Palmisano, Nicholas J. et al. (2013) International Worm Meeting "Screen for endocytic genes required for dauer development and autophagy."

Melendez, Alicia, Palmisano, Nicholas J., Jimenez, David

[International Worm Meeting, 2013]

Autophagy and endocytosis are two cellular pathways that are vital to proper development and tissue homeostasis. Autophagy is a dynamic and catabolic process used by eukaryotic cells, involving the de novo formation of a double-membrane vesicle called the autophagosome that engulfs long-lived proteins and organelles. The autophagosome then fuses with the lysosome forming an autolysosome, resulting in the degradation and recycling of the engulfed material. Endocytosis involves the uptake of extracellular material into the cell through the formation of intracellular vesicles termed endosomes. Crosstalk between endocytosis and autophagy is evident by multiple findings. Autophagosomes can directly fuse with lysosomes, or can initially fuse with endosomes to form hybrid organelles called amphisomes, which then fuse with lysosomes. Morphological studies in mammals show that amphisomes consist of early autophagosomes and late endosomes/multivesicular bodies (MVB). Additionally, loss of the Endosomal Sorting Complex Required for Transport (ESCRT), a series of protein complexes required for MVB formation, results in the accumulation of autophagosomes. To further study the relationship between autophagy and endocytosis and identify endocytic genes which function in autophagy, we utilized daf-2(e1370) mutants expressing a transgene with an autophagy marker, GFP::LGG-1. In C. elegans, daf-2 encodes the insulin-like receptor and daf-2(e1370) mutants are dauer constitutive at 25 deg C, long lived at the 15 deg C, and have increased autophagy levels in both. Furthermore, defects in both autophagy and endocytosis affect the longevity phenotype of daf-2(e1370) mutants. Previous data showed that knockdown of autophagy genes in daf-2(e1370) mutants results in defects in normal dauer morphology, increased global fluorescent intensity of GFP::LGG-1, and the formation of GFP::LGG-1 positive aggregates. Therefore, to identify endocytic genes that may affect autophagy, we have performed a screen for endocytic genes that when knocked down by RNAi, result in the lack of dauer phenotypes and presence of GFP::LGG-1 aggregates. Here, we present data that identify several endocytic genes important for autophagy function and dauer development.

Palmisano, Nicholas J et al. (2011) International Worm Meeting "Molecular and genetic characterization of suppressor of bec-1 lethality (sub) mutants."

Palmisano, Nicholas J, Melendez, Alicia

[International Worm Meeting, 2011]

Autophagy is the major intracellular pathway for the degradation and recycling of long-lived proteins and organelles and is thus crucial for cell homeostasis. It is induced by stress, over-crowding, or starvation conditions. C. elegans bec-1 is the ortholog of beclin1/ATG6/VPS30 in mammals and yeast, an important regulator of autophagy. Yeast Atg6/Vps30p was identified in a genetic screen for mutants that are starvation sensitive or defective in vacuolar protein sorting. In mammals, beclin 1 has been shown to be a haploinsufficient tumor suppressor gene. Human Beclin 1 was identified as a protein interacting with the anti-apoptotic protein Bcl-2 in a yeast two-hybrid assay and is monoallelically deleted in up to 75% of various human cancers. Therefore, insights on the function of bec-1 in C. elegans will likely shed light on its role in tumorigenesis in humans. We and others have found that bec-1 functions in various biological processes, including survival, longevity, fat accumulation, dauer and reproductive development. In addition, we have recently shown that bec-1 is important for the proper localization of MIG-14 to the Golgi network and is required for germ cell corpse clearance, implicating a role for bec-1 in phagosome maturation. Thus, bec-1 serves a role in both autophagy and endocytosis. Complete loss of bec-1 results in lethality, therefore highlighting the importance of bec-1 in development. Whereas many functions and interactions of Atg6/Beclin 1/BEC-1 have been described, little is known about the genes that regulate or act downstream of Atg6/Beclin 1/BEC-1 during development. A complete loss of function mutation of C. elegans bec-1(ok691) is essential for viability. We have isolated two dominant suppressor mutations that suppress the bec-1 lethal phenotype, and named these mutants sub for suppressor of bec-1 lethality. Importantly, both sub mutations suppress all bec-1 loss of function phenotypes tested, including the decrease in fat accumulation in bec-1 homozygous mutant animals, as well as the shortening of lifespan associated with heterozygous bec-1 mutants. Using whole genome sequencing combined with SNP mapping, we have found various candidate mutations for sub-1 and sub-2. Since both mutations act dominantly in their suppression for bec-1, they may represent either gain of function or haploinsufficient mutations. We will report on the molecular and genetic characterization of the sub mutations. We hypothesize that these suppressor mutations represent novel loci that may regulate bec-1 mediated activity. Such novel loci would further our knowledge of BEC-1 and its role in development as well as provide new therapeutic targets for cancer treatment and age-related diseases.

