Reports that single-emryo lysates (prepared using our microfluidic lysis chips) contain sufficient material to observe 3D structures of native protein complexes. We visualized ribosomes and proteasomes from single-embryo lysates as a proof of principle.
The Auxin-Inducible Degron (AID) system is emerging as a powerful tool for controlled depletion of proteins of interest. This system requires expressing a ubiquitin ligase, TIR1, that derives from plants. We created a TIR1 expression construct that contains a built-in BFP::AID reporter, allowing TIR1 expression and activity to be monitored without using the GFP or RFP imaging channels. The lab was brand new at the time and we didn't have the bandwidth to further develop this ourselves. We shared the constructs and strategy with the Ward and Reiner labs, who developed a really nice toolkit based on our idea. Kudos to their groups for building an awesome resource.
Ras-dependent cell fate decisions are reinforced by the RAP-1 small GTPase in Caenorhabditis elegans.
Rasmussen NR, Dickinson DJ, Reiner DJ (2018). Genetics 210(4): 1339-54.
One of my side projects as a postdoc was devising an algorithm to design protein-coding sequences that would evade silencing in the C. elegans. I contributed this algorithm to an optogenetics project in the van den Heuvel lab; it was used to design sequences that allowed expression of LOV domain-based optogenetic tools in C. elegans.
Light-dependent cytoplasmic recruitment enhances the dynamic range of a nuclear import photoswitch.
Yumerefendi H, Wang H, Dickinson DJ, Lerner AM, Malkus P et al. (2018). Chembiochem 19(12): 1319-25.
A CRISPR tagging-based screen reveals localized players in Wnt-directed asymmetric cell division.
Heppert JK, Pani AM, Roberts AM, Dickinson DJ, Goldstein B. (2018). Genetics 208(3): 1147-64.
Reports an approach for analyzing protein-protein interactions in single, staged C. elegans zygotes. Cells are lysed in nanoliter volumes using microfluidics, and contents analyzed using single-molecule pull-down. This approach was used to discover that oligomerization of the PAR-3 protein is surprisingly dynamic: it is upregulated specifically during polarity establishment. PAR-3 oligomerization is essential for proper polarity establishment and is linked to the cell cycle via direct phosphorylation of PAR-3 by PLK-1.
Results of a collaboration between Dan and Alakanada Das, who was a graduate student in Kevin Slep's lab at the time. Alka solved the crystal structure of a key microtubule-binding domain in the Crescerin protein. Dan helped her learn how to work with worms, and designed and constructed some targeted mutants in the worm Crescerin gene che-12 to test specific hypotheses about its function.
Results of a collaboration between Dan and Hayretin Yumerefendi, a postdoc in Brian Kuhlman's lab. Hayretin engineered a small protein tag that localizes to the cytoplasm in the dark but translocates reversibly to the nucleus on stimulation with blue light. Dan designed and carried experiments to test whether this switch can be used to control transcription factor activity in vivo (spoiler alert - it can).
Reports a strategy for modifying the C. elegans genome, which was designed and optimized to minimize hands-on labor. The key innovation was a new self-excising cassette (SEC) for drug selection. The SEC works in a wild-type background, produces a visible phenotype to facilitate mutant isolation, and can be removed after use by simply heat shocking the worms. The SEC strategy is particularly effective for fluorescent protein tagging, but can also be used to produce point mutations.
Reports that the CRISPR/Cas9 system can be used to trigger homologous recombination in C. elegans. By supplying an appropriate homologous repair template, a variety of designer mutations were produced, including endogenous GFP fusions and targeted phosphorylation site mutations. This was one of the first papers reporting that CRISPR could be used in C. elegans.
Presents a new hypothesis for the evolution of metazoan multicellularity from unicellular ancestors. Some modern organisms, including Dictyostelium and some fungi, are "facultative" multicellular organisms - that is, they become multicellular only under certain conditions. Based on similarities between epithelial tissues in Dicty and animals, it is proposed that this facultative mode of multicellularity may be ancestral.
A philosophical essay that argues for a more experimental approach towards understanding protein evolution that considers protein function in the context of a cell. Read carefully and you'll see some early traces of the ideas that fueled Dan's postdoctoral work.
Reports the identification of a polarized epithelial tissue during multicellular development of the social amoeba Dictyostelium. Epithelial cells are organized into a tube at the tip of the developing fruiting body, where they secrete cellulose and ECM proteins that surround the stalk. A newly-identified alpha-catenin homolog, together with a previously-identified beta-catenin homolog, are essential for normal epithelial organization and polarity, suggesting that aspect of animal multicellularity may have been present in the common ancestor of Dicty and animals.