Weaver, Benjamin et al. (2015) International Worm Meeting "Beyond Cell Death: Comprehensive Analyses of Non-apoptotic CED-3 Caspase Functions."

Weaver, Benjamin, Weaver, Yi, Han, Min

[International Worm Meeting, 2015]

Recent findings from several labs, including ours, have implicated important non-apoptotic functions for the canonical apoptotic regulatory pathway in C. elegans. We have recently reported a miRISC enhancer screen that identified 126 genetic interactors. We further carried out extensive analyses of one of them, the CED-3 caspase previously known for its essential role in apoptosis (Weaver, et al. 2014). Our results indicated that a non-apoptotic caspase activity of CED-3 negatively regulates the expression of LIN-28 and likely also LIN-14 and DISL-2 (ribonuclease) in the LIN-28 pluripotency pathway in late larval development to support proper cell fate patterning. Following these findings, we have carried out two sets of experiments to address two important questions related to this non-apoptotic function of CED-3. First, we examined whether CED-3 cleavage renders the target protein more susceptible to other proteolysis pathways. Our data show that compromising the N-end rule ubiquitin (NRU) pathway delays the downregulation of LIN-28 protein and enhances the miRISC developmental timing defect including the appearance of supernumerary seam cells by early adulthood similar to our findings for CED-3 and miRISC. However, compromising the NRU pathway does not enhance ced-3(lf) phenotypes, suggesting that the NRU pathway may act with CED-3 cleavage to repress the expression of LIN-28 and possibly other factors in the heterochronic pathway. Second, to systematically investigate the extent of non-apoptotic functions for the CED-3 caspase beyond temporal cell fate patterning, we have performed a ced-3(lf) enhancer screen using a genome-wide RNAi approach. We identified over 500 candidate genes in the primary screen and confirmed more than 100 of these in a secondary screen. In combination with ced-3(lf), RNAi of the candidate genes resulted in pleiotropic developmental defects. These genes encode proteins of diverse functional categories including protein stability regulators, signal transduction factors, RNA-binding proteins and transcriptional regulators, thus revealing the involvement of CED-3 caspase in a wide-range of non-apoptotic developmental regulatory pathways. Additional network and pathway analyses have implicated several proteins that are potential common targets of CED-3 and specific regulators identified in the above ced-3(lf) enhancer screen. CED-3 cleavage assays, expression analyses and genetic analyses are being performed to characterize the interaction network and its functional significance. Altogether, our data suggest that the CED-3 caspase is widely used in non-apoptotic cellular processes to enhance the robustness of dynamic gene expression patterns of diverse regulatory pathways.

Podbilewicz B (1994) Worm Breeder's Gazette "What Snakes and Worms Have In Common: Disintegrins"

Podbilewicz B

[Worm Breeder's Gazette, 1994]

WHAT SNAKES AND WORMS HAVE IN COMMON: DISINTEGRINS Benjamin Podbilewicz, MRC Laboratory of Molecular Biology, Cambridge, UK

Yuan, Wang et al. (2021) International Worm Meeting "Wide-Spread Non-Canonical CED-3 Caspase Activities Regulate Gene Expression Dynamics Including Antagonizing PMK-1 p38 MAPK Stress-Priming Function to Support Development"

Yuan, Wang, Weaver, Benjamin, Weaver, Yi

[International Worm Meeting, 2021]