Palmisano, Nicholas et al. (2021) International Worm Meeting "The conserved histone deacetylase, HDA-1, functions in cell cycle-dependent and independent roles to promote invasive differentiation"

Medwig-Kinney, Taylor, Palmisano, Nicholas, Saldanha, Joanne, Moore, Frances E. Q., Matus, David Q., Smith, Jayson, Martinez, Michael A. Q., Zhang, Wan, Sirota, Sydney S.

[International Worm Meeting, 2021]

Cell invasion occurs naturally during development; however, it also contributes to tumor progression. Although understudied, data from different cancer subtypes suggests that a dichotomy exists between invasive and proliferative behavior. Our lab uses the C. elegans anchor cell (AC) to study this proliferative-invasive switch during uterine-vulval development. Post-embryonically, the AC invades into the vulval epithelium to form the mature vulva. We have shown that the AC exists in G0/G1-cell cycle arrest during invasion, which requires the nuclear hormone transcription factor, nhr-67 (tailless/Tlx). Loss of nhr-67 results in mitotic, non-invasive ACs. nhr-67 maintains AC arrest by regulating the expression of the cyclin-dependent kinase inhibitor, cki-1 (p21/p27). Induced expression of CKI-1 is sufficient to restore invasion in an nhr-67-depleted background, suggesting that the G1/G0 state is required for invasive activity. Differentiation of invasive behavior is also regulated by chromatin modifiers that act downstream and/or parallel to G0/G1 arrest. We have found that the histone deacetylase, hda-1, promotes AC invasion by positively regulating pro-invasive gene expression; however, whether hda-1 regulates invasion by controlling cell cycle arrest is unknown. We and others have recently shown that key pro-invasive transcription factors, nhr-67/Tlx, hlh-2/E, fos-1a/Fos, and egl-43/Evi1, maintain the AC in a post-mitotic, pro-invasive state by acting as part of gene regulatory network composed of two subcircuits. nhr-67, egl-43, and hlh-2 function in a type 1 coherent loop with positive feedback to maintain the AC in a post-mitotic state and facilitate invasion, while fos-1 acts in a cell cycle-independent subcircuit to promote invasion. Using genetic approaches, CRISPR-Cas9 genome engineering and high-resolution imaging, we explored the regulatory relationships between HDA-1 and these key transcription factors. We find that hda-1 functions in the AC to maintain G1/G0 arrest, and that loss and depletion of hda-1 results in mitotic, non-invasive ACs. We show that in the AC, hda-1 regulates the activity of NHR-67, FOS-1a, and HLH-2. Interestingly, induced expression of CKI-1 fails to restore invasion in hda-1-deficient ACs, but is able to rescue the mitotic defect of hda-1-depleted animals. These results suggest that hda-1 functions in both cell cycle-dependent and independent subcircuits to maintain the post-mitotic, pro-invasive state of the AC.

