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Rivera Gomez, K. A., Muller Reichert, T., Fabig, G., Schvarzstein, M., Clarke, E., Villeneuve, A. M.
[
International Worm Meeting,
2017]
Crossovers (COs) are integral in enabling the ordered partitioning of the duplicated genome during the two meiotic divisions. Studies have focused on understanding the meiotic prophase I steps leading to formation of COs connecting each maternal homologous chromosome to its paternal counterpart. However it is understudied how different perturbations in prophase I translate into specific chromosome segregation defects. Observations of the product of meiotic mutant divisions by Severson et al. suggest that mutants lacking COs segregate homologous chromosomes in the first division in one of two different patterns, depending on whether mutant chromosomes have the cohesin component REC-8 required for sister chromatid co-orientation in meiosis I. Our live imaging analysis of these meiotic mutants revealed the pattern of chromosomes segregation in the two meiotic divisions. Meiotic mutants that lack the meiosis cohesin component REC-8 segregate sister chromatids away from each other in the first division, as they would in the second division in wild type meiosis. These mutants fail to partition chromatids in the second division resulting in the formation of diploid gametes. These findings are the basis of a scheme designed to derive viable and stable tetraploids from any C. elegans strain in order to query the roles of genome size on cell division, development, and evolution. Meiotic mutants with REC-8 include
him-3,
syp-1,
syp-2 and
spo-11 that are defective at different steps in CO formation. These mutants keep sister chromatids together as the wild type yet fail to partition in the first meiotic division. Their centrosomes, however, continue to progress through the cell cycle giving rise to spermatocytes with transient tetrapolar spindles. The chromatids eventually segregate to each of the four spindle poles yielding aneuploid sperm. Interestingly, analysis of altered karyotype and special meiotic mutant spermatocytes suggests that a single bi-oriented homologous chromosome pair is sufficient to suppress the formation of the transient tetrapolar spindles. We will report on the mechanism by which a single homologous chromosome pair might prevent formation of tetrapolar spindles. Together these studies will lead to a better understanding of fundamental mechanisms promoting accurate chromosome inheritance in normal and pathological meiosis.
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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.
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[
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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[
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.
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[
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.
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[
International Worm Meeting,
2003]
All organisms studied so far, except for the nematode Caenorhabditis elegans, have been shown to have developed an import system for peroxisomal proteins containing a peroxisomal targeting signal type 2 (PTS2). The currently accepted consensus sequence of this amino-terminal nonapeptide is (R/K)(L/V/I)X5(H/Q)(L/A). Some C. elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by a PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the ORFs D1053.2 and W10G11.11. Two proteins (CeMeK and the product of ORF W10G11.11) localized to the cytosol, while the protein encoded by D1053.2 was found in nucleoli. The three putative PTS2s did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. After extensive analysis of available experimental data on both functional and nonfunctional PTS2s, we propose two re-evaluated PTS2 consensus sequences. The first, R(L/V/I)XX(L/V/I)(S/A/L/K)X(H/Q)(L/A), describes the most common PTS2 variants, while the second (R/K)(L/V/I/Q)XX(L/V/I/H/Q)(S/A/L/K/G)X(H/Q)(L/A/F) includes all rare variants of PTS2. Our study also confirms the finding that C. elegans apparently lacks any PTS2-dependant peroxisomal protein import.
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[
International Worm Meeting,
2011]
A large population of K+ channels provides a way for organisms to maintain and modify cell excitability, allowing for appropriate behavioral responses to various stimuli. C. elegans male mating is a complex behavior that requires precise regulation to be successfully completed. Males must locate a hermaphrodite, properly position their tales on the hermaphrodite cuticle, locate the vulva, insert their copulatory spicules, and transfer sperm. We focus on the spicule insertion step of mating, and have identified three K+ channels expressed in the sex circuit that regulate the timing of insertion: ether-a-go-go (EAG)/EGL-2, EAG-related gene (ERG)/UNC-103, and big current (BK)/SLO-1. 79% of a population of males lacking all three channels displays spontaneous spicule protraction (Prc) in the absence of mating cues. Our research indicates the function of these three K+ channels is modified by the presence or absence of the others. The loss of SLO-1 alone results in 70% instance of Prc, while a
slo-1(lf)
egl-2(0) double mutant is 30% Prc. This decrease in the mutant phenotype is due to an up-regulation of
unc-103 in the absence of
egl-2, as a transgenically expressed dominant-negative form of
unc-103 returns the Prc phenotype to 72%. Additionally, the
rg432 allele of
slo-1 affects the Prc penetrance in an
unc-103(0);
egl-2(0) background. 58% of
unc-103(0);
egl-2(0) males are Prc; that percentage is reduced to 35% in
unc-103(0);
slo-1(
rg432)
egl-2(0) males.
