From The Dickinson Lab @ UT Austin

Rapid extraction and kinetic analysis of protein complexes from single cells

Sarıkaya S, Dickinson DJ. (2021)

bioRxiv 2021.07.07.451367; doi: 10.1101/2021.07.07.451367

Our 2017 method for single-cell biochemistry was a powerful approach for measuring protein-protein interactions in vivo. But an important limitation was that the time from (manual) cell lysis to data collection was 3-5 minutes – too slow for protein complexes that have weaker affinity. We found that we could use a pulsed infrared laser to lyse cells and then begin collecting data immediately, allowing us to measure the kinetics of protein complex dissociation. Laser lysis also improves reproducibility and allows us to document the stage at which each cell or embryo is lysed. 

A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization

Chang Y, Dickinson DJ.  (2021)

bioRxiv 2021.06.07.447386;  doi: 10.1101/2021.06.07.447386

PAR-3 oligomers are essential for C. elegans zygote polarization, but since oligomers have a range of sizes, it was difficult to determine at the biophysical level how different-sized oligomers contribute to polarity. Ivy address this challenge by engineering PAR-3 oligomers of defined sizes and quantitatively analyzing their diffusion. She found that oligomers as small as a trimer undergo directed motion due to cortical flow and can support cell polarization. She also verified this by developing an innovative dual-labeling strategy that allows simultaneous tracking and size measurement of endogenous PAR-3.

Electron microscopy snapshots of single particles from single cells.

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.

Work from others that we've contributed to

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

Ashley GE, Duong T, Levenson MT, Martinez MAQ,  Johnson LC, Hibshman JD, Saeger HN, Palmisano NJ, Doonan R, Martinez-Mendez R, Davidson BR,  Zhang W, Ragle JM, Medwig-Kinney TN, Sirota SS,  Goldstein B, Matus DQ, Dickinson DJ, Reiner DJ, Ward JD. Genetics, Volume 217, Issue 3, March 2021, iyab006,

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.

From Dan's Postdoc

Goldstein Lab @ UNC Chapel Hill

Optogenetic dissection of mitotic spindle positioning in vivo. 

Fielmich LE, Schmidt R, Dickinson DJ, Goldstein B, Akhmanova A & van den Heuvel S. (2018). eLife e38198.

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.

A single-cell biochemistry approach reveals PAR complex dynamics during cell polarization.

Dickinson DJ*, Schwager F, Pintard L, Gotta M, Goldstein B. (2017). Developmental Cell 42(4): 416-34.

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. 

CRISPR-based methods for genome engineering.

Dickinson DJ, Goldstein B. (2016). Genetics 202(3): 885-901.

Comprehensive review of CRISPR methods for C. elegans, including (still current!) recommendations for which strategies are most appropriate for which kinds of modifications.

Crescerin uses a TOG domain array to regulate microtubules in the primary cilium.

Das A, Dickinson DJ, Wood C, Goldstein B, Slep KC. (2015). Molecular Biology of the Cell 26(23): 4248-64.

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. 

Control of protein activity and cell fate specification via light-mediated nuclear translocation.

Yumerefendi H‡, Dickinson DJ‡, Wang H, Zimmerman SP, Bear JE et al. (2015). PLoS One 10(6): e0128443.

‡Equal Contribution

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). 

Streamlined genome engineering with a self-excising drug selection cassette.

Dickinson DJ*, Pani AM, Heppert JK, Higgins CD, Goldstein B. (2015). Genetics 200(4): 1035-49. 

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. 

Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.

Dickinson DJ*, Ward JD, Reiner DJ, Goldstein B. (2013). Nature Methods 10: 1028-34. 

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. 

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From Dan's Ph.D.

Nelson and Weis labs @ Stanford

An epithelial tissue in Dictyostelium challenges the traditional origin of metazoan multicellularity.

Dickinson DJ*, Nelson WJ, Weis WI. (2012). Bioessays 34: 833-40.

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.

Protein evolution in cell and tissue development: Going beyond sequence and transcriptional analysis.

Dickinson DJ, Weis WI, Nelson WJ. (2011). Developmental Cell 21: 32-34. 

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. 

A polarized epithelium organized by beta- and alpha-catenin predates cadherin and metazoan origins.

Dickinson DJ, Nelson WJ, Weis WI. (2011). Science 331: 1336-39.

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. 

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