Dickinson Lab

From The Dickinson Lab @ UT Austin

Non-invasive chimeric HaloTag labeling to study clustering and diffusion of membrane proteins

Chang Y, Dickinson DJ.  (2022)

STAR Protocols 3(4): 101857

PubMed  Full Text 

In our Cell Reports paper published earlier this year, we showed how we could use dual-colored labeling of HaloTag to simultaneously estimate the sizes of PAR-3 clusters and track their diffusion on the cortex. Here we provided a protocol for this dual-labeling strategy, which has potential applications for many proteins beyond PAR-3.

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Shi Y, Kopparapu N, Ohler L, Dickinson DJ. (2022)

bioRxiv 2022.10.10.511307


Endogenous gene tagging has been a cornerstone of our experimental toolkit since 2013, when Dan developed the first fluorescent protein knock-in protocol for C. elegans. A decade later, we’ve extended this strategy to a new model system: mouse embryonic stem cells (mESCs). Although the techniques required for gene tagging in mESCs are different from C. elegans, the outcome is the same: We can perform live imaging of endogenous proteins – and coming soon, single-cell biochemistry! – without overexpression artifacts.

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

Chang Y, Dickinson DJ.  (2022)

Cell Reports 39(2): 110652

PubMed  Full Text  bioRxiv

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 addressed 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.

Rapid extraction and kinetic analysis of protein complexes from single cells

Sarıkaya S, Dickinson DJ. (2021)

Biophysical Journal 120(22):5018-31.

PubMed  Full Text  bioRxiv

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. 

Single-cell single-molecule pull down (sc-SiMPull) for detection of protein complexes from embryonic lysates

Stolpner N, Dickinson DJ. (2022)

Methods in Mol. Biol. 2438: 59-81.

PubMed  Full Text  

Presents a protocol for performing sc-SiMPull using mechanical lysis of C. elegans embryos, as first developed in Dan’s 2017 Developmental Cell paper. See later papers from our group for updates and improvements.

Electron microscopy snapshots of single particles from single cells

Yi X, Verbeke EJ, Chang Y, Dickinson DJ*, Taylor DW*. (2019)

J. Biol. Chem 294(5): 1602-08.

Pubmed   Full Text

Reports that  single-embryo 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.

From our “Glow Worms” Freshman Research stream

Highly improved cloning efficiency for plasmid-based CRISPR knock-in in C. elegans

DeMott E, Dickinson DJ, Doonan, R.  (2021)

microPublication Biology 10.17912/micropub.biology.000499.

Cloning is often a rate-limiting step in molecular biology research, and genome editing is no exception.  Our team extensively optimized the DNA concentrations and conditions for assembling CRISPR/Cas9 targeting vectors and repair templates for C. elegans, resulting in nearly 100% efficiency. 

Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans

Huang G, de Jesus B, Koh A, Blanco S, Rettmann A, DeMott E, Sylvester M, Ren C, Meng C, Waterland S, Rhodes A, Alicea P,  Flynn A, Dickinson DJ, Doonan, R.  (2021)

microPublication Biology 10.17912/micropub.biology.000460.

The Self-excising cassette is a selectable marker, invented by Dan in 2015, that enables streamlined and robust generation of genome-edited C. elegans strains.  Our team of freshman researchers, led by Assistant Professor of Practice and staff scientist Ryan Doonan, has extensively optimized this protocol and achieved a more than 10-fold increase in efficiency compared to our original procedure.

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. (2021)

Genetics 217(3): iyab006.

Pubmed   Full Text

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

Pubmed  Full Text

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.
Pubmed  Full Text

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.

SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette

Dickinson DJ, Slabodnick MM, Chen AH, Goldstein B. (2018). microPublication Biology 10.17912/W2KTON
Pubmed  Full Text

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.
Pubmed  Full Text

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.
Pubmed  Full Text

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.
Pubmed  Full Text

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. 

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

Heppert JK, Dickinson DJ, Pani AM, Higgins CD, Steward A. et al. (2016). Molecular Biology of the Cell 27(22): 3385-94.
Pubmed  Full Text

Quantitative head-to-head comparison of our favorite green and red fluorescent proteins for imaging C. elegans embryos.

MRCK-1 drives apical constriction in C. elegans by linking developmental patterning to force generation

Marston DJ, Higgins CD, Peters KA, Cupp TD, Dickinson DJ et al. (2016). Current Biology 26(16): 2079-89.
Pubmed  Full Text

CRISPR-based methods for genome engineering

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

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

Identifying regulators of morphogenesis common to vertebrate neural tube closure and Caenorhabditis elegans gastrulation

Sullivan-Brown J, Tandon P, Bird KE, Dickinson DJ, Heppert JK et al. (2016) Genetics 202(1): 123-39.
Pubmed  Full Text

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.
Pubmed  Full Text

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
Pubmed  Full Text

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. 
Pubmed  Full Text

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. 
Pubmed  Full Text

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. 

From Dan’s Ph.D.

Nelson and Weis labs @ Stanford

Measuring Protein Binding to F-actin by Co-sedimentation. 

Heier JA, Dickinson DJ, Kwiatkowski AV. (2017). Journal of Visualized Experiments. (123). doi: 10.3791/55613.

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.

Alpha-catenin and IQGAP regulate myosin localization to control epithelial tube morphogenesis in Dictyostelium.

Dickinson DJ, Robinson DN, Nelson WJ, Weis WI. (2012). Developmental Cell 23: 533-46. 

Identifies a link between alpha-catenin, IQGAP and myosin during multicellular development of Dictyostelium. Alpha-catenin recruits IQGAP to the basolateral membrane of Dicty epithelial cells, and IQGAP in turn acts to exclude myosin from the basolateral cortex. This results in apical actomyosin enrichment, which controls the diameter of the epithelial tube and thereby controls the shape of the entire fruiting body. 

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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