Shin N et al. (2020) Sci Rep "Micelle-stabilized Olfactory Receptors for a Bioelectronic Nose Detecting Butter Flavors in Real Fermented Alcoholic Beverages."

Park TH, Lee SH, Shin N, Pham Ba VA, Hong S

[Sci Rep, 2020]

A bioelectronic nose device based on micelle-stabilized olfactory receptors is developed for the selective discrimination of a butter flavor substance in commercial fermented alcoholic beverages. In this work, we have successfully overexpressed ODR-10, a type of olfactory receptor, from Caenorhabditis elegans using a bacterial expression system at a low cost and high productivity. The highly-purified ODR-10 was stabilized in micelle structures, and it was immobilized on a carbon nanotube field-effect transistor to build a bioelectronic nose for the detection of diacetyl, a butter flavor substance, via the specific interaction between diacetyl and ODR-10. The bioelectronic nose device can sensitively detect diacetyl down to 10 fM, and selectively discriminate it from other substances. In addition, this sensor could directly evaluate diacetyl levels in a variety of real fermented alcoholic beverages such as beer, wine, and makgeolli (fermented Korean wine), while the sensor did not respond to soju (Korean style liquor without diacetyl). In this respect, our sensor should be a powerful tool for versatile food industrial applications such as the quality control of alcoholic beverages and foods.

Zhang T et al. (2021) Food Funct "Differentiated 4,4-dimethylsterols from vegetable oils reduce fat deposition depending on the NHR-49/SCD pathway in Caenorhabditis elegans."

Chang M, Zhang T, Xie L, Wang X, Jin Q, Liu R

[Food Funct, 2021]

Consumption of 4-desmethylsterols has been claimed to have many beneficial effects, but the benefits of 4,4-dimethylsterols are less appreciated. We utilized a nematode model, Caenorhabditis elegans (C. elegans), to explore the anti-obesity effects of different classes of 4,4-dimethylsterols purified from rice bran oil (RST) and shea nut butter (SST). Both SST and RST significantly reduced fat deposition in C. elegans with smaller sizes and numbers of lipid droplets. But the food intake was not significantly affected. Metabolomics analysis indicated a significantly altered pathway after treatment with 4,4-dimethylsterols. Finally, it was found that 4,4-dimethylsterols targeted stearoyl-CoA desaturases (SCD) and nuclear hormone receptor-49 (NHR-49), resulting in a reduced desaturation index as proved by a lower ratio of oleic acid (C18:1n-9) to stearic acid (C18:0). Overall, 4,4-dimethylsterols can inhibit fat deposition via regulating the NHR-49/SCD pathway in C. elegans.

Placentino, Maria et al. (2021) International Worm Meeting "Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the Caenorhabditis elegans embryo"

Butter, Falk, Isolehto, Ida, Placentino, Maria, Dietz, Sabrina, Ketting, Rene, de Jesus Domingues, Antonio Miguel, Hellmann, Svenja, de Albuquerque, Bruno FM, Schreier, Jan

[International Worm Meeting, 2021]

Argonaute proteins (AGOs) are involved in many gene regulatory processes and use small RNAs as guides to find their target. C. elegans employs several small RNA mediated pathways to initiate and maintain proper gene regulation throughout development. In germ cells, 21U RNAs and associated Argonautes are particularly important to maintain an immortal germline. 21U RNAs trigger a silencing mechanism that is dependent of amplification of 22G RNAs by RNA dependent RNA polymerase. Strikingly, silencing responses can become independent of triggering 21U RNAs and maintained across generations, a mechanism called RNA epigenetic memory (RNAe). Components of small RNA pathways often localize to perinuclear phase separated condensates termed germ granules. The exact functions of germ granules are not clear, but it is known that they are necessary for proper function of small RNA pathways and inheritance of small RNAs. Intrinsically disordered regions (IDRs) can mediate multivalent protein-protein interactions and are often important for phase separation. We show that maternally provided 21U RNAs are sufficient to initiate RNAe in embryos and that the IDR containing protein PID-2/ZSP-1 is required for this process. PID-2/ZSP-1 localizes to and affects the appearance and number of Z-granules[1][2]. Furthermore, we found that PID-2/ZSP-1 mutually exclusively interacts with PID-4 and PID-5. Mutants lacking PID-4 and PID-5 partially phenocopy pid-2 mutants. Interestingly, even though PID-4 and PID-5 interact with PID-2, they localize to the adjacent P-granules rather than Z-granules. We identify distinct interacting proteins for PID-4 and PID-5. For instance, PID-5 interacts with the X-prolyl aminopeptidase APP-1 and PID-5 itself has an APP-1 related domain that is likely catalytically inactive. Several AGOs contain intrinsically disordered N-terminal tails and are predicted substrates for APP-1, suggesting a role for N-terminal processing in small RNA pathways and germ granule dynamics. Taken together, we have found a protein complex that affects small RNA mediated regulation and germ granules, potentially providing a mode of communication between different granules, and are on the route to understand the functional relationship between small RNA mediated gene regulation, epigenetic inheritance, and phase separation. [1] Placentino et al. EMBO J, 2021 [2] Wan et al. EMBO J, 2021