Medwig-Kinney, Taylor et al. (2021) International Worm Meeting "Coordinating proliferative and invasive cellular fates: insights from C. elegans somatic gonad development"

Tank, Sujata, Moore, Frances, Palmisano, Nicholas, Somineni, Neha, Sirota, Sydney, Martinez, Michael, Matus, David, Saldanha, Joanne, Medwig-Kinney, Taylor

[International Worm Meeting, 2021]

The decision between proliferation and invasive differentiation is a key part of several developmental events as well as cancer; however, how cells switch between these states is not well understood. Two cell types in the C. elegans somatic gonad, the anchor cell (AC) and ventral uterine (VU) cells, both specified from initially equipotent cells through a Notch-mediated cell fate decision, serve as an excellent model of this dichotomy in cellular behavior. The post-mitotic AC, the default state of the AC/VU decision, goes on to invade the underlying basement membrane during development of the reproductive system, while the VUs remain proliferative. We have previously shown that four transcription factors, fos-1 (Fos), egl-43 (EVI1/MEL), hlh-2 (E/Daughterless), and nhr-67 (NR2E1/Tailless/TLX), are necessary for AC invasion, with the latter three playing a role in regulating cell cycle arrest. We next examined whether any of these transcription factors are sufficient for invasion and find that ectopic expression of NHR-67, which is enriched in the AC compared to its neighbors, results in transdifferentiation of VUs into invasive ACs. We hypothesize that post-translational degradation of HLH-2 in the VU results in lower levels of NHR-67 and that a transcriptional repressive mechanism functions in the VU to prevent remaining NHR-67 activity from inappropriately activating AC-specific targets. We find that knockdown of unc-37 and lsy-22, homologs of the transcriptional repressor Groucho, results in a low penetrance of animals with multiple ACs. Others have previously demonstrated that Groucho forms a repressive complex with histone deacetylase (hda-1) and TCF/LEF (pop-1) to restrict endoderm differentiation in the early embryo. Unlike hda-1 or Groucho, which are not differentially expressed between the two cell types, pop-1 is enriched in the VUs compared to the AC, but does not appear to activate transcription based on quantification of a transgenic POP-1 activity reporter. Furthermore, we find that depletion of the pro-invasive TFs associated with cell cycle arrest results in ectopic expression of pop-1 in the AC. Here, we investigate whether differential pop-1 levels are necessary for AC/VU fate maintenance as well as whether this process is downstream or synergistic with Notch signaling and/or cell cycle dynamics. Together, these results provide new insights into how cells can establish and maintain cellular programs necessary for growth and morphogenesis.

Delia M O'Rourke et al. (2005) International Worm Meeting "Analysis of gene expression changes in C. elegans following infection with Microbacterium nematophilum."

Delia M O'Rourke, Maria Demidova, Jonathan Hodgkin, Richard Mott, Dilair Baban

[International Worm Meeting, 2005]

Microbacterium nematophilum is a bacterial pathogen of C. elegans (Hodgkin et al, 2000) that adheres to the rectum and post-anal region of the worm causing swelling of the underlying hypodermal tissue, mild constipation and slow growth rates. Affected worms are described as having a Dar (Deformed Anal Region) phenotype. C. elegans responds to this infection in part through activation of an ERK MAP kinase cascade which mediates tail swelling and prevents severe constipation (Nicholas and Hodgkin, 2004). To identify the downstream transcriptional changes that occur in C. elegans during this host/pathogen interaction we have conducted a genome wide analysis of gene expression using Affymetrix gene chips. We infected a synchronised population of larval C. elegans in liquid culture for 6hrs before harvesting the worms and extracting RNA. Our control sample was an identical experiment using an avirulent M. nematophilum generated in our laboratory by Tanya Akimkina and Steve Curnock. Analysis of data from triplicate microarray experiments has identified a number of statistically significant gene clusters whose expression changes upon infection. Clusters containing up-regulated genes are located on chromosomes IV and V and include C-type lectins, lysozyme-like proteins and proteins containing metridin-like ShK toxin and DUF141 domains. An RNAi feeding screen of these and other induced genes has identified a number that affect the Dar response. The microarray data suggest that although similar functional domains are found in proteins induced by both M. nematophilum and other pathogens, the response of C. elegans to M. nematophilum is specific. Hodgkin, J. Kuwabara, P. E. Corneliussen, B. Current Biology 200010 ;1615-1618 Nicholas, HR, Hodgkin, J. Current Biology 2004 14 ;1256-1261