Recent studies across diverse animal phyla have revealed non-canonical activities of apoptotic caspases involving specific modulation of gene expression, such as our finding of LIN-28 inactivation in worms and others findings of Nanog and Pax7 inactivation in mice to limit pluripotency of stem-like cell types to promote differentiation. We still do not know how broadly these non-canonical caspase functions span across various biological processes or their underlying molecular mechanisms. Using a forward genetic screen, we identified the CED-3 caspase working in diverse biological processes in C. elegans. Specifically, we recently showed that CED-3 caspase negatively regulates an epidermal p38 stress-responsive MAPK pathway to promote development. We also showed that PMK-1 (p38 MAPK) functions to prime animals for adverse environmental conditions prior to experiencing the stress. However, this priming function comes with the cost of retarding post-embryonic development. CED-3 caspase counters this stress-priming function by inactivating PMK-1 through proteolytic cleavage thereby promoting development. Strikingly, using RNA-seq and ribosomal profiling, we find that CED-3 and PMK-1 inversely regulate a key sub-set of more than 300 genes. A key enrichment amongst these genes were stress-responsive and pathogen resistance factors. Now, our ongoing studies are working to identify the molecular and physiological mechanisms of CED-3 and PMK-1 antagonism in regulating cell signaling and gene expression dynamics during development and stress-response states.

Yarychkivska O et al. (2020) Dev Cell "Development or Disease: Caspases Balance Growth and Immunity in C.elegans."

Shaham S, Yarychkivska O

[Dev Cell, 2020]

Caspase proteases execute apoptosis but also function in development. In this issue of Developmental Cell, Weaver etal. report that C.elegans CED-3 caspase promotes animal growth through PMK-1/p38 kinase cleavage, and at the expense of pathogen and stress immunity, revealing an unexpected homeostatic relationship between development and disease.

Conradt B (2017) Dev Cell "Partners in Crime."

Conradt B

[Dev Cell, 2017]

Caspases have apoptotic and non-apoptotic functions, both of which depend on their abilities to cleave proteins at specific sites. What distinguishes apoptotic from non-apoptotic substrates has so far been unclear. In this issue of Developmental Cell, Weaver etal. (2017) now provide an answer to this crucial question.

Chimal-Monroy JDS et al. (2020) Int J Dev Biol "Cell fusion and fusogens - an interview with Benjamin Podbilewicz."

Escalante-Alcalde DM, Chimal-Monroy JDS

[Int J Dev Biol, 2020]

Cell fusion is a process in which cells unite their membranes and cytoplasm and is fundamental for sexual reproduction and embryonic development. Among the best-known cell fusion processes during animal development are fertilization, myoblast fusion, osteoclast generation, and vulva formation in Caenorhabditis elegans. Although it is involved in many other functions in unicellular and multicellular organisms, little is known about the mechanisms of cell fusion and the genes that code for the proteins participating in this process. Benjamin Podbilewicz has dedicated many years to understanding the process and mechanisms of cell fusion. In this interview, he spoke to us about how he began his studies of this process, his contributions to this exciting field, scientific ties with Ibero-America and his strategies for a well-balanced scientific/personal life.

Rocco V et al. (1996) Worm Breeder's Gazette "Cloning of the Rad51 gene in C. elegans"

Ederle S, La Volpe A, Valenzi M, Rocco V, De Martino A

[Worm Breeder's Gazette, 1996]

We are interested in the mechanisms of homologous recombination in the nematode C. elegans. In prokaryotes, the RecA protein has a pivotal role in homologous recombination, and in vitro it has been demonstrated to catalyse the transfer of a DNA strand on a homologous molecule. In different eukaryotes (as yeast, mouse, man, and others) structural homologues of the bacterial RecA protein have been found, suggesting a certain level of conservation in some recombination pathways among living organisms (Shinohara et al. 1993 Cell 4: 239). In yeast an ATP- dependent DNA binding activity and, more recently, a strand transfer activity of the Rad51 gene product have been shown, whereas for the other Rad51 homologues the strand transfer activity has not yet been demonstrated (Sung and Robberson 1995 Cell 82). Experimental evidences in yeast (Milne and Weaver 1993 Genes Dev. 7:1755) suggest that the Rad51 product acts in a multiprotein complex in eukaryotes.

Cram EJ (2014) Prog Mol Biol Transl Sci "Mechanotransduction in C. elegans morphogenesis and tissue function."

