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Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method
Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocyti...
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/PMC4230612/ https://www.ncbi.nlm.nih.gov/pubmed/25143395 http://dx.doi.org/10.1091/mbc.E13-01-0004 |
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author | Berro, Julien Pollard, Thomas D. |
author_facet | Berro, Julien Pollard, Thomas D. |
author_sort | Berro, Julien |
collection | PubMed |
description | Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocytic patches in fission yeast to increase the quality of the data extracted from quantitative microscopy movies. We present a universal method to achieve “temporal superresolution” by aligning temporal data sets with higher temporal resolution than the measurement intervals. These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP. We show that the time course of actin assembly and disassembly varies <600 ms between patches. Actin polymerizes during vesicle formation, but we show that polymerization does not participate in vesicle movement other than to limit the complex diffusive motions of newly formed endocytic vesicles, which move faster as the surrounding actin meshwork decreases in size over time. Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated. |
format | Online Article Text |
id | pubmed-4230612 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-42306122015-01-20 Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method Berro, Julien Pollard, Thomas D. Mol Biol Cell Articles Quantitative microscopy is a valuable tool for inferring molecular mechanisms of cellular processes such as clathrin-mediated endocytosis, but, for quantitative microscopy to reach its potential, both data collection and analysis needed improvement. We introduce new tools to track and count endocytic patches in fission yeast to increase the quality of the data extracted from quantitative microscopy movies. We present a universal method to achieve “temporal superresolution” by aligning temporal data sets with higher temporal resolution than the measurement intervals. These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP. We show that the time course of actin assembly and disassembly varies <600 ms between patches. Actin polymerizes during vesicle formation, but we show that polymerization does not participate in vesicle movement other than to limit the complex diffusive motions of newly formed endocytic vesicles, which move faster as the surrounding actin meshwork decreases in size over time. Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated. The American Society for Cell Biology 2014-11-05 /pmc/articles/PMC4230612/ /pubmed/25143395 http://dx.doi.org/10.1091/mbc.E13-01-0004 Text en © 2014 Berro and Pollard. 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 for Cell Biology. |
spellingShingle | Articles Berro, Julien Pollard, Thomas D. Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title | Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title_full | Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title_fullStr | Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title_full_unstemmed | Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title_short | Local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
title_sort | local and global analysis of endocytic patch dynamics in fission yeast using a new “temporal superresolution” realignment method |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4230612/ https://www.ncbi.nlm.nih.gov/pubmed/25143395 http://dx.doi.org/10.1091/mbc.E13-01-0004 |
work_keys_str_mv | AT berrojulien localandglobalanalysisofendocyticpatchdynamicsinfissionyeastusinganewtemporalsuperresolutionrealignmentmethod AT pollardthomasd localandglobalanalysisofendocyticpatchdynamicsinfissionyeastusinganewtemporalsuperresolutionrealignmentmethod |