Partridge, Frederick A. et al. (2009) International Worm Meeting "Investigation of the mechanism of infection induced tail swelling."

Partridge, Frederick A., Hodgkin, Jonathan

[International Worm Meeting, 2009]

When C. elegans and certain other rhabditid nematodes are infected by various bacterial pathogens, notably the coryneform Microbacterium nematophilum, they respond by altering the development of the hindgut, producing a swollen tail. This is a protective innate immune response [1]. Our lab has previously shown that tail swelling involves activation of the ERK MAPK cascade in the rectal epithelial cells. The mechanism of tail swelling has been hard to address in genetic screens as mutants that abolish infectability are much more common than mutants affecting the response to infection. To avoid this problem we have stably expressed activated MAP kinase pathway genes in the hindgut, which phenocopies tail swelling in the absence of infection. This has allowed us to screen for modifiers of tail swelling without the confounding effects of susceptibility to infection. Results of RNAi and forward genetic screens will be presented. [1] Nicholas and Hodgkin (2004) Curr Biol 14 1256-61.

Statzer, C.A. et al. (2017) International Worm Meeting "Assessing collagen levels during aging and longevity."

Statzer, C.A., Ewald, C.Y.

[International Worm Meeting, 2017]

Collagens are a major component of the extracellular matrix (ECM) and account for 30% of the total human protein mass. As we grow older our collagen mass declines 1% per year in our skin. This decrease is likely due to damage and degradation of collagen as well as transcriptional down-regulation of collagen expression with advanced age. Here, we determine the total collagen content and its decline during aging in C. elegans. We exploit the common post-translation modification of collagen fibers, which contain a hydroxylated form of the amino acid proline. The hydroxylation stabilizes the helical fold of collagens and can be utilized to specifically quantify their abundance. Interestingly, long-lived C. elegans show higher collagen protein levels compared to wild type. We are using publicly available expression profiles to identify collagens that show a progressive decline in mRNA expression levels during aging in wild type, but an upregulation in long-lived animals. We are employing the lifespan machine as developed by Dr. Nicholas Stroustrup to assess the requirements for these candidate collagens for longevity. Taken together, this work aims to elucidate how core ECM components are differentially regulated in normal and long-lived C.elegans and how the ECM itself is modulating the lifespan of an organism.

Coleman-Hulbert, A.L. et al. (2017) International Worm Meeting "Automation of the Caenorhabditis Intervention Testing Program."

Garret, T., Fulger, A.C., Lithgow, G.J., Johnson, E., Kish, J.L., Banse, S., Sedore, C.A., Presley, M.P., Driscoll, M., Harinath, G., Phillips, P.C., Lucanic, M., Falkowski, R., Chen, E., Blue, B.W., Crist, A.B., Hall, D., Coleman-Hulbert, A.L., Plummer, T.W.

