Pena, Salvador et al. (2015) International Worm Meeting "Hypoxia and the Mitochondrial Unfolded Protein Response."

Pena, Salvador, Sherman, Teresa, Brookes, Paul, Nehrke, Keith

[International Worm Meeting, 2015]

The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway induced by diverse stressors including reactive oxygen species, respiratory chain deficits, and mitochondrial DNA damage. Its activation helps to restore proteostasis and to protect against stresses caused by statin toxicity and bacterial infection. The transcription factor ATFS-1 is required for UPRmt signaling and the balance between ATFS-1 trafficking to the mitochondria and the nucleus regulates its signaling output. Consistent with a central role for mitochondria in mammalian ischemia-reperfusion injury, we found that either pharmacological or genetic activation of the UPRmt was sufficient to protect worms from anoxic death and cell damage. We found that atfs-1 was necessary for this protection and that in the absence of atfs-1, mitochondrial stress exacerbated anoxic death. In contrast, a gain-of-function (gf) mutation in atfs-1 was endogenously protected against anoxia. We then created a genetic model to study the cell specific effects of atfs-1. Our model utilizes a FLP-FRT-wCherry promoter interruption of an atfs-1 single copy MosSCI transgene to restrict the expression of either the wild type or gf allele to specific cells. Expression in this model via the native promoter at physiological levels is expected to more closely recapitulate endogenous regulatory mechanisms. Since mitochondrial stress has been suggested to result in non-cell autonomous signaling, we were motivated to use our model to determine whether the UPRmt could also protect cells from anoxic damage through similar mechanisms. We tested whether selective expression of atfs-1 was sufficient to activate the UPRmt in specific tissues and whether this elicited global adaptation to anoxia. In addition to allowing us to rigorously define the role of atfs-1 in regulating anoxic sensitivity, this model will be useful in studying how UPRmt influences other cellular and systems outputs.

Pena-Ramos O et al. (2023) STAR Protoc "Measuring the acidification of the phagosomal lumen in live C. elegans embryos."

Zhou Z, Pena-Ramos O

[STAR Protoc, 2023]

In metazoans, the acidification of the phagosomal lumen is essential for the efficient degradation of cargoes. Here, we present a protocol for measuring the rate of acidification inside phagosomal lumen containing apoptotic cells in living C. elegans embryos. We describe steps for generating a worm population, selecting embryos, and mounting embryos on agar pads. We then detail live imaging of embryos and data analysis. This protocol is applicable to any organism in which real-time fluorescence imaging can be performed. For complete details on the use and execution of this protocol, please refer to Pena-Ramos et al. (2022).¹.

Iwasa H et al. (2013) Exp Cell Res "Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1."

Hata Y, Ikeda M, Iwasa H, Kuroyanagi H, Nakagawa K, Maimaiti S

[Exp Cell Res, 2013]

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Junsu Kang et al. (2006) East Asia C. elegans Meeting "LATS kinase in a new signaling complex involved in development"

Junsu Kang, Junho Lee

[East Asia C. elegans Meeting, 2006]

LATS kinases are a member of tumor suppressor genes, which were shown to be involved in cell cycle regulation in mammalian cells. LATS1 mutant mice develop several tumors and exhibit developmental defects. Mice deficient of LATS2 show embryonic lethality. It is also known from Drosophila studies that several tumor suppressor genes such as NF2, Hippo, and Salvador, act upstream of LATS kinase for regulating cell proliferation and apoptosis. However, molecular mechanism underlying developmental defects and embryonic lethality caused by the loss of LATS function has not been elucidated. To address this, we used C. elegans as a model system. There is one homolog of LATS kinase in C. elegans, CeLATS. Surprisingly, we found that CeLATS was localized to the plasma membrane when adherens junctions were formed. Expression studies revealed that CeLATS colocalized with HMP2, a beta catenin homolog, on the membrane in the hypodermis and intestine. RNAi and mutant analysis revealed that CeLATS plays essential roles in the hypodermis, pharynx and intestine, loss of which caused a larval arrest, lethal phenotype. A main cause of the lethality was a defect in the intestine, because intestinal expression of CeLATS rescued the lethality. These results suggest that the LATS pathway acting in Drosophila might be conserved in the nematode for different purposes. It is possible that the nematode LATS protein has a novel function not yet revealed in mammalian LATS kinases.

Pena-Gonzalez, I. et al. (2013) International Worm Meeting "Suppressors of TDP-1 toxicity in Caenorhadbitis elegans."

Link, CD., Pena-Gonzalez, I.

[International Worm Meeting, 2013]

Suppressors of TDP-1 toxicity in Caenorhadbitis elegans Ilana Pena-Gonzalez1 and Christopher D Link1, 2 1 Integrative Physiology, University of Colorado, Boulder, CO 80309, USA 2 Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA RNA binding protein TDP-43 forms damaging aggregates in multiple neurodegenerative diseases. We have found that the deletion of tdp-1, the C. elegans ortholog of TDP-43, leads to increased accumulation of double stranded RNA. TDP-1 is not believed to bind to dsRNA itself; however the marked increase in dsRNA accumulation in worms deleted for tdp-1 implies TDP-1 normally participates in limiting the stability and structure of dsRNA. Although worms deleted for TDP-1 do not have a severe phenotype, overexpressed nuclear TDP-1 is toxic in worms. One possible explanation for this is that too much TDP-1 leads to excessive disruption of structured RNAs needed for normal RNA metabolism. Making use of this overexpression model we have established a heat shock induction system to help identify components associated with TDP-1's prevention of dsRNA accumulation. Identifying mutations that suppress the neurotoxic effects of overexpressed TDP-1 could help identify others factors contributing to the complex mechanism of disassembling dsRNA. A mutagenesis screen has identified four mutant strains that partially suppress the toxicity of overexpressed TDP-1. We have also tested suppressors of other toxic proteins. Further identifying the pieces of the mechanism regulating dsRNA breakdown could help better explain the pathogenic pathway of TDP-1 and consequently TDP-43 in disease.