Cram EJ

[Prog Mol Biol Transl Sci, 2014]

Mechanobiology is an emerging field that investigates how living cells sense and respond to their physical surroundings. Recent interest in the field has been sparked by the finding that stem cells differentiate along different lineages based on the stiffness of the cell surroundings (Engler et al., 2006), and that metastatic behavior of cancer cells is strongly influenced by the mechanical properties of the surrounding tissue (Kumar and Weaver, 2009). Many questions remain about how cells convert mechanical information, such as viscosity, stiffness of the substrate, or stretch state of the cells, into the biochemical signals that control tissue function. Caenorhabditis elegans researchers are making significant contributions to the understanding of mechanotransduction in vivo. This review summarizes recent insights into the role of mechanical forces in morphogenesis and tissue function. Examples of mechanical regulation across length scales, from the single-celled zygote, to the intercellular coordination that enables cohesive tissue function, to the mechanical influences between tissues, are considered. The power of the C. elegans system as a gene discovery and in vivo quantitative bioimaging platform is enabling an important discoveries in this exciting field.

Uccelletti D et al. (2008) Mol Biol Cell "APY-1, a novel Caenorhabditis elegans apyrase involved in unfolded protein response signalling and stress responses."

Palleschi C, Alberti A, Farina F, Mancini P, Hirschberg CB, Uccelletti D, Pascoli A

[Mol Biol Cell, 2008]

Monitoring Editor: Benjamin Glick Protein glycosylation modulates a wide variety of intracellular events and dysfunction of the glycosylation pathway has been reported in a variety of human pathologies. Endo-apyrases have been suggested to have critical roles in protein glycosylation and sugar metabolism. However, deciphering the physiological relevance of Endo-apyrases activity has actually proved difficult, owing to their complexity and the functional redundancy within the family. We report here that an UDP/GDPase, homologous to the human apyrase Scan-1, is present in the membranes of Caenorhabditis elegans, encoded by the ORF F08C6.6 and hereinafter-named APY-1. We showed that ER stress induced by tunicamycin or high temperature resulted in increased transcription of apy-1. This increase was not observed in C.elegans mutants defective in ire-1 or atf-6, demonstrating the requirement of both ER stress sensors for up-regulation of apy-1. Depletion of APY-1 resulted in constitutively activated UPR. Defects in the pharynx, and impaired organization of thin fibers in muscle cells, were observed in adult worms depleted of APY-1. Some of the apy-1(RNAi) phenotypes are suggestive of premature aging, since these animals also showed accumulation of lipofuscin and reduced lifespan that was not dependent on the functioning of DAF-2, the receptor of the insulin/IGF-1 signaling pathway.

Larsen MK et al. (2006) Mol Biol Cell "MAA-1, a novel acyl-CoA-binding protein involved in endosomal vesicle transport in Caenorhabditis elegans."

Tuck S, Larsen MK, Faergeman NJ, Knudsen J

[Mol Biol Cell, 2006]

Monitoring Editor: Benjamin Glick The budding and fission of vesicles during membrane trafficking requires many proteins including those that coat the vesicles, adaptor proteins that recruit components of the coat, and small GTPases that initiate vesicle formation. In addition, vesicle formation in vitro is promoted by the hydrolysis of acyl-CoA lipid esters. The mechanisms by which these lipid esters are directed to the appropriate membranes in vivo, and what precisely their roles are in vesicle biogenesis is not yet understood. Here we present the first report on MAA-1, a novel membrane-associated member of the acyl-CoA-binding protein family. We show that in C. elegans, MAA-1 localizes to intracellular membrane organelles in the secretory and endocytic pathway, and that mutations in maa-1 reduce the rate of endosomal recycling. A lack of maa-1 activity causes a change in endosomal morphology. Whereas in wild type, many endosomal organelles have long tubular protrusions, loss of MAA-1 activity results in loss of the tubular domains, suggesting the maa-1 is required the generation or maintenance of these domains. Furthermore, we demonstrate that MAA-1 binds fatty acyl-CoA in vitro and that this ligand binding ability is important for its function in vivo. Our results are consistent with a role for MAA-1 in an acyl-CoA-dependent process during vesicle formation.