title: A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity.
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.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115 Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Kaeling
authors.initials: K
authors.lastname: Tan
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Roberts
authors.forename: Mark
authors.initials: M
authors.lastname: Chonofsky
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Egan
authors.forename: Samara L
authors.initials: SL
authors.lastname: Reck-Peterson
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport; D056445:Cytoplasmic Dyneins / Q000235:genetics / Q000502:physiology; D000072159:Dynactin Complex; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D006836:Hydro-Lyases / Q000235:genetics; D016547:Kinesins / Q000235:genetics / Q000502:physiology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000502:physiology; D008870:Microtubules / Q000378:metabolism; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D020675:Peroxisomes; D013696:Temperature
chemical_list: D000072159:Dynactin Complex; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D056445:Cytoplasmic Dyneins; D004398:Dyneins; D016547:Kinesins; C109850:Arp1 protein, Aspergillus fumagatus; D006836:Hydro-Lyases