Davies JP et al. (2000) Science "Transmembrane molecular pump activity of Niemann-Pick C1 protein."

Davies JP, Ioannou YA, Chen FW

[Science, 2000]

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.

Nishino A et al. (2015) Comp Biochem Physiol A Mol Integr Physiol "Extension of the established period of diacetyl adaptation by oxygen intermediates in the nematode Caenorhabditis elegans."

Matsuura T, Sato F, Ito K, Nishino A

[Comp Biochem Physiol A Mol Integr Physiol, 2015]

After pre-exposure to the odorant diacetyl, the nematode Caenorhabditis elegans showed a decline in chemotactic responses to diacetyl, a phenomenon known as diacetyl adaptation. In the present study, we found that the established period of diacetyl adaptation in nematodes increased with the breeding temperature. When wild-type (N2) nematodes were bred at 15C, adaptation was observed from the young adult (YA) to the 3-day-old adult that is reached 3 days after the YA stage. On breeding nematodes at 20C and 25C, adaptation was observed between the YA and 5-day-old adult and between the YA and the 7-day-old adult, respectively. Breeding temperature has been shown to correlate with the rate of aging in nematodes, which is related to the level of oxygen consumption. Accordingly, long-lived isp-1 and clk-1 mutants that demonstrate decreased levels of oxygen consumption showed a shorter established period of adaptation than N2 nematodes, whereas short-lived gas-1 and mev-1 mutants that have a hypersensitive response to oxygen showed a longer period of adaptation than the N2. Moreover, the established period of diacetyl adaptation in N2 nematodes was shortened by the antioxidant -lipoic acid. These results suggest that oxygen intermediates, which are produced by oxygen consumption, play a significant role in diacetyl adaptation. Although this is only one of many factors that regulate diacetyl adaptation, such as the release of neurotransmitters and changes in intracellular conditions, the acquisition of this adaptation requires an increase in the intensity of moderate oxygen signals.

Matsuura T et al. (2015) Biosci Biotechnol Biochem "Chronic nicotine exposure augments gustatory plasticity in Caenorhabditis elegans: involvement of dopamine signaling."

Matsuura T, Urushihata T

[Biosci Biotechnol Biochem, 2015]

The chemotaxis of wild-type NaCl-conditioned nematodes exposed to 100 mM NaCl, maintained on a growth medium containing 0.3 mM nicotine from first larva to young adult (YA) hermaphrodite, was significantly weaker than the chemotaxis of those maintained on a medium without nicotine. The result indicates that chronic nicotine exposure augments gustatory plasticity. The gustatory plasticity was also augmented when tph-1 mutants, with a defect in serotonin biosynthesis, were maintained on a medium containing nicotine until the YA stage. Chronic nicotine exposure did not augment gustatory plasticity in bas-1 mutants, which had defects in both serotonin and dopamine biosynthesis, and in cat-2 mutants, which had a defect in dopamine biosynthesis. However, augmentation of gustatory plasticity was observed when bas-1 and cat-2 mutants were maintained on a growth medium containing nicotine along with dopamine, suggesting that dopamine signaling is involved in the augmentation of gustatory plasticity due to chronic nicotine exposure.

Matsuura T et al. (2007) Comp Biochem Physiol A Mol Integr Physiol "Developmental changes in chemotactic response and choice of two attractants, sodium acetate and diacetyl, in the nematode Caenorhabditis elegans."

Ichinose M, Endo S, Matsuura T, Takahashi H, Iwamoto R

[Comp Biochem Physiol A Mol Integr Physiol, 2007]

