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A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity
Tan K.2; Roberts A.J.2; Chonofsky M.2; Egan M.J.2; Reck-Peterson S.L.2
2014-03-01
Source PublicationMolecular Biology of the Cell
ISSN10591524 19394586
Volume25Issue:5Pages: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 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. © 2014 Tan et al.

DOI10.1091/mbc.E13-09-0557
URLView the original
Indexed BySCI
WOS Research AreaCell Biology
WOS SubjectCell Biology
WOS IDWOS:000339629100011
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Cited Times [WOS]:13   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Affiliation1.University of Leeds
2.Harvard Medical School
Recommended Citation
GB/T 7714
Tan K.,Roberts A.J.,Chonofsky M.,et al. A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity[J]. Molecular Biology of the Cell,2014,25(5):669-678.
APA Tan K.,Roberts A.J.,Chonofsky M.,Egan M.J.,&Reck-Peterson S.L..(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.
MLA Tan K.,et al."A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity".Molecular Biology of the Cell 25.5(2014):669-678.
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