Tan, K, Roberts, AJ, Chonofsky, M et al. (2 more authors) (2014) A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity. Molecular Biology of the Cell, 25 (5). 669 - 678.
Abstract
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 7 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 velocity requirements.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2014 Tan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). 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). |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 03 Mar 2014 10:39 |
Last Modified: | 18 Jan 2018 13:08 |
Published Version: | http://dx.doi.org/10.1091/mbc.E13-09-0557 |
Status: | Published |
Publisher: | American Society for Cell Biology |
Refereed: | Yes |
Identification Number: | 10.1091/mbc.E13-09-0557 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:77931 |