The chemotactic behavior of the nematode Caenorhabditis elegans to chemical attractants, water-soluble sodium acetate and odorant diacetyl, was investigated using nematodes at various developmental stages to examine the effects of postembryonic development on chemotactic response and spontaneous locomotion. The chemotactic responses to attractants increased as development progressed, and the largest responses to either 1.0 M sodium acetate or 0.1% diacetyl were seen at the young adult (YA) or day adult (A1) stage, respectively. Responses to the chemicals declined thereafter in-line with increasing age. The chemotaxis indices for attractants correlated with activity of spontaneous locomotion (p<0.01), suggesting that a change in spontaneous locomotion is one of the factors involved with the change in chemotactic responses during development. We also investigated the effect of aging on attractant choice by the simultaneous presentation of 0.6 M sodium acetate and 0.1% diacetyl. In the presence of both attractants, the fraction of larval animals at the sodium acetate location was greater than that at the diacetyl location (p<0.05). The fractions of YA animals that gathered at either location were almost identical, whereas the fraction of adult animals at the diacetyl location was greater than that at the sodium acetate location (p<0.05). The patterns of attractant choice of the long-lived daf-2 mutants and short lifespan mev-1 mutants showed the same tendency as those of wild type nematodes in the presence of both attractants. These results suggest that a change in the neuronal mechanisms controlling attractant choice and preference occurs during developmental progression.

Yamaguchi M et al. (2016) Biochim Biophys Acta "NF-Y in invertebrates."

Yoshida H, Ly LL, Yamaguchi M, Ali MS, Yoshioka Y

[Biochim Biophys Acta, 2016]

BothDrosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of egl-5 and tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized.

Dolfini D et al. (2012) Crit Rev Biochem Mol Biol "NF-Y and the transcriptional activation of CCAAT promoters."

Gatta R, Mantovani R, Dolfini D

[Crit Rev Biochem Mol Biol, 2012]

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Marino-Ramirez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Hakkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

Fatma Kaplan et al. (2007) International Worm Meeting "Behavioral/Developmental Metabolomics: Isolation and Characterization of Mating Pheromones from C. elegans."

Paul W. Sternberg, Peter Teal, Cherian Zachariah, Aaron Dossey, Fatma Kaplan, Arthur E. Edison, Michael Stadler, Jagan Srinivasan, Jim Rocca, Hans Alborn, Ramazan Ajredini

[International Worm Meeting, 2007]

Although C. elegans, is one of the best-studied model systems, very little is known about the chemistry that C. elegans uses to communicate between individuals and its environment. Several structures that secrete material to the outside have been described in nematodes, including the pharyngeal gland and the excretory/secretory system, and the intestine. However the roles of these secretions in animal communication are still not well understood. We hypothesize that C. elegans utilizes an extensive ‘chemical language that allows individuals to communicate and regulates basic behaviors such as feeding, mating, and population density control. As a first bioassay to determine at what developmental stage hermaphrodites release a mating cue that is sensed by males, conditioned water of different life stages of C. elegans (Egg, L1, L2, L2D, L3, L4, YA and Adult) were collected. We assayed the conditioned water with Cel-him-5 males and wild type hermaphrodites. To have a high-throughput system, we adapted the ‘holding assay as previously described (Simon and Sternberg, PNAS, 2002) to assay multiple animals in a single assay. This ‘population holding assay estimates the time the animals spend at the spot where the conditioned water was placed versus a control spot. Our results indicate that C. elegans starts releasing mating cue from L4 stage till Adult stage. The response to the conditioned water is sex-specific as him-5 males respond more robustly than hermaphrodites. We are currently testing the sub-fractions of the young adult conditioned water for activity. Further, we are pursuing the purification of the conditioned water in two independent directions. First, a ‘traditional forward approach of collecting large amounts of YA conditioned water is being conducted. Compounds that are released to the water are separated by various chromatographic methods, tested for biological activity, further purified, and identified by mass spectrometry and nuclear magnetic resonance (NMR). The second method is a reverse approach by using NMR to compare worm conditioned water of different developmental stages of worms. NMR signals that are unique to stages where mating cues are produced are identified from the mixture of compounds. These unique signals will guide HPLC purification, testing for biological activity, and identification with NMR and MS. Updates from both directions of purifications and behavioral assays will be presented. *FK and JS are co-first authors.

Merrihew, Gennifer E. et al. (2011) International Worm Meeting "Measuring Protein Abundance across Developmental Stages of C. elegans via Molecular Weight Fractionation and High-Resolution Mass Spectrometry."

