(2024) Development "The people behind the papers - Nadia Manzi and Daniel Dickinson."

[Development, 2024]

The mitotic kinase Aurora A has been shown to regulate the anterior-posterior polarity in developing Caenorhabditis elegans embryos. In a new study, Daniel Dickinson and colleagues find that Aurora A has temporally distinct roles in coordinating the localization of Partitioning defective (PAR) proteins to establish cell polarity during development. To find out more about the story behind the paper, we caught up with first author Nadia Manzi and corresponding author Daniel Dickinson, Assistant Professor at the University of Texas at Austin.

Huang, George et al. (2021) MicroPubl Biol

Rettmann, Aubrie, Waterland, Skye, Huang, George, DeMott, Ella, Sylvester, Melynda, Dickinson, Daniel J, Rhodes, Anita, Flynn, Abbey, Alicea, Persephone, Blanco, Sara, Ren, Cassie, Koh, Alex, Doonan, Ryan, de Jesus, Bailey, Meng, Carrie

[MicroPubl Biol, 2021]

The self-excising cassette (SEC) knock-in approach uses hygromycin selection and a visible roller phenotype to identify knock-ins, followed by a heat-shock induced excision of these visible markers to yield a seamless insertion of a fluorescent protein into the genome (Dickinson et al. 2015). Compared to protocols that utilize Cas9 protein and linear DNA repair templates (Paix et al. 2015; Dokshin et al. 2018; Ghanta and Mello 2020), the plasmid-based SEC approach employs a simpler screening strategy but requires more worms to be injected (Dickinson and Goldstein 2016).

Jinah Kim et al. (2001) International C. elegans Meeting "Identification and characterization of nic-1, a gene involved in regulation of the nAChR."

Kari Dickinson, Lisa Moore, William Schafer, Jinah Kim

[International C. elegans Meeting, 2001]

Long-term exposure to nicotine causes long-lasting changes in behavior which reflect in part changes in the abundance and activity of nicotinic acetylcholine receptors. The molecular mechanisms underlying this adaptation to nicotine are not well understood in any organism. To address these questions, we have been investigating nicotine adaptation in C. elegans . Acute treatment with nicotinic agonists such as nicotine and levamisole leads to body muscle hypercontraction and paralysis, increased pharyngeal pumping, and stimulation of egg laying under conditions inhibitory for egg laying. Long-term treatment with nicotine confers recovery from these effects, a process we have termed adaptation: worms regain movement, decrease their rate of pharyngeal pumping, and fail to be stimulated to lay eggs under inhibitory conditions. At least some of these behavioral changes (in particular those related to egg-laying) occur at least in part through downregulation of nicotinic receptor abundance. To identify molecules involved in the regulation of nicotinic receptor abundance or activity, we have conducted genetic screens for mutants that are hypersensitive or fail to adapt to nicotine. Among the genes identified in these screens is a new gene, nic-1 . nic-1 mutants are strongly nicotine hypersensitive: they undergo paralysis and are stimulated to lay eggs at doses of nicotine three-fold lower than wild type. In addition, these mutants exhibited phenotypes that support nic-1 's possible role in the regulation of cholinergic neurotransmission. For example, nic-1 animals were hyperactive for egg-laying and showed an egg-laying pattern reminiscent of wild-type animals treated with cholinergic agonists. Moreover, they displayed abnormalities in locomotion and male mating, two behaviors known to be controlled by cholinergic motor neurons. Experiments with UNC-29::GFP chimeras indicated that nic-1 mutants actually had reduced levels of the levamisole receptor in vulval muscles and normal levels in body muscles; thus, we believe the hypersensitivity of nic-1 animals reflects a change in receptor activity rather than abundance. To gain more information about the effect of nic-1 on nicotinic receptor activity, we are collaborating with Janet Richmond to obtain electrical recordings from the body muscle and egg-laying NMJs of nic-1 animals. We have also mapped nic-1 to an approximately a 100kb interval on LGX; thus, we are optimistic about the prospect of understanding the molecular identity of nic-1 very soon, and hope to report our findings at the meeting.

Munro E (2017) Dev Cell "Protein Clustering Shapes Polarity Protein Gradients."

Munro E

[Dev Cell, 2017]

In this issue of Developmental Cell, Dickinson etal. (2017) and Rodriguez etal. (2017), along with Wang etal. (2017) in Nature Cell Biology, show how PAR protein oligomerization can dynamically couple protein diffusion and transport by cortical flow to control kinase activity gradients and polarity in the C.elegans zygote.

