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[
International Worm Meeting,
2021]
Measurement of neuronal activities in non-invasive and unanesthetized condition is important for understanding neuronal function in intact animals. Ca2+ imaging by fluorescent gene encoded calcium indicators (GECI) are a powerful way to measure neuronal activities in C. elegans. Although Ca2+ imaging revealed important aspects in neuronal functions, the measurement of neuronal membrane voltage is important to understand the neuronal functions. Furthermore, the relations of change of membrane voltages and changes of Ca2+ has not been fully understood. Recently, several types of gene encoded voltage indicators (GEVI) that are derived from 7TM proteins used for optogenetics has been developed to measure changes of membrane voltage in living animals. Even though the fluorescence of these GEVIs is dim, they showed fast time constants and relatively high fluorescent change depend on voltages. Among those GEVIs, we use paQuasAr3 for the voltage measurement, because it shows relatively higher fluorescence with other superior characteristics. Since AWA, one of the olfactory sensory neurons, which is responsible for diacetyl sensation, was reported to show all-or-none action potentials (Liu et al. 2018), we firstly analyzed AWA voltage changes induced by diacetyl. We found that fluorescence of paQuasAr3 expressed in AWA cell body is changed in response to diacetyl stimulation with high reproducibility. At the beginning of the stimulation, the transient increase and decrease of fluorescence intensity was observed, whereas the relatively higher fluorescence intensity was sustained during the stimulation. To elucidate relations between the Ca2+ responses and the voltage responses, we made wild-type animals expressing paQuasAr3 and GCaMP6f in AWA neurons, and measured both fluorescence at a cell body simultaneously. We found that the changes of paQuasAr3 started faster than the changes of GCaMP. These analyses will give insights on the neuronal functions in informational processing.
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[
International Worm Meeting,
2021]
Chromosomes that have undergone crossing over in meiotic prophase must maintain sister chromatid cohesion somewhere along their length between the first and second meiotic divisions. To accomplish this, the holocentric organism Caenorhabditis elegans creates two chromosome domains of unequal length termed the short arm and long arm, which become the first and second site of cohesion loss at meiosis I and II. The mechanisms that confer distinct functions to the short and long arm domains remain poorly understood. Previously we and others have shown that phosphorylation of SYP-1, a central element of the synaptonemal complex (SC), at Thr452 provides a binding site for a Polo-like kinase PLK-2, and phosphorylated SYP-1 and PLK-2 cooperatively localize to the short arm to guide downstream factors triggering cohesin degradation at the short arm (Sato-Carlton et al. 2017; Brandt et al. 2020). Previous studies have shown that Polo kinase is activated via phosphorylation of its activation loop by Aurora kinase, and this interaction is promoted by Bora/SPAT-1 in mitosis (Tavernier et al. 2015). To understand the mode of Polo-like kinase regulation during meiotic prophase, we examined the effect of
spat-1 knockdown during oogenesis. We found that PLK-2 failed to spread to short arms but instead was confined at crossover designation sites in
spat-1 RNAi gonads. In addition, we found that homologous chromosome synapsis and crossover formation are impaired in
spat-1 RNAi animals. Interestingly, excess crossover designation, ranging from 6 to 13 sites per nucleus, was found in
spat-1 RNAi animals. Computational tracing of three-dimensional chromosome images revealed the presence of multiple crossover designation sites on the same chromosome, suggesting that crossover interference is impaired. These observations are reminiscent of
plk-2 mutant phenotypes, and suggest the possibility that SPAT-1 regulates PLK-2 during meiotic prophase. Brandt J. N., K. A. Hussey, and Y. Kim, 2020 Spatial and temporal control of targeting Polo-like kinase during meiotic prophase. J. Cell Biol. 219 Sato-Carlton A, Nakamura-Tabuchi C, Chartrand SK, Uchino T, Carlton PM (2018) Phosphorylation of the synaptonemal complex protein SYP-1 promotes meiotic chromosome segregation. J Cell Biol 217:555-570 Tavernier N., A. Noatynska, C. Panbianco, L. Martino, L. Van Hove, et al., 2015 Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos. J. Cell Biol. 208: 661-669.
