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Nat Methods. 2014 Jun;11(6):677-82. doi: 10.1038/nmeth.2928. Epub 2014 Apr 13.

Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos.

Author information

1
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA.
2
1] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [2] Biophysics Graduate Program, University of Chicago, Chicago, Illinois, USA.
3
1] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [2] Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois, USA. [3] Computation Institute, University of Chicago, Chicago, Illinois, USA. [4].

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.

PMID:
24727651
PMCID:
PMC4046709
DOI:
10.1038/nmeth.2928
[Indexed for MEDLINE]
Free PMC Article

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