat Methods 11, 677–682 (2014). https://doi.org/10.1038/nmeth.2928
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DOI: https://doi.org/10.1038/nmeth.2928
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