From The Dickinson Lab @ UT Austin
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.
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.
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, https://doi.org/10.1093/genetics/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.