Changes in cGMP Levels Affect the Localization of EGL-4 in AWC in Caenorhabditis elegans

The Protein Kinase G, EGL-4, is required within the C. elegans AWC sensory neurons to promote olfactory adaptation. After prolonged stimulation of these neurons, EGL-4 translocates from the cytosol to the nuclei of the AWC. This nuclear translocation event is both necessary and sufficient for adaptation of the AWC neuron to odor. A cGMP binding motif within EGL-4 and the Gα protein ODR-3 are both required for this translocation event, while loss of the guanylyl cyclase ODR-1 was shown to result in constitutively nuclear localization of EGL-4. However, the molecular changes that are integrated over time to produce a stably adapted response in the AWC are unknown. Here we show that odor-induced fluctuations in cGMP levels in the adult cilia may be responsible in part for sending EGL-4 into the AWC nucleus to produce long-term adaptation. We found that reductions in cGMP that result from mutations in the genes encoding the cilia-localized guanylyl cyclases ODR-1 and DAF-11 result in constitutively nuclear EGL-4 even in naive animals. Conversely, increases in cGMP levels that result from mutations in cGMP phosphodiesterases block EGL-4 nuclear entry even after prolonged odor exposure. Expression of a single phosphodiesterase in adult, naive animals was sufficient to modestly increase the number of animals with nuclear EGL-4. Further, coincident acute treatment of animals with odor and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) decreased the number of animals with nuclear EGL-4. These data suggest that reducing cGMP levels in AWC is necessary and even partially sufficient for nuclear translocation of EGL-4 and adaptation as a result of prolonged odor exposure. Our genetic analysis and chemical treatment of C. elegans further indicate that cilia morphology, as defined by fluorescent microscopic observation of the sensory endings, may allow for odor-induced fluctuations in cGMP levels and this fluctuation may be responsible for sending EGL-4 into the AWC nucleus

Regulators of AWC-Mediated Olfactory Plasticity in Caenorhabditis elegans

While most sensory neurons will adapt to prolonged stimulation by down-regulating their responsiveness to the signal, it is not clear which events initiate long-lasting sensory adaptation. Likewise, we are just beginning to understand how the physiology of the adapted cell is altered. Caenorhabditis elegans is inherently attracted to specific odors that are sensed by the paired AWC olfactory sensory neurons. The attraction diminishes if the animal experiences these odors for a prolonged period of time in the absence of food. The AWC neuron responds acutely to odor-exposure by closing calcium channels. While odortaxis requires a Gα subunit protein, cGMP-gated channels, and guanylyl cyclases, adaptation to prolonged odor exposure requires nuclear entry of the cGMP-dependent protein kinase, EGL-4. We asked which candidate members of the olfactory signal transduction pathway promote nuclear entry of EGL-4 and which molecules might induce long-term adaptation downstream of EGL-4 nuclear entry. We found that initiation of long-term adaptation, as assessed by nuclear entry of EGL-4, is dependent on G-protein mediated signaling but is independent of fluxes in calcium levels. We show that long-term adaptation requires polyunsaturated fatty acids (PUFAs) that may act on the transient receptor potential (TRP) channel type V OSM-9 downstream of EGL-4 nuclear entry. We also present evidence that high diacylglycerol (DAG) levels block long-term adaptation without affecting EGL-4 nuclear entry. Our analysis provides a model for the process of long-term adaptation that occurs within the AWC neuron of C. elegans: G-protein signaling initiates long-lasting olfactory adaptation by promoting the nuclear entry of EGL-4, and once EGL-4 has entered the nucleus, processes such as PUFA activation of the TRP channel OSM-9 may dampen the output of the AWC neuron

Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor

SummaryMost eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation.PaperFlic

Subcellular localization of GFP::EGL-4 in AWC does not affect cilia morphology.