Merrihew, Gennifer E., MacCoss, Michael J., Angelo, Giana

[International Worm Meeting, 2011]

A vast majority of the genome annotation of "protein coding genes" is based on experimental data collected on the transcript level. While powerful, the analysis of some proteins will only be possible at the protein level. Mass spectrometry is a powerful experimental tool that can contribute significantly to the annotation of genomes at the protein level. Combining mass spectrometry with molecular weight fractionation techniques strengthens our ability to identify more proteins at varying abundances and can resolve different protein isoforms. In addition, analyzing different developmental stages of an organism will improve our knowledge of changes of protein abundance during development. We are currently analyzing 12 developmental stages of C. elegans - N2 mixed embryo, N2 L1, N2 L2, N2 L3, N2 L4, N2 YA, N2 dauer, spe-9 L4, spe-9 YA, spe-9 adult, spe-9 adult reproductive diapause (ARD) and him- 8. The developmental stages are separated into 16 molecular weight fractions ranging from 3.5 - 500 kDa using the GelFree 8100 fractionation system (Protein Discoveries). An unfractionated set is also analyzed for all developmental stages. The peptides from each fraction are analyzed using a 120-minute LC-MS/MS run on a Thermo LTQ-Orbi-Velos mass spectrometer. A biological and analytical replicate is performed for each sample. The MS/MS spectra are assigned to peptides using the SEQUEST algorithm, q-values are assigned at the peptide level using Percolator and the peptides are assembled into proteins using an in-house implementation of IDPicker. Presently, we have analyzed the unfractionated samples and the half of the first molecular weight fraction (3.5-18 kDa). We identified 2736 non-redundant proteins and 14558 peptides in the unfractionated sample set and 1828 non-redundant proteins and 7122 peptides in the first molecular weight fraction at a very stringent peptide q-value cut-off of 0.01. Extrapolation of the current results out to all biochemical fractions suggests we will have sufficient depth of coverage to provide quantitative measures of protein abundance and confirmation of protein coding genes for approximately half the protein coding genes in the genome. Additionally, because we have data about the molecular weight fraction that the tryptic peptides were identified in, we will show examples of proteolytic processing of the protein coding gene to a smaller functional protein.

Lourenco AB et al. (2015) Biochim Biophys Acta "Analysis of the effect of the mitochondrial prohibitin complex, a context-dependent modulator of longevity, on the C. elegans metabolome."

Munoz-Jimenez C, Artal-Sanz M, Venegas-Caleron M, Lourenco AB

[Biochim Biophys Acta, 2015]

The mitochondrial prohibitin complex, composed of two proteins, PHB-1 and PHB-2, is a context-dependent modulator of longevity. Specifically, prohibitin deficiency shortens the lifespan of otherwise wild type worms, while it dramatically extends the lifespan under compromised metabolic conditions. This extremely intriguingly phenotype has been linked to alterations in mitochondrial function and in fat metabolism. However, the true function of the mitochondrial prohibitin complex remains elusive. Here, we used gas chromatography coupled to a flame ionization detector (GC/FID) and H NMR spectroscopy to gain molecular insights into the effect of prohibitin depletion on the Caenorhabditis elegans metabolome. We analysed the effect of prohibitin deficiency in two different developmental stages and under two different conditions, which result in opposing longevity phenotypes, namely wild type worms and daf-2(e1370) insulin signalling deficient mutants. Prohibitin depletion was shown to alter the fatty acid (GC/FID) and H NMR metabolic profiles of wild type animals both at the fourth larval stage of development (L4) and at the young adult (YA) stage, while being more pronounced at the later stage. Furthermore, wild type and the diapause mutant daf-2(e1370), either expressing or not prohibitin, were clearly distinguishable based on their metabolic profiles, revealing changes in fatty acid composition, as well as in carbohydrate and amino acid metabolism. Moreover, the metabolic data indicate that daf-2(e1370) mutants are more robust than the wild type animals to changes induced by prohibitin depletion. The impact of prohibitin depletion on the C. elegans metabolome will be discussed herein in the scope of its effect on longevity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging. Guest Editor: Aleksandra Trifunovic.

Paupard M-C et al. (2000) East Coast Worm Meeting "Improved Tissue Preservation Using Metal Mirror Fixation or High Pressure Freezing for TEM"

Macaluso F, Stephney G, Paupard M-C, Hall DH

[East Coast Worm Meeting, 2000]

Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.