Schreier, Jan et al. (2021) International Worm Meeting "A novel sperm-specific compartment secures a cytoplasmic Argonaute protein for paternal epigenetic inheritance of small RNA-mediated gene silencing"

Boermel, Mandy, Butter, Falk, Dietz, Sabrina, Schreier, Jan, Ketting, Rene, Seistrup, Ann-Sophie, Domingues, Antonio, Bronkhorst, Alfred, Oorschot, Viola, L'Hernault, Steven, Phillips, Carolyn, Nguyen, Dieu

[International Worm Meeting, 2021]

Germ cells possess specialized perinuclear, phase-separated compartments, also named condensates. Amongst others, they contain the mRNA surveillance machinery responsible for transposon silencing and fertility. In the nematode Caenorhabditis elegans, three condensates, P granules, Z granules and Mutator foci, are home to RNA interference-related pathways, driven by a highly diversified Argonaute sub-clade (WAGO) that mediates gene silencing. Intriguingly, it has been shown that WAGO-mediated gene silencing can be inherited via both oocyte and sperm. Especially the inheritance via sperm is remarkable, since significant amounts of cellular material, including Argonaute proteins, are expelled from maturing spermatids into so-called residual bodies. How then does sperm-mediated inheritance of cytoplasmic RNAi work? We genetically identify WAGO-3 as a major Argonaute protein required for the paternal inheritance of endogenous small RNAs. Just like other Argonaute proteins, like WAGO-1 and ALG-3, WAGO-3 localizes to P granules in naive germ cells. During spermatogenesis, however, P granules disappear and WAGO-3, but not WAGO-1 and ALG-3, accumulates in a newly identified condensate, the PEI granule. In contrast to P granules, PEI granules remain stable during later stages of spermatogenesis. They are retained within maturing spermatids and selectively keep WAGO-3 from accumulating in the residual body. Using immunoprecipitation experiments followed by label-free quantitative mass spectrometry, we identified two uncharacterized proteins: PEI-1 and PEI-2. Both proteins are specifically expressed during spermatogenesis and we dissect their roles in PEI granule transport and function. Based on correlative light and electron microscopy (CLEM) and genetic studies, proper segregation of PEI granules in mature sperm is coupled, likely via S-palmitoylation, to the myosin-driven transport of membranous organelles. Our results identify a new sperm-specific condensate, which we call PEI granules. While not essential for spermatogenesis, PEI granules are required for paternal inheritance of small RNAs and we reveal a novel mechanism for the subcellular sorting of condensates through coupling to transport of membranous structures. pei-like genes are also found in human and often expressed in testis, suggesting that the here identified mechanism of subcellular transport of membraneless organelles may be more broadly conserved.