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[
International Worm Meeting,
2009]
Accurate segregation of homologous chromosomes during meiosis is necessary for the faithful transmission of the genome from parent to progeny. To segregate properly, homologs must first undergo pairing, synapsis, and recombination. The mechanism of homologous chromosome pairing remains a major mystery of meiosis. In C. elegans, as in many other eukaryotes, pairing is accompanied by a global rearrangement of chromosomes. We have found that this rearrangement is driven through the association of special chromosome regions known as Pairing Centers (PC) with nuclear envelope proteins and cytoskeletal motors (Phillips et al. 2005, Sato A. unpublished). Using fluorescent markers for nuclear envelope attachment sites and Pairing Centers, we are analyzing chromosome dynamics through real-time imaging and quantitative motion tracking. Our results reveal a dramatic increase in chromosome motion at the onset of chromosome pairing that persists after homologous loci are paired. We show that this increased mobility is dependent on the conserved cell cycle checkpoint kinase, CHK-2, and that motion slows following knockdown of cytoplasmic dynein. Our data supports a model in which homolog pairing is promoted by a small number of fast, motor-driven movements that augment the smaller, Brownian motions seen prior to meiosis. The observation that fast motions persist well after pairing is completed suggests additional roles in chromosome synapsis or recombination.
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[
International Worm Meeting,
2021]
To carry out the two successive chromosome disjunction events of meiosis, the holocentric chromosomes of C. elegans use the position of the single off-center meiotic crossover to define two functional domains: the short arm, which loses cohesion in meiosis I, and the long arm, which loses cohesion in meiosis II. These two domains must be detected and defined on each chromosome at each meiosis, since crossovers can occur anywhere. The domains accumulate distinct sets of proteins or protein modifications, whose roles in mediating the timing of cohesion loss have recently begun to be uncovered. In contrast to the downstream mechanisms that lead to cohesin loss or protection, the mechanisms that initially sense the length difference of the two domains remains less well-understood. We have previously shown that the accumulation of the synaptonemal complex (SC) central element protein SYP-1 phosphorylated at T452 on the short arm during the pachynema stage is one of the earliest signs of distinction between the short and long arms (Sato-Carlton et al. 2018). Phosphorylated SYP-1 localizes to the entire SC at the beginning of meiotic prophase and quickly accumulates on the short arm upon crossover designation, while departing the long arm entirely. In order to understand the mechanism of this short arm confinement, we have tagged non-phosphorylatable SYP-1 with an HA tag and compared its localization with phosphorylated SYP-1. We found that non-phosphorylatable SYP-1 also becomes enriched on the short arm, but does so more slowly than phosphorylated SYP-1. To gain further insight into the principles determining short arms, we have analyzed the formation of short arm-like domains enriched for phospho-SYP-1 on the fusion chromosome meT7 (Hillers and Villeneuve 2003; Martinez-Perez et al. 2008), a "megasome" that combines chromosomes III, X, and IV, and which frequently receives two or three crossovers. On meT7 chromosomes with two crossover designation sites at pachynema, domains of phospho-SYP-1 can occur in one of three modes: (1) a short arm domain at each end; (2) a single short arm domain in the middle segment, and (3) one short arm domain at one end, and one short arm domain in a small part of the middle segment. We present a model that predicts these outcomes from the strength of a putative accumulating signal emanating bidirectionally from crossover designation sites. Hillers K. J., and A. M. Villeneuve, 2003 Curr. Biol. 13: 1641-1647. Martinez-Perez E., M. Schvarzstein, C. Barroso, J. Lightfoot, A. F. Dernburg, et al., 2008 Genes Dev. 22: 2886-2901. Sato-Carlton A., C. Nakamura-Tabuchi, S. K. Chartrand, T. Uchino, and P. M. Carlton, 2018 J. Cell Biol. 217: 555-570.