slo-1(
rg432) is a point mutation in an intron located in a region that contains variable splicing and could result in up-regulation of specific isoforms that preferentially reduce male sex muscle excitability. In support of isoform-specific regulation, we determined that particular isoforms of
slo-1 are more capable of rescuing the Prc defect than others. Our work suggests that while the male sex muscles attempt to compensate for the loss of one K+ channel by up-regulating others, various factors exist that determine the success of such mechanisms. Future work will explore how K+ channel expression changes over time due to loss of channel function. This will allow us to dissect the relationship between the K+ channels that control appropriate sensory-motor responses.
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[
International Worm Meeting,
2015]
The highly conserved ether-a-go-go (EAG) K+ channel (also known as Kv10.1 and KCHN1) contains an n-terminal Per-Arnt-Sim (PAS) domain that interacts with a cyclic nucleotide-binding homology (CNBh) domain. These channels regulate egg-laying, chemotaxis and male mating in C. elegans and are involved in cognition, seizures, development and cancer in humans; however the functional role for the PAS and CNBh domains remain unidentified. In other contexts, the PAS domain has different functions, one of which is a nutrient sensor. Thus, we are interested in the role of the PAS domain in the
egl-2-encoded EAG K+ channel. We are utilizing a variety of molecular and behavioral approaches to address this question. Using CRISPR, we tagged the genomic EAG K+ channel gene
egl-2 by inserting YFP to a region between the CNBh domain and the c-terminus. The cells that express the CRISPR-engineered K+ channel in
egl-2:YFP worms match transgenic animals that express promoter:YFP constructs. Our previous research identified
egl-2 up-regulation as an important aspect of both aging and response to transient food deprivation. To confirm this construct is useful in measuring variable metabolic states, we measured fluorescent levels in both aging and transiently food-deprived males. We found that fluorescence increases with age or after a transient period of food deprivation, similar to our previous reports using transgenic promoter:marker constructs. Interestingly, while EGL-2:YFP is expressed uniformly in sex muscles, the protein is trafficked to the ciliated tips of sensory neurons in both the head and tail. This suggests EAG K+ channels regulate membrane excitability in cilia, perhaps responding to environmental signals through their PAS domains. To address this possibility, we are constructing a version of EAG with the channel removed and the circular permutated GFP inserted in its place, in an attempt to create a construct that will change fluorescence when the animal experiences different nutrient conditions. .
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[
International C. elegans Meeting,
1999]
Two putative homologues of large conductance Ca2+-activated K+ channel alpha-subunit gene (slowpoke or slo) were found by C. elegans genome sequencing project. These genes (Y51A2D.h and F08B12.3) bear similar structural features of Slo family K+ channels previously identified ranging from Drosophila to human. One of the two genes, F08B12.3, show somewhat a lower sequence similarity and lacks the key functional domains (Calcium-bowl and charged S4 segment) found in all Slo proteins. Thus, C. elegans F08B12.3 may encode a subfamily of Slo channels with distinct functional characteristics or even a member of a new K+ channel family. We first investigated the expression pattern of F08B12.3 using a reporter gene, green fluorescence protein (GFP). A construct containing in-frame fusion of gfp with about 2 kb 5'- upstream region and 1 kb of F08B12.3 coding sequence was made and wild type worms (N2) were transformed with the reporter construct. Green fluorescence was detected in various neuronal cells, especially those at the nerve ring, the ventral tract of the body, and the tail, suggesting some functional roles of this gene product in C. elegans nervous system. A full-length cDNA of F08B12.3 was cloned by screening cDNA library for functional studies. The gene encodes a protein containing six putative transmembrane segments with a putative K+-selective pore region (GYG motif) and a large C-terminal cytosolic domain as predicted by genome sequencing. The C-terminal region of F08B12.3 was over-expressed in bacteria and the antibody against this region was obtained from rats. Whole mount immunostaining further confirmed its expression in these neuronal cells detected by GFP experiments. Currently, we endeavor to find a deletion mutant based on chemically mutagenized worm library. A putative mammalian counterpart of C. elegans F08B12.3 was found from a human brain EST database. Deduced amino acid sequences of human EST #180888 have 50 % identities and 70 % similarities to the C-terminal sequences of F08B12.3. Northern blot and mRNA dot blot analysis using the human EST clone as a prove revealed a strong and specific expression of this gene in the brain and the skeletal muscle.
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[
International C. elegans Meeting,
1999]
Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.