Davis, K. C. et al. (2019) International Worm Meeting "Beauty sleep: Skin collagens regulate sleep in response to cell stress."

Davis, K. C., Raizen, D. M.

[International Worm Meeting, 2019]

When animals are sick or injured from an exposure that results in cell stress, they respond by sleeping; such behavior is called sickness or stress induced sleep (SIS). The pathway regulating SIS may be conserved from nematodes to vertebrates, and is relevant to fatigue behavior in humans during sickness. Cellular stress leads to activation of the epidermal growth factor (EGF) receptor on the ALA neuron, which releases neuropeptides. ALA neuropeptides induce SIS behavior, which consists of movment and feeding quiescence, elevated arousal threshold, and rapid reversibility. While much is known about ALA activation and its down stream mechanisms, the SIS pathway upstream of EGF remains poorly understood. This is the focus of our work. Mutants disrupting the cuticular collagens DPY-5, DPY-10, or DPY-13 showed impaired feeding and movement quiescence following exposures to ultraviolet (UV) irradiation, heat shock, or viral infection. Each of the three dpy mutants showed normal or enhanced SIS following EGF overexpression, suggesting that these genes act upstream of or in parallel to EGF. To determine if mutants with a Dpy phenotype due to mutations other than collagen genes are important for SIS, we tested mutants in dpy-19, which encodes a C-mannosyltransferase. dpy-19 mutants had normal SIS following both UV and heat shock, suggesting that specifically collagen gene disruption and not the Dpy phenotype explains the impairment in SIS in the dpy-5, -10, and -13 mutants. To determine whether disruption of the cuticle without disrupting collagens affects SIS, we tested bus-8 mutants, which encodes a glycosyltransferase that is important for cuticle integrity. bus-8 mutants are not Dpy. bus-8 mutants were not defective in SIS, suggesting that disrupting the cuticle is not sufficient to impair SIS, but that collagen disruption specifically is important. We propose that effects of skin collagen disruption on sleepiness are relevant to commmon complaint of fatigue in patients with connective tissue disorders.

MW Davis et al. (1999) International C. elegans Meeting "EXP-2 is a K+ channel that repolarizes the pharyngeal muscle"

R Joho, R Fleischhauer, MW Davis, JA Dent, L Avery

[International C. elegans Meeting, 1999]

We have found that exp-2 encodes a K + channel involved in the repolarization of pharyngeal muscles. Electropharyngeograms and intracellular current clamp recordings show that exp-2 gain-of-function pharyngeal muscles have short action potentials. Loss-of-function muscles have long action potentials with very slow repolarization. These electrophysiological defects are what we expect if exp-2 encodes a subunit of the C. elegans negative spike channel (NSC), a K + current in Ascaris pharyngeal muscles that is responsible for the fast repolarization at the end of the action potential (Byerly and Masuda, 1979, J Physiol 288, 263). We have expressed the EXP-2 channel in Xenopus oocytes. Its properties closely resemble those of the NSC. Upon depolarization, the channel activates slowly ( t =56ms at +20mV) and inactivates rapidly. Repolarization then rapidly removes inactivation and allows K + current to flow until the channel deactivates. These properties result in a channel that conducts rapidly when the voltage drops below a threshold after a long depolarization and make EXP-2 well suited to repolarize the muscle quickly at the end of the action potential. The exp-2 gain-of-function mutation is a cys to tyr mutation in the sixth transmembrane segment of the protein. It causes the channel to activate at potentials at least 100mV more negative than the wild-type channel. This results in a substantial fraction of open channels at -80mV, and would explain many of the phenotypes of the gain-of-function mutant.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[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.

Starr, D. et al. (2015) International Worm Meeting "UC Davis-University of Maryland Eastern Shore Graduate Admissions Pathway: Collaborating to broaden participation in molecular and cellular biology."

Starr, D., Hom, C.

