Teaming up: from motors to people.

When I reflect on how I became a cell biologist and why I love being one today, one thing that comes to mind is the many terrific collaborations I have had. The science I am most proud of from my graduate and postdoctoral training would not have been possible without working in teams with other scientists. Now, in my own group, much of our best work is being done collaboratively, both within the lab and with other labs. In this essay, I will highlight my experiences working in teams as a trainee, the role teamwork has played in my own research group, and how important I think collaborative science is for the future of biological research

Reconstitution of dynein transport to the microtubule plus end by kinesin

Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track. DOI: http://dx.doi.org/10.7554/eLife.02641.00

Characterization of the Mutagenic Spectrum of 4-Nitroquinoline 1-Oxide (4-NQO) in Aspergillus nidulans by Whole Genome Sequencing

4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO−based genetic screens have been used to study diverse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO−induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing

Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the "tail" of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3's ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors

Dynein Achieves Processive Motion Using both Stochastic and Coordinated Stepping

Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this, we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use \u27hand-over-hand\u27 mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for its diverse cellular functions

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

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