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Arnold, Meghan, Toth, Marton, Melentijevic, Ilija, Driscoll, Monica, Harinath, Girish, Smart, Joelle, Guasp, Ryan
[
International Worm Meeting,
2017]
Mitochondria provide energy, execute key steps of metabolism, control calcium, and modulate cellular decisions for life/death. Given these critical functions in cell, tissue, and organism health, it is not surprising that mitochondrial functionality plays an essential role in neuronal maintenance in everyday biology, aging, and late-onset neurodegenerative disease. Mitochondrial quality control is thought to be primarily executed through cell-internal elimination via mitophagy and lysosome degradation. However, the Driscoll lab has discovered, and recently published (Melentijevic, 2017 Nature 542:367) that mitochondria can be thrown out of neurons in large membrane bound vesicles we call exophers. Mitochondria in exophers budding from C. elegans touch neurons tend to have elevated oxidation of mitoROGFP reporters localized to the matrix. Genetic and pharmacological treatments that compromise mitochondria can increase numbers of exophers produced by touch neurons, suggesting that throwing away defective mitochondria may be a mechanism for neuronal quality control. Indeed, some mammalian neurons can throw out their mitochondria for degradation by neighboring astrocytes (Davis, PNAS 11:9633), suggesting relevance across phyla. In C. elegans, beautiful work on degradation of sperm mitochondria upon fertilization have been published (Sato, Science 334:1141). We will present data on our initial efforts to characterize mito-exopher production and the factors that prompt neurons to extrude mitochondria. Our hope is that our findings will be relevant to understanding neuronal maintenance and neuronal degeneration, especially as associated with perturbed mitochondrial quality as may occur in Parkinson's disease and many other human disorders.
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[
Development & Evolution Meeting,
2008]
Accurate segregation of chromosomes during meiosis is necessary for the faithful transmission of the genome from parent to progeny. Conversely, meiotic missegregation leads to impaired viability and fertility, as well as aneuploid progeny that can have dramatic developmental defects, such as Down syndrome in humans. To segregate properly, homologs must first undergo pairing, synapsis, and recombination. Despite its central role in meiosis, pairing of homologous chromosomes remains poorly understood. In C. elegans, as in many other eukaryotes, this process is accompanied by a global rearrangement of chromosomes. We have found that this rearrangement is driven through the association of special chromosome regions, known as Homolog Recognition Regions or Pairing Centers, with the nuclear envelope. In other organisms, telomeres establish similar attachments to the NE during "bouquet stage" of meiosis, but the function of this configuration has not been established in any system. Chromosomes are not merely tethered at the nuclear envelope, but instead associate with specific nuclear envelope proteins and cytoskeletal motors (Phillips et al. 2005, Sato A. unpublished). Using ZYG-12:GFP (Malone et al. 2003) as a marker for the chromosome attachment sites, we have observed that they undergo rapid movements throughout the pairing process. Disruption of cytoplasmic dynein causes a dramatic decrease in patch motion, supporting a model in which pairing dynamics are driven by cytoskeletal components outside the nucleus. These data suggest that meiotic chromosome pairing is an active process rather than being purely a result of a diffusion-based mechanism.
