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High-density single-molecule analysis of cell surface dynamics in C. elegans embryos

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 im...

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Detalles Bibliográficos
Autores principales: Robin, Francois B., McFadden, William M., Yao, Baixue, Munro, Edwin M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046709/
https://www.ncbi.nlm.nih.gov/pubmed/24727651
http://dx.doi.org/10.1038/nmeth.2928
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author Robin, Francois B.
McFadden, William M.
Yao, Baixue
Munro, Edwin M.
author_facet Robin, Francois B.
McFadden, William M.
Yao, Baixue
Munro, Edwin M.
author_sort Robin, Francois B.
collection PubMed
description 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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spelling pubmed-40467092014-12-01 High-density single-molecule analysis of cell surface dynamics in C. elegans embryos Robin, Francois B. McFadden, William M. Yao, Baixue Munro, Edwin M. Nat Methods Article 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. 2014-04-13 2014-06 /pmc/articles/PMC4046709/ /pubmed/24727651 http://dx.doi.org/10.1038/nmeth.2928 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Robin, Francois B.
McFadden, William M.
Yao, Baixue
Munro, Edwin M.
High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title_full High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title_fullStr High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title_full_unstemmed High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title_short High-density single-molecule analysis of cell surface dynamics in C. elegans embryos
title_sort high-density single-molecule analysis of cell surface dynamics in c. elegans embryos
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046709/
https://www.ncbi.nlm.nih.gov/pubmed/24727651
http://dx.doi.org/10.1038/nmeth.2928
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