Jinah Kim et al. (2002) West Coast Worm Meeting "nic-1 encodes a glycosyltransferase that affects nAChR activity"

Jinah Kim, William Schafer, Stuart Haslam, Kari Dickinson

[West Coast Worm Meeting, 2002]

Exposure to nicotinic agonists such as nicotine and levamisole leads to body muscle hypercontraction and paralysis, increased pharyngeal pumping, spicule protraction in males, and stimulation of egg laying under conditions inhibitory for egg laying. Long-term treatment with nicotine leads to recovery from these effects, a process we have termed adaptation: worms regain movement, decrease their rate of pharyngeal pumping, and fail to be stimulated to lay eggs under inhibitory conditions. The molecular mechanisms underlying these processes involving the nicotinic acetylcholine receptor are not well understood in any organism. To address this question, we have been investigating the mechanisms of nicotinic receptor regulation in C. elegans; one way we have been doing this is by searching for genes that confer resistance or hypersensitivity to the effects of nicotine.

DeMott, Ella et al. (2021) MicroPubl Biol

Doonan, Ryan, DeMott, Ella, Dickinson, Daniel J

[MicroPubl Biol, 2021]

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.

Chang Y et al. (2022) STAR Protoc "Non-invasive chimeric HaloTag labeling to study clustering and diffusion of membrane proteins."

Chang Y, Dickinson DJ

[STAR Protoc, 2022]

As live imaging plays an increasingly critical role in cell biology research, the desire to label and track individual protein molecules in vivo has been growing. To address this, in this protocol we describe steps for sparse labeling using two different HaloTag ligand dyes in C. elegans. This labeling approach is simple, is non-invasive, and preserves the view of the bulk protein population. We further describe how to carry out single-particle tracking experiments and extract information about particle diffusion behavior. For complete details on the use and execution of this protocol, please refer to Chang and Dickinson (2022).¹.

Otsuka AJ (1990) Worm Breeder's Gazette "ALTERATIONS IN UNC-44 ALLELES"

Otsuka AJ

[Worm Breeder's Gazette, 1990]

Preliminary analysis of six spontaneous unc-44 alleles indicates that alterations occur over a fairly large region in the genomic DNA. Southern analysis of two alleles from E. Hedgecock (rh1013 and rf1042) and four alleles (mn259, mn339, q331, and st200) from R. Herman and other labs (kindly provided by C. Kari, R. Herman, W. Li, and J. Shaw) indicated that restriction fragment differences from wild type are associated with all six alleles (see Franco et al., C. elegans Meeting abstracts, 1989). The construction of preliminary restriction maps of unc-44 cosmid (B0350 and C44A, kindly provided by A. Coulson and J. Sulston) and phage (DD#LRF1 and DD#LRF6) clones allows the approximate positioning of mutant changes. The absolute orientations of the cosmid and phage clones are based on positioning of these clones on the cosmid map. The overlapping of the cosmid and phage clones has not yet been demonstrated. Unfortunately, the cosmid isolate that could quickly clarify the situation (C02C3) deleted during growth. [See Figure 1]

Dickinson DJ et al. (2018) MicroPubl Biol "SapTrap assembly of repair templates for Cas9-triggered homologous recombination with a self-excising cassette"

Goldstein B, Dickinson DJ, Chen AH, Slabodnick MM

[MicroPubl Biol, 2018]