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[
International Worm Meeting,
2015]
The Gram-negative bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora. EPN invades insect host and releases the symbiotic bacteria into the hemocoel to kill the host by bacterial toxins. Even though several toxins of P. luminescens are under active investigation, the underlying mechanism of its virulence remains unclear. P. luminescens behaves as a pathogen against not only insects but also non-mutualistic nematodes including Caenorhabditis elegans. We revealed a novel molecular mechanism of P. luminescens virulence toward a model host C. elegans;
p38 MAPK pathway and Insulin/IGF-1 signaling pathway in C. elegans are required for defense response against P. luminescens, but Insulin/IGF-1 signaling pathway is inactivated by P. luminescens through the overexpression of insulin-like gene (Sato et al., 2014). In addition to the lethal effect of P. luminescens on C. elegans adult, we also found its growth inhibitory effect on C. elegans larva.To reveal the new virulence mechanism we constructed transposon mutagenized library of P. luminescens and screened for attenuation of growth inhibitory effect on C. elegans larva. We transferred synchronized L1-stage C. elegans on P. luminescens mutants and checked the C. elegans growth after several days. Until now we isolated three virulence-attenuated mutants by screening approximately 700 independent transposon-inserted mutants. We sequenced transposon insertion sites of the two of these mutants and confirmed the two genes pdxB (encodes Erythronate-4-phosphate dehydrogenase) and
plu3602 (unknown functional gene) were required for full pathogenicity toward C. elegans. On the avirulent mutants, C. elegans could develop from L1 to adulthood and produce a lot of brood, and C. elegans lifespans from the adult stage were about 12.7% (on pdxB mutant) and 11.9 % (on
plu3602 mutant) longer than those when grown on the wild type P. luminescens. Further phenotypic and genetic analyses of these mutants would give us a new insight of the P. luminescens virulence mechanism.Sato et al. SpringerPlus, 2014, 3, 274.
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[
East Asia C. elegans Meeting,
2006]
Mutants Having AlteredPreference of Chemotaxis in Simultaneous Presentation of Two Attractants Lin Lin, <SUP>a</SUP> Tokumitsu WAKABAYASHI, <SUP>b</SUP> Tomohiro OIKAWA,<SUP>a</SUP>Tsutomu SATO,<SUP>b</SUP> Tarou OGURUSU <SUP>a, b</SUP> and Ryuzo SHINGAI<SUP>a,b,</SUP> <SUP>a</SUP>Department of Computer and Information Science, Graduate School of Engineering, Iwate University, 4-3-5 Ueda, Morioka, 020-8551 Japan <SUP>b</SUP>Laboratory of Bioscience, Faculty of Engineering, Iwate University, 4-3-5 Ueda, Morioka, 020-8551 Japan Upon presentation of two distinct chemoattractants such as sodium acetate and diacetyl simultaneously, the worms were preferentially attracted by one of these chemoattractants. We isolated two mutants having altered preference of chemotaxis behavior toward simultaneous presentation of sodium acetate and diacetyl.
chep-1(
qr1) (for chemosensory preference) mutant preferred sodium acetate to diacetyl, while
chep-2(
qr2) mutant preferred diacetyl to sodium acetate in simultaneous presentation of these chemoattractants.
chep-1(
qr1) showed decreased preference toward diacetyl in the simultaneous two-spot presentation in all conditions tested. The decreased preference toward diacetyl could be explained by the sole effect of the defective chemotaxis toward diacetyl. The chemotaxis behavior of
chep-2(
qr2) mutant in simultaneous presentation suggested the function of
chep-2 gene products within the chemosensory informational integration pathway as well as in the chemosensory pathway. Sodium acetate is detected by the ASEL chemosensory neuron and the neuron sends its synaptic output onto AIA, AIB, AIY interneurons and several other sensory neurons. A low concentration of diacetyl is detected by a bilateral pair of AWA sensory neurons and send their synaptic outputs onto AIY and AIZ neurons and several sensory neurons including ASE. Multiple information detected separately by ASEL and AWA neurons could be integrated directly within the ASEL neuron and within the AIY interneurons. The integration within the AIA, AIB and/or AIZ interneurons were also possible.
chep-2 gene product may function in one to several of these neurons to regulate the informational integration pathway.
