Michael C Fitzgerald et al. (2001) International C. elegans Meeting "Role of MEX-3 in Patterning PAL-1 Expression"

Michael C Fitzgerald, Craig P Hunter

[International C. elegans Meeting, 2001]

PAL-1 is a caudal-like homeodomain protein that is first expressed at the 4-cell stage in the two posterior blastomeres. This spatial and temporal patterning of PAL-1 expression is dependent on the putative RNA binding protein MEX-3 and the pal-1 3' untranslated region (UTR). A large body of circumstantial evidence suggests that MEX-3 may repress pal-1 translation by binding to the pal-1 3' UTR. We are taking a biochemical approach to test this possibility directly. Current efforts focus on using reversible RNA-protein cross-linking, followed by immunoprecipitation of MEX-3, and RT-PCR analysis of the co-precipitated RNA. Positive results in this assay will be followed by experiments to investigate the mechanisms by which MEX-3 patterns PAL-1 expression.

Michael C Fitzgerald et al. (2005) International Worm Meeting "SID-1 dependent intercellular RNA transport: substrates, kinetics, and mechanism"

Michael C Fitzgerald, Craig P Hunter, Joseph D Shih

[International Worm Meeting, 2005]

RNA interference (RNAi) is a phenomenon in which introduction of double stranded RNA (dsRNA) triggers gene-specific post-transcriptional gene silencing. There is tremendous potential for RNAi in therapeutic applications because of its promise to silence specific genes without adverse side affects. However efficient delivery of dsRNA into target cells and tissues is crucial for the success of such therapies. In C. elegans, silencing readily spreads between cells and tissues, a process termed systemic RNAi. However, the mechanisms behind systemic RNAi are not yet understood. To address this problem, the Hunter lab conducted a screen using a C. elegans strain that visually differentiates systemic silencing from cell autonomous RNAi. Five different systemic interference defective (sid) mutants were identified that define three different pathways for RNA transport: intercellular transport (sid-1 dependent), dsRNA uptake from the environment (sid-2 dependent), and signal export or transport from the intestine to other tissues (sid-3 dependent).Recent work by the Hunter lab with transiently transfected Drosophila S2 cells indicates that SID-1, the protein predicted to be the channel responsible for transport of the RNAi silencing signal between cells, is a nucleic acid transporter capable of mediating the entry of long double-stranded nucleic acids into cells2. This process occurs on the seconds timescale for dsRNA and does not require ATP in S2 cells. Additional studies have revealed that although silencing efficiency is strongly dependent on dsRNA length, 100bp and longer dsRNA are transported with similar kinetics. Poisoning assays using a missense mutant SID-1 reveal that functional SID-1 is most likely a homomultimer. Further studies seek to place SID-1 in a defined transporter class (ligand gated, co-transporter ion, etc.) for future patch clamp studies.

Michael C Fitzgerald et al. (2004) East Coast Worm Meeting "Specificity of the C. elegans Putative Transmembrane Channel SID-1"

Craig P Hunter, Michael C Fitzgerald

[East Coast Worm Meeting, 2004]

SID-1 was identified in a genetic screen for mutants capable of cell autonomous RNAi but deficient for systemic RNAi (Winston et al., 2002). The systemic RNAi defect is likely due to the inability of cells lacking SID-1 to import double-stranded RNA (dsRNA) from neighboring cells. The nature of SID-1 activity was elucidated using a heterologous system whereby either wild-type SID-1 or, as a negative control, a missense mutant form of SID-1 was transiently expressed in Drosophila S2 cells (Feinberg & Hunter 2003). These investigations showed that SID-1 enables efficient RNAi by adding dsRNA to the media of transfected S2 cells (soaking RNAi) and uptake of labeled dsRNA into cells. Furthermore, long dsRNA was shown to be more effective than short dsRNA for SID-1 mediated soaking RNAi in S2 cells and for systemic RNAi in C. elegans . We are investigating the length dependence of silencing as well as substrate specificity of the SID-1 channel. We have shown that SID-1 is extremely efficient, enabling soaking RNAi with less than one molecule of dsRNA per transfected cell and will present evidence that transport is extremely rapid. We will also report the results of ongoing analyses of length-dependent transport and channel selectivity, which may have implications for the use of SID-1 as a tool for molecular biology. W. M. Winston, C. Molodowitch, C. P. Hunter, Science 295 , 2456-59 (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. E. H. Feinberg, C. P. Hunter, Science 301 , 1545-7 (2003). Transport of dsRNA into cells by the transmembrane protein SID-1.

Colonnello A et al. (2020) Neurotox Res "Correction to: Comparing the Neuroprotective Effects of Caffeic Acid in Rat Cortical Slices and Caenorhabditis elegans: Involvement of Nrf2 and SKN-1 Signaling Pathways."

Tunez I, Rubio-Lopez LC, Aguilera-Portillo G, Silva-Palacios A, Evaristo-Priego Y, Santamaria A, Galvan-Arzate S, Cortez-Nunez S, Aschner M, Rangel-Lopez E, Chen P, Colonnello A, Robles-Banuelos B, Garcia-Contreras R

[Neurotox Res, 2020]

One of the authors has incorrect family name spelling in the original article_. Michael Ashner should read as Michael Aschner.

