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A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity
The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation us...
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/PMC3937092/ https://www.ncbi.nlm.nih.gov/pubmed/24403603 http://dx.doi.org/10.1091/mbc.E13-09-0557 |
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author | Tan, Kaeling Roberts, Anthony J. Chonofsky, Mark Egan, Martin J. Reck-Peterson, Samara L. |
author_facet | Tan, Kaeling Roberts, Anthony J. Chonofsky, Mark Egan, Martin J. Reck-Peterson, Samara L. |
author_sort | Tan, Kaeling |
collection | PubMed |
description | The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. |
format | Online Article Text |
id | pubmed-3937092 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-39370922014-05-16 A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity Tan, Kaeling Roberts, Anthony J. Chonofsky, Mark Egan, Martin J. Reck-Peterson, Samara L. Mol Biol Cell Articles The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance. The American Society for Cell Biology 2014-03-01 /pmc/articles/PMC3937092/ /pubmed/24403603 http://dx.doi.org/10.1091/mbc.E13-09-0557 Text en © 2014 Tan 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 Tan, Kaeling Roberts, Anthony J. Chonofsky, Mark Egan, Martin J. Reck-Peterson, Samara L. A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title | A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title_full | A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title_fullStr | A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title_full_unstemmed | A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title_short | A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
title_sort | microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937092/ https://www.ncbi.nlm.nih.gov/pubmed/24403603 http://dx.doi.org/10.1091/mbc.E13-09-0557 |
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