Tali Melkman et al. (2002) East Coast Worm Meeting "The paired-like homeodomain protein SNS-10 is required for the development of the AWA olfactory neurons"

Piali Sengupta, Tali Melkman

[East Coast Worm Meeting, 2002]

C. elegans is attracted to the butter-like smell of diacetyl and almond-like odor of benzaldehyde, but is repelled by 2-nonanone. These odorants are sensed by the olfactory neuron pairs AWA, AWC and AWB respectively. All three neuron pairs respond to volatile chemicals but do so using distinctive specialized cilia, and different signaling pathways. How do the similarities and differences between these (and other) neurons arise? In particular, what molecular mechanisms are at play? By comparing and contrasting the developmenal processes required in different sensory neurons we hope to understand how diversity of function is generated in the nervous system. A number of factors have been identified as playing a role in specifying the fate of the AWA olfactory neurons. odr-7 , a nuclear hormone receptor transcription factor, is expressed specifically in AWA and controls its specification, such that in odr-7 mutants AWA-specific gene expression and function is lost (Sengupta et al, 1994). lin-11, a LIM HD transcription factor, is required to initiate odr-7 expression (Sarafi-Reinach et al., 2001), and in lin-11 mutants, the development of both the AWA neurons and their lineal sisters, the ASG neurons is affected. Restriction of odr-7 expression to the AWA neurons is regulated by the forkhead transcription factor UNC-130 which is expressed in the AWA/ASG precursor cells (Sarafi-Reinach and Sengupta, 2000). In a screen aimed at identifying additional genes required for AWA specification, I isolated two alleles of the gene sns-10. In sns-10 mutants, the expression of both AWA-specific genes, such as odr-7, and ASG-specific genes is lost. I also observe occasional ectopic expression of ODR-7. sns-10 mutants exhibit various pleiotropies suggesting that sns-10 is involved in the development and/or function of additional cells. sns-10 encodes a paired-like homeodomain transcription factor with high homology to the Drosophila gene Aristaless. Interestingly, Aristaless is involved in the formation of the aristae sensory organs (Schneitz et al. 1993).

Dwyer ND et al. (1996) West Coast Worm Meeting "odr-4 ENCODES A NOVEL MEMBRANE PROTEIN REQUIRED FOR LOCALIZATION OF AN ODORANT RECEPTOR, odr-10"

Bargmann CI, Sengupta P, Dwyer ND

[West Coast Worm Meeting, 1996]

The simplicity of the worm chemosensory system provides an excellent model for signal transduction and general neuronal function. Through behavioral screening, our laboratory has identified many genes involved in chemosensation. One of these genes, odr-4, encodes a novel membrane protein that may be involved in targeting specific odorant receptors to the cilia of the neuron in which they are expressed. odr-4, odr-8, and odr-10 are all required for the response to diacetyl, a butter-like smell. While odr-10 is specific for diacetyl, odr-4 and odr-8 mutants are also defective in responding to benzaldehyde (sensed by AWC) and trimethylthiazole (sensed by AWA and AWC). An odr-4;odr-8 double mutant shows no new chemotaxis defects. This indicates that odr-4 and odr-8 are in the same pathway, perhaps upstream or downstream of odr-10 in a diacetyl-sensing pathway. Recently odr-10 was cloned (see Sengupta et al, Cell, vol. 10:899-909). It encodes a seven transmembrane domain receptor that localizes to the cilia of AWA. Therefore it is very likely to be the receptor for the odorant diacetyl. Interestingly, odr-4 and odr-8 mutants fail to localize gfp-tagged Odr-10 to the cilia of AWA. Instead, the receptor remains in the cell body at reduced levels. The AWA cilia are morphologically normal in odr-4 mutants, so this defect is not caused by a general defect in cilium development. We mapped odr-4 genetically to a small region on LGIII, and rescued the mutant with cosmid C28A6. Rescuing fragments were subcloned and used to screen a cDNA library. This yielded several full-length cDNA's representing two genes, and we made frameshifts to identify the odr-4 gene. Odr-4 appears to be a novel protein of 445 amino acids with no signal sequence and a transmembrane domain at the C-terminus. Two types of proteins share this unusual structure: syntaxins and ninaA. Syntaxins are conserved from yeast to mammals, and are thought to be involved in targeting membrane vesicles to specific cell membrane compartments. ninaA is a Drosophila cyclophilin, required for folding and localization of specific rhodopsins. We are testing models for odr-4 function based on these similarities. Using a gfp translational fusion, we found that odr-4 is expressed in a subset of chemosensory neurons, including AWA and AWC. We are currently undertaking experiments to determine subcellular localization, and whether odr-4 is required to localize other receptors or transduction machinery.