Wilkinson HA et al. (1994) Worm Breeder's Gazette "Reciprocal Changes in Expression of lin-12 and lag-2 Prior to Commitment of Z1.ppp and Z4.aaa."

Greenwald IS, Wilkinson HA, Fitzgerald K

[Worm Breeder's Gazette, 1994]

Reciprocal changes in expression of lin-12 and lag-2 prior to commitment of Z1.ppp and Z4.aaa. Hilary A. Wilkinson*, Kevin Fitzgerald and Iva Greenwald, HHMI and Dept. of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; *Current address: Merck Research Laboratories, Rahway, NJ 07065

Hengartner MO et al. (1992) Worm Breeder's Gazette "unc-50, a Gene Required for Acetylcholine Receptor Function, Encodes an Unfamiliar Protein"

Horvitz HR, Tsung N, Hengartner MO, Lewis JA

[Worm Breeder's Gazette, 1992]

unc-50, a gene requiired for acetylcholine receptor function, encodes an unfamiliar protein Michael Hengartner, Nancy Tsung, Jim Lewist, and Bob Horvitz HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA. t Division of Life Sciences, U. of Texas at San Antonio, San Antonio, IX 78249

Holway AH et al. (2007) J Cell Biol "Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos"

Kim SH, Michael WM, La Volpe A, Holway AH

[J Cell Biol, 2007]

After publication of this manuscript, the authors noticed that two images (Fig. 4 B, top left and middle) had been duplicated from a previous publication (Holway, A.H., C. Hung, and W.M. Michael. 2005. Genetics. 169:14511460). Here we provide new images corresponding to these control experiments. The authors apologize for this oversight.

Zhou MH et al. (1996) Worm Breeder's Gazette "Liquid Culture of the Worms with a Fermentor"

Zhou MH, Yang H, Walthall WW

[Worm Breeder's Gazette, 1996]

The yield of C. elegans and their food (E.coli) are relatively low in the large liquid cultures growth (Koelle et al.,1994) and the whole growth process is tedious. Here we report the successful growth of C.elegans with a fermentor. The following protocol is based on the protocol by Michael Koelle and Tory Herman (1994) from Internet.

Michael WM (2017) MicroPubl Biol "Asymmetric distribution of cyb-3 in 4-cell stage embryos."

Michael WM

[MicroPubl Biol, 2017]

Early embryos were fixed and stained with Mab F2F4 (green), shown to recognize CYB-3 (Michael, 2016), and DAPI, to illuminate the DNA (blue). Either wild type or par-4 mutant embryos were examined, after 24-hour incubation at 25C (the non-permissive temperature for the it47 allele of par-4). Anterior is to the left in all images. The data presented here reveals previously not shown data that depicts CYB-3 as asymmetrically distributed at the 4-cell stage. These data further support reported findings in Michael, 2016. There is more CYB-3 in the AB cell relative to its sister P1. In 4-cell embryos there is more CYB-3 in the EMS cell relative to its sister, P2. Thus, during P-lineage divisions, CYB-3 is asymmetrically distributed such that the somatic precursor receives more than its germline precursor sister cell. This asymmetry is abolished in par-4 mutant embryos, where all blastomeres contain equivalent amounts of CYB-3.

Grooms, Noa et al. (2021) International Worm Meeting "Expression of trx-1 correlates with intrinsic regenerative capacity"

Urena, Samuel, Chung, Samuel, Grooms, Noa, Schulting, Leilani, Fitzgerald, Michael

[International Worm Meeting, 2021]

A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals. Lesion conditioning could be utilized to drive powerful therapies for neuroinjuries. Despite being studied for >30 years, lesion conditioned regeneration remains poorly understood, and progress is severely limited by low throughput in vertebrate models. To expedite research in the field, we are developing a model for lesion-conditioned regeneration in C. elegans. Our model employs green fluorescent protein (GFP) to label the ASJ neuron. When we condition the neuron, we see increased fluorescence in the ASJ, indicating a correlation between GFP expression and regenerative capacity. Meanwhile, following prior work from our laboratory, disruptions to the sensory pathway can also chronically condition the neuron, increasing regenerative capacity without the need of a conditioning lesion. We saw increased fluorescence in chronically conditioned strains, further supporting the correlation between GFP expression and regenerative capacity. We used ethyl methanesulfonate to stochastically introduce mutations into a chronically conditioned strain and selected for offspring with decreased ASJ fluorescence, indicating a mutation in a gene potentially in the conditioning pathway. We isolated twelve strains, originating from six distinct F1s. Six of these strains show reduced frequency of ectopic axon outgrowths compared to the pre-mutagenized strain. A reduction in ectopic outgrowths suggests a disruption in the conditioning pathway, as previously characterized by our laboratory. To quantify the fluorescent correlation, we imaged the mutagenized strains with calibrated fluorescent beads and compared fluorescent intensities of the ASJ neurons to strains with increased outgrowths and regenerative potential. We found significantly decreased brightness in cell bodies and dendrites in strains with reduced regenerative potential compared to chronically conditioned strains. This correlation provides a powerful proxy for evaluating regenerative capacity and to identify genes potentially implicated in the lesion conditioned pathway.