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Mazzochette, Eileen, Loizeau, Frederic, Goodman, Miriam, Pruitt, Beth, Fechner, Sylvia, Nekimken, Adam, Vergassola, Massimo
[
International Worm Meeting,
2015]
Gentle touch receptor neurons (TRNs) of C. elegans share many response dynamics with sensory neurons found in Pacinian and Meissner corpuscles in mammals [1]. For example, they show rapid adaptation and symmetrical on and off responses to the application and removal of mechanical cues [2]. Whether TRNs are frequency-dependent remain however unknown. In Pacinian corpuscles, the onion-like lamellar capsule acts as a mechanical band-pass filter between 20 Hz and 400 Hz [3]. Here, we ask whether the C. elegans body, and in particular the tissues engulfing the TRNs, is viscoelastic and how this property might play a role in the transduction of mechanical cues at low and high frequencies to the TRNs. To reach this goal, we developed a custom instrument capable of delivering defined force or displacement profiles to the worm body within the nN-mN or nm-mm range, respectively [4]. To apply the mechanical cues, silicon micro-cantilevers were fabricated using manufacturing processes derived from the integrated circuits industry. We integrated a piezoresistive strain gauge at their surface to detect the deflection of the micro-cantilever. Hence, the known dimensions of the micro-cantilevers coupled with a defined contact area with the worm body allow us to measure the applied force and indentation. The movement of the micro-cantilever is provided by a piezoelectric actuator controlled using a custom LabView interface and a field-programmable gate array. The controller manages data acquisition up to 10 kHz and feedback control in either displacement, i.e. displacement-clamp configuration, or force, i.e. force-clamp configuration. We achieved force and displacement step profiles with rising times below 3 ms and 1 ms, respectively. Sine waves have also been applied up to 200 Hz in force-clamp and up to 400 Hz in displacement-clamp. With sine stimuli of various frequencies, we are currently investigating the viscoelasticity of the worm body and its role in the mechanosensation of C. elegans.[1] A. Zimmerman, et al., Science, 346, 6212, 950-954, 2014[2] R. O'Hagan, et al., Nat. Neurosci., 8, 1, 43-50, 2005.[3] M. Sato, J. Physiol., 159, 3, 391-409, 1961.[4] S.-J. Park, et al., Rev. Sci. Instrum., 82, 4, 043703, 2011.
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[
International Worm Meeting,
2013]
FBF-1 and FBF-2 are translational regulators maintaining germline stem cells in C. elegans (Crittenden et al., 2002). We previously reported that the localization and function of FBF-2 depends on the integrity of P granules, perinuclear RNA granules of germ cells (Strome and Wood, 1982; Pitt et al., 2000; Voronina et al., 2012). To understand the role of cofactors in FBF-2 function, we are characterizing FBF-2 interactome by mass-spectroscopy. To select the components of FBF-2 RNP contributing to FBF-2-mediated regulation, we employed a genetic interaction assay. We depleted candidate FBF-2-interacting partners by RNAi in N2 worms, as well as in
fbf-1(lf) mutant (Lamont et al., 2004), in which the worm relies solely on FBF-2 for stem cell maintenance, translational regulation, and fertility. Cofactors specific for FBF-2 should be required for FBF-dependent regulation in
fbf-1(lf) mutant, but not in a wild-type strain, where FBF-1 can compensate. The screen identified C. elegans homolog of La protein (provisionally named LHP-1) and dynein light chain DLC-1 as candidate FBF-2 cofactors. La protein contributes to multiple steps in RNA biogenesis in the nucleus, and has been recently implicated in the regulated translation of several mRNAs in the cytoplasm (Bayfield et al., 2012). DLC-1 is a cargo-binding component of dynein motor complex required for cell division (Gonczy et al., 1999), meiotic chromosomal synapsis (Sato et al., 2009), and regulation of meiotic entry (Dorsett and Schedl, 2009). Similar to
pgl-1(lf), both
lhp-1(RNAi) and
dlc-1(RNAi) in the
fbf-1(lf) background lead to derepression of FBF target reporter in the distal mitotic region and masculinization of germline; the phenotypes not observed after same RNAi treatments of the wild type or
fbf-2(lf) worms. Localization of FBF-2 to the nuclear periphery is maintained after
lhp-1(RNAi), but is lost after
dlc-1(RNAi), even when perinuclear P granules are normal. Our data is consistent with DLC-1 affecting FBF-2 subcellular distribution, and LHP-1 contributing to FBF-2 function downstream of its localization to P granules.