<p>(A) Fluorescent confocal image of the AWC neuron in a wildtype animal. Inset is a magnified view of the AWC cilia. (B) Fluorescent confocal image of wildtype animal expressing a constitutively nuclear form of GFP::EGL-4. The animal displays normal AWC cilia. Inset is a magnified view of the AWC cilia. (C) An <i>egl-4(n479)</i> mutant animal expressing constitutively cytosolic GFP::EGL-4 displays normal AWC cilia. Inset is a magnified view of the AWC cilia. (D) Though the <i>py825</i> mutant strain has constitutively nuclear EGL-4, expressing a cytosolic version of GFP::EGL-4 does not rescue the ciliopathy of <i>py825</i> mutant animals. (E) Quantification of the localization of GFP::EGL-4 in AWC. In a wildtype population very few animals display nuclear GFP::EGL-4. In the mutant <i>che-3(e1124),</i> GFP::EGL-4 is in the nucleus of all animals. Mutating a key residue in the cGMP binding site of EGL-4 prevents the nuclear entry of GFP::EGL-4 in <i>che-3(e1124)</i> mutant animals.** indicates statistical significance at <i>p</i><0.005 between wildtype and <i>che-3</i> mutant animals For all images anterior is to the left, and white dotted lines indicate outline of animal's head region. <i>P</i> values calculated using the Student's <i>t</i>-test. Error bars represent the S.E.M.</p

Decreases in cGMP levels direct the nuclear entry of GFP::EGL-4 in AWC and promote adaptation.

<p>(A) Percent of animals displaying nuclear GFP::EGL-4 in AWC of naive (black bars), benzaldehyde-exposed (gray bars) and butanone-exposed (white bars) treatments. **Indicates <i>p</i>≤0.005 significant differences between nuclear GFP::EGL-4 values of wildtype unadapted animals and <i>odr-1</i> or <i>daf-11</i> mutant unadapted animals, and also significant differences between wildtype adapted and <i>pde</i> quadruple mutant adapted values. (B) Chemotaxis response of PDE mutants and the guanylyl cyclase mutants <i>daf-11</i> and <i>odr-1</i> to the AWC sensed odor benzaldehyde. “−” indicates unexposed animals and “+” indicates exposed animals. **Indicates <i>p</i>≤0.005 significant differences between chemotaxis index (CI) values between wildtype unexposed animals and mutant unexposed animals. *Indicates <i>p</i>≤0.05 significant differences between wildtype odor-exposed CI values and mutant odor-exposed CI values. (C) Populations of GFP::EGL-4 (<i>pyIs500</i>) expressing animals were exposed to the odor benzaldehyde with or without the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Populations exposed to 10 mM concentrations of IBMX displayed a reduced number of animals exhibiting nuclear GFP::EGL-4 in AWC at 80 minutes post exposure to benzaldehyde and IBMX. *Indicates <i>p</i>≤0.05 significant differences between odor treated animals with IBMX versus odor treated animals without IBMX. (D) Expression of the cGMP phosphodiesterase PDE-3 under a heat-inducible promoter causes some increase in the number of animals displaying nuclear GFP::EGL-4. *Indicates <i>p</i>≤0.05 significant differences between wildtype and transgenic animals after heat induction; **Indicates <i>p</i>≤0.005 significant differences between transgenic animals with and without heat induction. “−” indicates no heat induction and “+” indicates after heat induction. <i>P</i> values calculated using the Student's <i>t</i>-test. Error bars represent the S.E.M.</p

Forward genetic screen revealed that cilia defective mutants show constitutively nuclear EGL-4.

