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Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in  C. elegans
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, article id 8410Article in journal (Refereed) Published
Abstract [en]

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Place, publisher, year, edition, pages
Springer Nature, 2023. Vol. 14, article id 8410
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:umu:diva-217489DOI: 10.1038/s41467-023-44177-zPubMedID: 38110404Scopus ID: 2-s2.0-85180225404OAI: oai:DiVA.org:umu-217489DiVA, id: diva2:1817185
Funder
Swedish Research Council, 2018-02914Swedish Research Council, 2021-06602Swedish Research Council, 2018-02216
Note

Originally included in thesis in manuscript form. 

Available from: 2023-12-05 Created: 2023-12-05 Last updated: 2024-01-05Bibliographically approved
In thesis
1. A molecular exploration of sensory responses in c. elegans
Open this publication in new window or tab >>A molecular exploration of sensory responses in c. elegans
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Sensation provides a pivotal ability, allowing animals to survive in complex environments. The cues sensed by animals are represented by external stimuli and internal signals. However, the mechanisms mediating sensations in molecular and cellular level are still not well-studied. In this thesis, by using free-living nematodes C. elegans with relatively simple nerve system, we are trying to get better understandings of molecular mechanisms by which animals sense and interpret external cues and internal signals.

G protein-coupled receptors (GPCRs), as one of the major families of transmembrane proteins, participate in a variety of physiological responses to both external stimuli and internal cues. Previous studies have shown that GPCR signals are broadly involved in many processes in C. elegans, such as olfactory sensing, nociceptive responses, social behavior, pathogen responses, and mating. However, the complexity and diversity of GPCRs pose significant challenges to systematic dissection of their function as well as identification of receptor-ligand pairs which play crucial roles for animals´ sensory behaviors. Interestingly, the genome of C. elegans encodes one of the largest GPCR repertoires among any sequenced organisms, indicating a dramatical expansion and high degree of gene redundancy. To comprehensively dissect GPCR signaling in C. elegans and gain more insights into their roles in sensations, we developed an approach by employing CRISPR/Cas9-based gene editing to mutate closely related GPCRs and neuropeptide genes (internal signals) in a single strain on a genome-wide scale, resulting in disrupting nearly all the GPCR and neuropeptide genes (more than 1800 genes in total) and eliminating high degree of gene redundancy as well. Then using these two genetic libraries, we successfully identified neuropeptide (FLP -1) and cognate receptors (DMSR-4, DMSR-7 and DMSR-8) required for hypoxia-evoked locomotory responses, obtained a set of novel regulators of the pathogen-induced immune response including FMI-1 and DOP-6, and especially identified receptors (SRX-64) in AWA neurons for the volatile odorant pyrazine and redundant receptors (SRX-1, SRX-2 and SRX-3) in AWCOFF neuron for 2,3-pentanedione.

In nature, animals often experience and sense constantly changing gas environments. And human bodies also generate internal gas as gasotransmitters for signal transduction, such as CO, NO and H2S. For the mechanism governing sensory and adaptive responses to different gaseous cues, extensive studies are still needed. Here, taking advantage of the robust locomotory responses to H2S in C. elegans, we delineated the molecular mechanisms of H2S sensation and adaptation. We found that C. elegans exhibited transiently increased locomotory and turning activity as a strategy to escape the noxious H2S. The behavioral responses to H2S were modulated by a complex network of signaling pathways, ranging from cyclic GMP signaling in ciliated sensory neurons, calcineurin, nuclear hormone receptors, to the major starvation regulators such as insulin and TGF-β signaling. Prolonged exposure to H2S robustly evoked H2S detoxification and reprogrammed gene expression, where genes involved in iron homeostasis, including ftn-1 and smf-3, were robustly modified, implying that labile iron levels are affected by H2S. In addition, the roles of labile iron for modulating H2S response were further investigated by using genetic studies and chemical applications. Interestingly, the response to H2S was substantially affected by the ambient O2 levels and their prior experience in low O2 environments, suggesting an intricate interplay between O2 and H2S sensing. The crosstalk is often seen between different experiences and sensations. In addition to the interplay between O2 and H2S sensing, we found hypoxia challenge could induce food leaving behavior in C. elegans. The alteration of food behavior by hypoxia experience was independent of the known mechanisms involved in O2 response, including pathways in acute hypoxia and HIF-1 signaling for chronic hypoxia response. The robust failure of induced food avoidance in egl-3 and egl-21 mutants suggested that neuropeptidergic signaling was required for this response. And future work is needed for comprehensively understanding the roles of neuropeptide signaling in the crosstalk between hypoxia experience and food leaving behavior.

In summary, our studies shed light on the molecular and cellular mechanisms of how animals sense and interpret the signals, allowing them to survive in a complex environment niche. More specifically, 1) we demonstrated the dissection of genetic landscape of GPCR signaling through phenotypic profiling in C. elegans. And as a powerful genetic resource, our libraries can greatly expedite the analyses of GPCR signaling in multiple additional contexts. 2) we provided molecular insights into how C. elegans detects and adapts its response to H2S and modulates behaviors through ambient environment and experience. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2023. p. 86
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2276
Keywords
C. elegans, sensation, CRISPR/Cas9, G protein-coupled receptors, neuropeptides, acute hypoxia, pathogen, chemosensation, hydrogen sulfide, HIF-1, iron
National Category
Neurosciences Genetics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-217493 (URN)978-91-8070-238-6 (ISBN)978-91-8070-239-3 (ISBN)
Public defence
2024-01-19, KBE301-Lilla hörsalen, KBC-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2023-12-21 Created: 2023-12-08 Last updated: 2023-12-11Bibliographically approved

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Pu, LongjunWang, JingLu, QiongxuanNilsson, LarsZhao, LinaMyint, Si LhyamWilliams, ChloeGilthorpe, Jonathan D.Wai, Sun NyuntHenriksson, JohanChen, Changchun

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Pu, LongjunWang, JingLu, QiongxuanNilsson, LarsZhao, LinaMyint, Si LhyamWilliams, ChloeGilthorpe, Jonathan D.Wai, Sun NyuntHenriksson, JohanChen, Changchun
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Umeå Centre for Molecular Medicine (UCMM)Department of Molecular Biology (Faculty of Medicine)Wallenberg Centre for Molecular Medicine at Umeå University (WCMM)Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)Department of Integrative Medical Biology (IMB)
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