[International Worm Meeting, 2015]

STEM professionals in the 21st century remain predominantly Caucasian/white, in spite of decades of work by professional societies, colleges and universities, and individual scientists to broaden participation. This multifaceted problem includes concerns among students and faculty at minority-serving institutions about the economics of career choice, family pressure to pursue a career in a biomedical field, and limited exposure to natural history. Further, institutional efforts in recruitment by research universities remain rooted in graduate fairs that target senior undergraduates from groups underrepresented in science, whereas connections made via shared research networks provide a more sure means to admission in molecular and cell biology. The UC Davis-University of Maryland Eastern Shore (UMES) Molecular and Cellular Biology Graduate Admissions Pathways (MCBGAP) program addresses this challenge via collaborations between faculty at the two institutions and a research co-mentoring program that brings UMES undergraduates to UC Davis for summer research. The program is funded by a grant from the University of California Office of the President and the UC Davis College of Biological Sciences.MCBGAP supported two cohorts of five UMES students in the summers of 2014 and 2015. The MCBGAP program consists of reciprocal student-faculty visits, close interactions between key UC Davis and UMES faculty, monthly Skype meetings that involve mentors and students, and research, professional development, and field trips in the summer. MCBGAP has catalyzed change both at UMES, where students are given the opportunity to self-identify as researchers at a tier 1 research university, and at UC Davis, where increased numbers of faculty recognize the need to be proactive in graduate recruiting and admissions, and multiple deans have committed time to mentor students and funds to support additional undergraduates from Historically Black Colleges and Universities for summer research and mentoring. The experience has also inspired us to apply for a Postbaccalaureate Research Education Program (PREP) from the NIH.

Franks CJ et al. (1997) International C. elegans Meeting "ACTION POTENTIALS RECORDED FROM THE PHARYNX OF CAENORHABDITIS ELEGANS."

Franks CJ, Holden-Dye L, Walker RJ

[International C. elegans Meeting, 1997]

Recently, Davis et al. (1995) have shown that it is possible to demonstrate phenotypic differences between wild-type and mutant C. elegans, using an electrophysiological approach. We have adapted their protocol in order to further characterise the wild-type phenotype, which will act as a basis for future comparative studies. We describe the properties of action potentials recorded from the terminal bulb of the pharyngeal muscle of wild type C. elegans. The anterior region of C. elegans was sectioned from the body and placed in a perfusion chamber on an inverted microscope stage in modified Dent's saline (composition in mM: NaCl 144, MgCl2 10, CaCl2 1, KCl 6, HEPES 5, pH 7.4). Intracellular recordings were made from pharyngeal muscle (60 MW microelectrodes;3M KAcetate), connected to an Axoclamp 2A amplifier. Drugs were applied by perfusion . Data are the mean +- S.E.M. The pharyngeal muscle membrane potential was -68.4 ! 1.5 mV (n=16). Action potentials had an amplitude of 85.5 ! 4.2 mV (n=16) with a duration of 0.26 ! 0.23 s, followed by a transient hyperpolarization of 7.5 ! 1.0 mV (mean of 4 to 10 action potentials from 16 cells; Figure 1). 5-HT (5!M; n=8) increased the frequency of firing in a reversible manner. Application of 4-aminopyridine (250!M) changed the firing pattern to bursting activity with a loss of the after hyperpolarization (n=6). This reversed on washing. We investigated the effect of changing external K+ concentrations (3-10 !M). Increasing [K+]out caused a decrease in the amplitude of the negative going after potential and concomitantly elicited a depolarisation of the membrane potential (n=6). Figure 1. Pharyngeal action potentials recorded from C. elegans. The membrane potential is indicated at the start of the trace. Reference. Davis et al. (1995) Mutations in the Caenorhabditis elegans Na, K-ATPase a-subunit gene, eat-6, disrupt excitable cell function. J. Neurosci. 15(12): 8408-8418.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[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.

Davis MW et al. (1995) International C. elegans Meeting "Mutations in the Na, K-ATPase a-subunit gene eat-6 disrupt excitable cell function"

Lee RYN, Somerville D, Fambrough DM, Davis MW, Avery L

[International C. elegans Meeting, 1995]

