A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity |
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Authors: | Kaeling Tan Anthony J Roberts Mark Chonofsky Martin J Egan Samara L Reck-Peterson |
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Institution: | University of Exeter;aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115;bAstbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom |
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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. |
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