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New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales
Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algori...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967967/ https://www.ncbi.nlm.nih.gov/pubmed/24501425 http://dx.doi.org/10.1091/mbc.E13-03-0162 |
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author | Yamashiro, Sawako Mizuno, Hiroaki Smith, Matthew B. Ryan, Gillian L. Kiuchi, Tai Vavylonis, Dimitrios Watanabe, Naoki |
author_facet | Yamashiro, Sawako Mizuno, Hiroaki Smith, Matthew B. Ryan, Gillian L. Kiuchi, Tai Vavylonis, Dimitrios Watanabe, Naoki |
author_sort | Yamashiro, Sawako |
collection | PubMed |
description | Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. |
format | Online Article Text |
id | pubmed-3967967 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-39679672014-06-16 New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales Yamashiro, Sawako Mizuno, Hiroaki Smith, Matthew B. Ryan, Gillian L. Kiuchi, Tai Vavylonis, Dimitrios Watanabe, Naoki Mol Biol Cell Articles Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. The American Society for Cell Biology 2014-04-01 /pmc/articles/PMC3967967/ /pubmed/24501425 http://dx.doi.org/10.1091/mbc.E13-03-0162 Text en © 2014 Yamashiro et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. |
spellingShingle | Articles Yamashiro, Sawako Mizuno, Hiroaki Smith, Matthew B. Ryan, Gillian L. Kiuchi, Tai Vavylonis, Dimitrios Watanabe, Naoki New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title | New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title_full | New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title_fullStr | New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title_full_unstemmed | New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title_short | New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
title_sort | new single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967967/ https://www.ncbi.nlm.nih.gov/pubmed/24501425 http://dx.doi.org/10.1091/mbc.E13-03-0162 |
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