We have cloned a Na, K-ATPase a-subunit and shown that eat-6 mutants can be rescued by a genomic fragment containing the ATPase coding region plus 1kb of non-coding sequence on each side of the ORF. Further, eat-6 pharynxes are hypersensitive to a Na,K ATPase inhibitor and this inhibitor phenocopies eat-6 when it is applied to wild-type pharynxes. These results together with the fact that the physical map position of the ATPase gene is consistent with the interpolated position of eat-6 suggest that eat-6 encodes this ATPase. eat-6 mutations cause slow, delayed relaxations of pharyngeal muscle. These defects are due to defects in the electrical properties of the muscle because currents generated when pharynxes from eat-6 worms relax are severely reduced, as we have shown using an extracellular electrical recording technique called the electropharyngeogram. Also, the eat-6 phenotype remains when 19/20 of the pharyngeal neurons are ablated, suggesting that the phenotype is not due entirely to a nervous system defect. Using intracellular recording, we have shown that the resting membrane potential of eat-6 mutant pharyngeal muscles is consistently more depolarized compared to wild-type (-20mV to -45mV in eat-6 compared to -40mV to -60mV in wild-type). Further, eat-6 action potentials are smaller, and the return to resting potential is slower. To explain these abnormalities, we propose that a reduction of Na, K-ATPase activity in eat-6 mutants leads to a reduction of the resting potential of the cell. This reduced potential would change the gating kinetics of many voltage dependent ion channels, such as a voltage gated K+ channel which con- tributes to repolarization at the end of the action potential (Beyery and Masuda, J. Physiol. 288, 263-284).

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[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.

Konop C et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Cellular Localization of AF19 and Related Peptides in the Cephalic Region of the Nematode Ascaris suum."

Reinitz C, Konop C, Jarecki J, Knickelbine J, Stretton A OW

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Over fortyAscaris FMRFamide-like (AF) peptides have been sequenced in the parasiticnematode Ascaris suum by the Strettonlab. Many of these peptides have beenshown to have potent effects on the A.suum locomotory nervous system (Davis & Stretton, 1996). Locomotion isvital to the survival of the parasite in its host so an in-depth knowledge ofpeptide localization and behavioral effects is essential to develop neweffective anthelmintics. The FMRFamide-like peptide AF19 (AEGLSSPLIRFamide)has been shown to have inhibitory effects on locomotion (Davis & Stretton,2001). Injection of the peptide in the head-restricted behavioral assayabolished all locomotory waveforms and their propagations (Reinitz &Stretton, 2000). Ananti-peptide antibody specific to AF19 reproducibly stains a subset of neuronsin the cephalic region of A. suum. ALA , one of the two cells in the dorsal ganglion , shows AF19-immunoreactivity. This isdata is corroborated by single cell mass spectrometric (MS) and MS/MS data. Cloning, based on th sequence ofa novel peptide in ALA, yieldedthe transcript that encodes AF19 (afp-13)and two other amidated peptides. In situ hybridizationexperiments are planned to further corroborate the cellular localization ofAF19. Supported by NIH grant AI15429.

Chen LS et al. (1997) International C. elegans Meeting "MOLECULAR AND GENETIC ANALYSIS OF C. ELEGANS NEF-1, A MEMBER OF THE IG/FNIII SUPERFAMILY OF CELL ADHESION MOLECULES"

Bennett V, Chen LS

[International C. elegans Meeting, 1997]

A cell's identity, its interactions with other cells, and its ablity to adapt to its environment are heavily dependent on the proteins associated with its plasma membrane. Ankyrin, an intracellular protein that couples several integral membrane proteins to the spectrin cytoskeleton, is believed to be instrumental in organizing many membrane proteins into specialized domains. Recently, a family of cell adhesion molecules belonging to the Ig/FnIII superfamily was shown to bind ankyrin; these proteins include neurofascin, L1, NrCam and NgCam in vertebrates and neuroglian in Drosophila (1-6). This family of ankyrin binding CAMs is believed to be involved in neurite outgrowth, axonal fasciculation and targeting, cell migration and synaptogenesis during embryonic and postnatal vertebrate development (1-4). A member of the family has been identified in C. elegans. The gene encoding the homologue has been designated nef-1. In addition to the Ig and FnIII domains, it contains a highly conserved ankyrin binding domain. We are interested in elucidating the role of nef-1 in C. elegans development and its interactions with the C. elegans ankyrin homologue, UNC-44. Interestingly, mutations in unc-44 causes defects in axon guidance (7). 1. Sonderegger and Rathgen, 1992. J.Cell Biol. 119:1387-1394. 2. Rathgen and Jessel, 1991. Sem. of Neurosci. 3:297-307. 3. Grumet, 1991. Curr. Opin. Neurobiol. 1:370-379. 4. Hortsch and Goodman, 1991. Ann. Rev. Cell Biol. 7:505-557. 5. Davis et al., 1993. J. Biol. Chem.232:121-133 6. Davis and Bennett, 1994. J. Biol. Chem.267:18955-18972. 7. Otsuka et al., 1995. J.Cell Biol. 129: 1089-1092.