The advent of CRISPR/Cas9 technology in C. elegans has enabled an unprecedented level of control over this organisms genome, which is facilitating research in a wide variety of fields. Several different experimental approaches exist for CRISPR in this organism (Dickinson and Goldstein 2016). One particularly powerful approach uses a drug selection with a Self-Excising Cassette (SEC) to isolate the desired genetically-modified animals following Cas9-triggered homologous recombination (Dickinson et al. 2015). The most labor-intensive step in this procedure is the construction of a plasmid-based repair template for homologous recombination. SapTrap (Schwartz and Jorgensen 2016) is a high-throughput cloning procedure that allows modular assembly of repair templates for CRISPR/Cas9-triggered homologous recombination. The original publication that described SapTrap cloning (Schwartz and Jorgensen 2016) used unc-119(+) as a positive selection marker for isolating recombinant strains. Compared to unc-119 selection, SEC selection is faster; can be used directly in a wild-type background; and eliminates an extra injection step followed by outcrossing to remove the selection markers after strain isolation (Dickinson et al. 2015). Because of these advantages of SEC selection over unc-119, we wished to incorporate the SEC into the SapTrap workflow. Here, we describe a toolkit of plasmids that allow building SEC-containing repair templates via SapTrap cloning.In brief, SapTrap links pieces of a repair template construct together by means of unique 3-bp overlaps generated by the type II restriction enzyme SapI. Constructs generated using SapTrap have the general form shown in Figure 1A. In a typical assembly reaction, the sgRNA target is provided as a pair of oligos, the homology arms are provided as PCR products, and a Fluorescent Protein (FP), SEC and linker fragments are provided by pre-existing donor vectors. These fragments are mixed together, along with the requisite enzymes, and are assembled in a single reaction. The linkers can be omitted from the final construct if desired, by designing the homology arms with 3-bp overlaps that anneal directly to the FP or SEC instead of to the linker.Using SapTrap to generate repair templates for SEC selection involves two modifications to the original approach described by Schwartz and Jorgensen. First, an AAG junction is added between the FP and SEC fragments (Figure 1A). Together, these two fragments replace the FP/unc-119(+) donors used by the Jorgensen lab. Second, it is important to ensure that a coding exon follows SEC in order to avoid nonsense-mediated decay. For this reason, we always include the NT-tag linker in our constructs, even for C-terminal tags. We generated and tested fluorescent protein donors covering the current best available fluorescent proteins for C. elegans imaging (Heppert et al. 2016 and our unpublished observations) along with SEC donors containing three different Lox sites that do not recombine with each other, for multiplex editing. These constructs have been deposited at Addgene and are listed in Table 1.Using various combinations of fluorescent protein, SEC and linker donors, we were routinely able to obtain the desired repair templates, although the cloning efficiency was lower compared to the ccdB/Gibson-based cloning approach described by Dickinson et al. (2015) (Figure 1B). Note that, for both the Gibson and SapTrap data reported in Figure 1B, we used PCR products 500-700 bp in length as the homology arms, enabling a direct comparison of the two cloning methods. We have not tested short homology arms formed from annealed oligonucleotides, which were used by Schwartz and Jorgensen (2016), with either cloning approach. Based on our extensive experience with both SapTrap and the ccdB/Gibson-based cloning procedure, we believe that each procedure has advantages. The advantages of SapTrap are:SapTrap is more modular: different fluorophores, epitope tags, and selection cassettes can be freely combined without the need to construct a new FPSEC vector each time.SapTrap does not require the use of ccdB-containing vectors, which are toxic to E. coli and therefore can be difficult to grow and maintain.

Roy, Vincent et al. MicroPubl Biol

Gagne, Olivier, Hamiche, Karim, Narbonne, Patrick, Labbe, Jean-Claude, Roy, Vincent

[MicroPubl Biol, 2018]

PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al. 2011), and metabolism (Narbonne and Roy 2009). Its expression pattern and subcellular localization has been determined in fixed animals by antibody staining (Watts et al. 2000). Here, we used CRISPR/Cas9-mediated genome editing to fluorescently label the two longest endogenous PAR-4 isoforms, PAR-4A and PAR-4C, with monomeric Neon Green (mNG) (Shaner et al. 2013), using the self-excising cassette (SEC) system (Dickinson et al. 2015) (Fig. 1A). We found ubiquitous mNG::par-4a & c expression and cytoplasmic and cortical enrichment of mNG::PAR-4A & C proteins in the germ line and early embryos (Fig. 1B-H), as previously described (Watts et al. 2000). Interestingly, we find that mNG::PAR-4A & C cortical enrichment is transiently lost in the pachytene area of the germ line (Fig. 1F), although it remains unclear whether this is functionally relevant. The intermediate strain that still contains the SEC (Dickinson et al. 2015) is predicted to be null for par-4a & c, potentially leaving the shorter par-4b isoform functional. To evaluate the requirement for par-4a & c we examined the embryonic lethality (at 25C) of the generated SEC-containing and SEC-excised strains. We found that the self-progeny of homozygous SEC-containing (e.g. par-4a & c null) animals is viable (Fig. 1H). This suggests that par-4b alone is sufficient to establish embryonic polarity and sustain the essential function of par-4. Consistent with this, to our knowledge, all existing par-4 alleles that impair embryonic development disrupt par-4b (Morton et al. 1992; Watts et al. 2000).