<p>(A) In wildtype naive animals GFP::EGL-4 is diffuse throughout the entire cytosol of AWC, in the mutant <i>py825</i>, GFP::EGL-4 is constitutively accumulated in the nucleus of AWC and GFP::EGL-4 is seen constitutively accumulated in the nucleus of AWC in the mutant <i>py827</i>. The constitutively nuclear GFP::EGL-4 mutants <i>py825</i> and <i>py827</i> are chemotaxis and dye filling defective. (B) Chemotaxis responses of wildtype animals and the mutants, <i>py825</i> and <i>py827</i> to the AWC sensed odors benzaldehyde (black bars) and isoamyl alcohol (gray bars). **Indicates <i>p</i>≤0.005, and *indicates <i>p</i>≤0.05 significant differences between mutants and wildtype animals for each odor. (C) Dye filling with the lipophillic dye DiD in wildtype, <i>py825</i>, and <i>py827</i> animals. The amphid neurons of the mutants <i>py825</i> and <i>py827</i> fail to dye fill. (D) <i>py825</i> encodes OSM-1, an ortholog of the intraflagellar transport (IFT) complex B component, IFT172. Three factor mapping with <i>dpy-6 unc-3</i> placed <i>py825</i> close to <i>unc-3</i>. The molecular lesion of <i>py825</i> is indicated by an arrowhead. (E) <i>py827</i> encodes CHE-3, the isoform 1b of the dynein heavy chain (DHC). Gene structure of <i>che-3</i> and molecular lesion of <i>py827</i>. The molecular lesion of <i>py827</i> is indicated in the gene structure by a black horizontal line. Three factor mapping with <i>dpy-5 unc-13</i> placed <i>py825</i> near <i>unc-13</i>. Exon intron graphic generated using the web application at: <a href="http://wormweb.org/exonintron" target="_blank">http://wormweb.org/exonintron</a>. (F) Cartoon illustrating the features of the (p)<i>odr-1</i>::DsRed expression pattern in the subsequent fluorescent confocal images. (G) Normal AWC cell and cilia morphology in wildtype animals. (H) AWC cilia defects displayed by <i>py825</i>. (I) AWC cilia defects observed in <i>py825/osm-1(pr816)</i> trans heterozygotes. (J) Normal cilia morphology observed in <i>py825</i> transgenic animals expressing a rescuing array of OSM-1. (K) AWC cilia defect displayed by <i>py827</i>. (L) AWC cilia defects observed in <i>py827/che-3(e1124)</i> trans heterozygotes. (M) Normal cilia morphology observed in <i>py827</i> transgenic animals expressing a rescuing array of CHE-3. (N) Quantification of the constitutively nuclear GFP::EGL-4 phenotype in <i>py825</i> and <i>py827</i> mutant animals. In a wildtype population very few animals display nuclear GFP::EGL-4. The mutants <i>py825</i> and <i>py827</i> both display nuclear GFP::EGL-4 in all animals. The nuclear GFP::EGL-4 mutant phenotype of <i>py825</i> is reduced to wildtype levels in transgenic mutant animals expressing the rescuing array containing OSM-1, and similarly for <i>py827</i> the nuclear GFP::EGL-4 mutant phenotype is reduced to wildtype levels in transgenic mutant animals expressing the rescuing array containing CHE-3.**Indicates <i>p</i>≤0.005 significant differences between wildtype and mutant animals. <i>P</i> values calculated using the Students <i>t</i>-test. Error bars represent the S.E.M. For all images anterior is to the left, and white dotted lines indicate outline of animal's head region in (C) and white dotted rectangles indicate cilia structural defects in (H), (I), (K) and (L).</p

Oncogene-regulated release of extracellular vesicles

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

miR-380-5p represses p53 to control cellular survival and is associated with poor outcome in MYCN-amplified neuroblastoma

A drawback of electrical stimulation for muscle control is that large, fatigable motor units are preferentially recruited before smaller motor units by the lowest-intensity electrical cuff stimulation. This phenomenon limits therapeutic applications because it is precisely the opposite of the normal physiological (orderly) recruitment pattern; therefore, a mechanism to achieve orderly recruitment has been a long-sought goal in physiology, medicine and engineering. Here we demonstrate a technology for reliable orderly recruitment in vivo. We find that under optical control with microbial opsins, recruitment of motor units proceeds in the physiological recruitment sequence, as indicated by multiple independent measures of motor unit recruitment including conduction latency, contraction and relaxation times, stimulation threshold and fatigue. As a result, we observed enhanced performance and reduced fatigue in vivo. These findings point to an unanticipated new modality of neural control with broad implications for nervous system and neuromuscular physiology, disease research and therapeutic innovation