[International Worm Meeting, 2017]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional consortium supported by the National Institutes of Aging and tasked with identifying robust life-extending compounds using nematodes from the genus Caenorhabditis. CITP partners at Rutgers University, the University of Oregon, and the Buck Institute for Aging Research have streamlined efforts and reproduced findings from 22 diverse strains of Caenorhabditis under ten separate compound interventions. Our work highlights the necessity of replication of longevity analysis, and our intention is to greatly expand the scale of our assays in order to keep up with demand from the aging research community. The C. elegans Lifespan Machine developed by Nicholas Stroustrup (Fontana Lab, Harvard) holds a great deal of promise as a high-throughput longevity approach. The C. elegans Lifespan Machine uses typical office scanners modified to hold petri plates containing nematodes. Through hourly image capture and later image processing, the Lifespan Machine automates the process of gathering lifespan data while providing tight temporal scaling. Here, we compare manual and automated lifespan measurements for multiple compounds across dozens of strains within each of the three laboratories. Overall, there are distinct differences in phenotype between manual and automated approaches for some strains and, especially, for some compounds. Lab to lab variation can also be a challenge. Nevertheless, the automated approach provides a great deal of value to the workflow of the CITP, and we plan to incorporate it into our future work, coupled with verification via manual lifespan assays.

Smith, Jayson et al. (2021) International Worm Meeting "The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo"

Chondhok Delos Reyes, Mana, Kratsios, Paschalis, Moore, Frances, Smith, Jayson, Martinez, Michael, Adikes, Rebecca, Zhang, Wan, Wen, Kailong, Xiao, Yutong, Kohrman, Abraham, Liu, Simeiyun, Matus, David, Palmisano, Nicholas, Bracht, Sydney, Parsan, Nithin, Medwig-Kinney, Taylor

[International Worm Meeting, 2021]

Chromatin remodeling complexes, such as the SWItching defective/Sucrose Non-Fermenting (SWI/SNF) ATP-dependent chromatin remodeling complex, coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors (CRFs) are associated with a variety of diseases including cancer metastasis, which co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and CRFs that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and RNA interference (RNAi), we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Specifically, we implicate the SWI/SNF BAF assembly in the regulation of the cell cycle, whereas our data suggests that the SWI/SNF PBAF assembly promotes AC attachment to the basement membrane (BM) and promotes the activation of the invasive machinery. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

Denise S. Walker et al. (2006) European Worm Meeting "ITR-1, the C. elegans IP3 Receptor, Functions in ASH-Mediated Responses"

Nicholas J.D. Gower, Denise S. Walker, Elizabeth Gregory, Howard A. Baylis

[European Worm Meeting, 2006]

Denise S. Walker, Elizabeth Gregory, Nicholas J.D. Gower and Howard A. Baylis. Inositol 1,4,5 trisphosphate receptors (IP3Rs) are ligand-gated Ca2+ channels that control Ca2+ release from intracellular stores. They are central to a wide range of cellular responses. IP3Rs in C. elegans are encoded by a single gene, itr-1, which is widely expressed, and for which a wide range of functions have been identified. To determine the role of IP3 receptors in the nervous system, we have used the neuronal unc-119 promoter1 to direct expression of an IP3 sponge2. In a parallel approach we have knocked down itr-1 by expressing an RNA inverted repeat3 under the control of the unc-119 promoter. We describe the role of itr-1 in the aversive response to nose touch, and we present evidence that both egl-8 (encoding phospholipase C beta) and plc-3 (encoding phospholipase C gamma) function upstream of itr-1 in this response. The response to nose touch is mediated by the ASH polymodal sensory neurons, which also mediate avoidance of high osmolarity and volatile and water-soluble repellents4,5. We demonstrate that while itr-1 does not function in the majority of these responses, it does function in avoidance of benzaldehyde. We are currently investigating the cellular basis of these functions, and the role of signalling pathway components that may function upstream.. 1. Maduro, M. & Pilgrim, D. (1995) Genetics 141, 977-988.. 2. Walker et al. (2002) Mol. Biol. Cell 13, 1329-1337.. 3. Tavernarakis et al. (2000) Nat. Genet. 24, 180-183.. 4. Hart et al. (1999) J. Neurosci. 19, 1952-1958.. 5. Hilliard et al. (2004) EMBO J. 23, 1101-1111.