A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

• Published in: Molecular biology of the cell Vol. 25; no. 5; pp. 669 - 678
• Main Authors: Tan, Kaeling, Roberts, Anthony J, Chonofsky, Mark, Egan, Martin J, Reck-Peterson, Samara L
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01-03-2014
• Subjects: Aspergillus nidulans - genetics; Aspergillus nidulans - metabolism; Aspergillus nidulans - ultrastructure; Biological Transport; Cytoplasmic Dyneins - genetics; Cytoplasmic Dyneins - physiology; Dynactin Complex; Dyneins - genetics; Dyneins - metabolism; Fungal Proteins - genetics; Fungal Proteins - physiology; Hydro-Lyases - genetics; Kinesin - genetics; Kinesin - physiology; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - physiology; Microtubules - metabolism; Organelles - metabolism; Organelles - ultrastructure; Peroxisomes; Temperature
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E13-09-0557

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.