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[
International Worm Meeting,
2007]
Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes
egl-1,
ced-3 and
ced-4 and anti-apoptotic genes
ced-9 and
icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene
eif-3.K. Loss-of-function by mutation or RNAi inactivation in
eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of
eif-3.K weakly but significantly increased cell corpses. Interestingly, the
eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of
egl-1 or
ced-4. This result suggests that
eif-3.K may act downstream of or in parallel to
egl-1 and
ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express
eif-3.k in touch neurons, we showed that
eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human
eif-3.K can functionally substitute C. elegans
eif-3.K in an
eif-3.K mutant, the function of
eif-3.K in apoptosis is likely conserved in evolution.
-
[
East Asia C. elegans Meeting,
2006]
Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes
egl-1,
ced-4 and
ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene
eif-3.K. A loss-of-function mutation or inactivation by RNA interference in
eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of
eif-3.K weakly but significantly promoted programmed cell death. In addition,
eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of
egl-1 and
ced-4. This result suggests that
eif-3.K may act downstream of or in parallel to
egl-1 and
ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that
eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of
eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and
eif-3.K mutants.
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[
West Coast Worm Meeting,
2004]
Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the
age-1 and
daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the
daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.
-
[
International Worm Meeting,
2017]
Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.
-
[
International Worm Meeting,
2021]
Anosmia is among the most prevalent symptom of SARS-CoV-2 infection. Interestingly though, the SARS-CoV-2 virus infects the olfactory supporting cells, rather than the primary sensory neurons themselves. These recent findings have underscored the importance of the supporting cells in olfaction. However, very little is known about the mechanisms underlying the regulation of olfaction by the supporting cells. In C. elegans, the Amphid sheath (Amsh) glial cells are the supportive cells of the amphid sensory apparatus, sharing with mammalian olfactory supporting cells general function and expression of the homologs of the SARS-CoV-2 viral entry proteins ACE2 and TMPRSS2. To understand the contribution of the Amsh glia to sensory function, our lab has taken the unbiased approach of sequencing the mRNA extracted from these cells. Among the ~1,000 glial-enriched genes, we identified 14 ion channel and transporter genes with 2.7- to 29.6-fold mRNA enrichment in Amsh glia as compared to other cells. To determine whether these channels and transporter genes are needed for sensory behaviors, we performed behavioral assays on knock-outs and Amsh cell specific knock-downs. Our results support the predominant requirement of glial K+ and Cl- channels and transporters for the response to isoamyl alcohol, octanol, diacetyl, and NaCl. Given, the recent report that Amsh glia function as odorant receptor cells, Ca2+ imaging experiments are underway to determine whether these channels and transporters alter the response of Amsh glia or sensory neurons (or both) to these sensory cues. Taken together, our findings expand on our understanding of the mechanisms underlying the contribution of Amsh glia to sensory perception in C. elegans; mechanisms that might be conserved from worm to man.
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[
International Worm Meeting,
2019]
Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.
-
[
International Worm Meeting,
2005]
Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans
gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that
rnp-8(RNAi) and
gld-2 mutants have similar defects during oogenesis. Both
gld-2 and
rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and
rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412
-
[
International Worm Meeting,
2015]
Sexual dimorphism is a widely acknowledged biological phenomenon, yet the mechanisms underlying specific developmental dimorphisms are largely unknown. As a result of the defined connectome available for both C. elegans males and hermaphrodites, it is clear that there are dimorphic wiring differences in shared neurons between the adult animals of the two sexes (1). Several of these dimorphisms are found in the phasmid sensory neurons, whose synapses onto the command interneurons are sexually dimorphic, suggesting that they have sex-specific functions. I am confirming the predicted sex-specific functions of the phasmid neurons using behavioral assays. To study how these dimorphic patterns of connectivity (and function) are established, I am using transsynaptic labeling (GRASP technology; 2) to visualize both male-specific and hermaphrodite-specific synaptic connections of phasmid neurons. Sexual determination is regulated across many invertebrate and vertebrate species by the highly-conserved doublesex/mab (DM) domain genes. In C. elegans, the DM domain class contains 11 paralogs, the majority of which have no known function. I analyzed expression patterns for 8 of the dmd genes in both larval and adult stages in the two sexes to identify dimorphisms, and identified
dmd-4 as a candidate for dimorphic regulation in the PHA and PHB phasmid neurons.
dmd-4 is an embryonic lethal gene, so I am generating a conditional knockout allele and perform mosaic rescue analysis to determine if
dmd-4 is part of the genetic regulatory system for dimorphism in the phasmid neurons, and if this function is cell-autonomous. Ultimately, we will seek to elucidate the genetic regulatory logic of dimorphisms in shared neurons.1. Jarrell TA, Wang Y, Bloniarz AE, Brittin CA, Xu M, Thomson JN, Albertson DG, Hall DH, Emmons SW. 2012. Science 337, 437-444.2. Feinberg EH, VanHoven MK, Bendesky A, Wang G, Fetter RD, Shen K, Bargmann CI. 2008. Neuron 57(3), 353-363.
-
[
International C. elegans Meeting,
2001]
Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.
-
[
International Worm Meeting,
2009]
Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .