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1.
Rapid Commun Mass Spectrom ; 38(17): e9850, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39034751

RESUMO

RATIONALE: Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation. METHODS: This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results. RESULTS: An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5 µm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data. CONCLUSIONS: This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.


Assuntos
Caenorhabditis elegans , Lipídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Animais , Caenorhabditis elegans/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Lipídeos/análise , Lipídeos/química , Cromatografia Líquida/métodos
2.
Anal Chim Acta ; 1317: 342913, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39030025

RESUMO

BACKGROUND: Lipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. RESULTS: A SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SIGNIFICANCE: This method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.


Assuntos
Caenorhabditis elegans , Cromatografia com Fluido Supercrítico , Espectrometria de Mobilidade Iônica , Lipidômica , Lipídeos , Cromatografia com Fluido Supercrítico/métodos , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Animais , Espectrometria de Mobilidade Iônica/métodos , Lipidômica/métodos , Lipídeos/análise , Lipídeos/química , Espectrometria de Massas/métodos
3.
Anal Chem ; 96(21): 8432-8440, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38709576

RESUMO

Cytoarchitectural staining is of great importance in disease diagnosis and cell biology research. This study developed user-friendly multifunctional red-emissive carbon dots (R-CDs) for rapid cell nucleus staining via targeting nuclear proteins. R-CDs, simply prepared by electrochemical treatment of 1,2,4-benzenetriamine, exhibit strong emission at 635 nm when excited at 507 nm. The R-CDs can rapidly stain the nucleus of human SH-SY5Y, HepG2, and HUH-7 cells with a high signal-to-noise ratio owing to fluorescence enhancement after entering the nucleus. Compared to conventional cytosolic dyes such as Hoechst and DAPI, R-CDs are cheaper, more highly dispersed in water, and more stable (requiring no stringent storage conditions). The R-CDs show stable optical properties with insignificant photobleaching over 7 days and salt resistance up to 2 M of NaCl. More importantly, R-CDs, possessing a positive charge, allow rapid staining of live cells (3 min) and dead cells (10 s) in saline. According to kinetic variation, R-CDs can distinguish live cells from dead cells. Staining exhibits high efficiency in onion epidermal cells, Aspergillus niger, Caenorhabditis elegans, and human spermatozoa. The mechanism for efficient staining is based on their fast accumulation in the nucleus due to their small size and positive charge and strong interaction with nuclear proteins at amino acid residues of histidine and arginine, resulting in fluorescence enhancement by dozens of times. The developed R-CDs do not bind to DNA and would not cause genetic damage and will find various safe applications in biological and medical fields.


Assuntos
Carbono , Núcleo Celular , Pontos Quânticos , Humanos , Carbono/química , Núcleo Celular/química , Núcleo Celular/metabolismo , Pontos Quânticos/química , Animais , Proteínas Nucleares/metabolismo , Proteínas Nucleares/análise , Corantes Fluorescentes/química , Coloração e Rotulagem , Caenorhabditis elegans/química , Cebolas/química , Cebolas/citologia
4.
STAR Protoc ; 4(1): 101956, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36856764

RESUMO

A major barrier to immunostaining Caenorhabditis elegans is the permeabilization of the worm's cuticle without distorting or damaging its body. We present here a gel-based immobilization protocol for fixed worms coupled with chemical and enzymatic permeabilization. The permeabilization is followed by antibody staining and fluorescent imaging. This protocol can be modified for different fixatives, permeabilizing reagents, or molecular readouts. Unlike previous immunostaining approaches, such as freeze cracking or dissection, this protocol enables immunostaining across the whole body of a well-preserved C. elegans.


Assuntos
Resinas Acrílicas , Caenorhabditis elegans , Animais , Caenorhabditis elegans/química , Fixadores , Anticorpos
5.
ACS Synth Biol ; 12(3): 700-708, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36802521

RESUMO

Optogenetic techniques have been intensively applied to the nematode Caenorhabditis elegans to investigate its neural functions. However, as most of these optogenetics are responsive to blue light and the animal exhibits avoidance behavior to blue light, the application of optogenetic tools responsive to longer wavelength light has been eagerly anticipated. In this study, we report the implementation in C. elegans of a phytochrome-based optogenetic tool that responds to red/near-infrared light and manipulates cell signaling. We first introduced the SynPCB system, which enabled us to synthesize phycocyanobilin (PCB), a chromophore for phytochrome, and confirmed the biosynthesis of PCB in neurons, muscles, and intestinal cells. We further confirmed that the amount of PCBs synthesized by the SynPCB system was sufficient for photoswitching of phytochrome B (PhyB)-phytochrome interacting factor 3 (PIF3). In addition, optogenetic elevation of intracellular Ca2+ levels in intestinal cells induced a defecation motor program. These SynPCB system and phytochrome-based optogenetic techniques would be of great value in elucidating the molecular mechanisms underlying C. elegans behaviors.


Assuntos
Fitocromo , Animais , Caenorhabditis elegans/química , Raios Infravermelhos , Optogenética , Transdução de Sinais/genética
6.
Nucleic Acids Res ; 51(4): 1512-1527, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36598924

RESUMO

Microprocessor (MP) is a complex involved in initiating the biogenesis of microRNAs (miRNAs) by cleaving primary microRNAs (pri-miRNAs). miRNAs are small single-stranded RNAs that play a key role in the post-transcriptional regulation of gene expression. Thus, understanding the molecular mechanism of MP is critical for interpreting the roles of miRNAs in normal cellular processes and during the onset of various diseases. MP comprises a ribonuclease enzyme, DROSHA, and a dimeric RNA-binding protein, which is called DGCR8 in humans and Pasha in Caenorhabditis elegans. DROSHA cleaves stem-loop structures located within pri-miRNAs to generate pre-miRNAs. Although the molecular mechanism of human MP (hMP; hDROSHA-DGCR8) is well understood, that of Caenorhabditis elegans MP (cMP; cDrosha-Pasha) is still largely unknown. Here, we reveal the molecular mechanism of cMP and show that it is distinct from that of hMP. We demonstrate that cDrosha and Pasha measure ∼16 and ∼25 bp along a pri-miRNA stem, respectively, and they work together to determine the site of cMP cleavage in pri-miRNAs. We also demonstrate the molecular basis for their substrate measurement. Thus, our findings reveal a previously unknown molecular mechanism of cMP; demonstrate the differences between the mechanisms of hMP and cMP; and provide a foundation for revealing the mechanisms regulating miRNA expression in different animal species.


The Microprocessor complex that initiates miRNA biogenesis was discovered in animals in 2004. However, the molecular mechanism of C. elegans Microprocessor (cMP) has remained elusive since its discovery 18 years ago. In this study, we revealed the unique molecular mechanism of cMP by conducting high-throughput pri-miRNA cleavage assays. We demonstrated that cMP, consisting of cDrosha and Pasha, each can measure the stem lengths of pri-miRNAs. cDrosha measures ∼16 bp of the lower stem length, whereas Pasha measures ∼25 bp of the upper stem in pri-miRNAs. In addition, we identified the cleavage sites and cleavage efficiency of cMP in C. elegans pri-miRNAs. These results will be helpful for future studies of miRNA biogenesis in C. elegans.


Assuntos
Caenorhabditis elegans , MicroRNAs , Proteínas de Ligação a RNA , Animais , Humanos , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Ribonuclease III/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo
7.
Nature ; 610(7933): 796-803, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36224384

RESUMO

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.


Assuntos
Caenorhabditis elegans , Microscopia Crioeletrônica , Canais Iônicos , Mecanotransdução Celular , Animais , Arrestinas/química , Arrestinas/metabolismo , Arrestinas/ultraestrutura , Caenorhabditis elegans/química , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestrutura , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/ultraestrutura , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Lipídeos
8.
RNA ; 28(1): 58-66, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34772788

RESUMO

Nuage are RNA-rich condensates that assemble around the nuclei of developing germ cells. Many proteins required for the biogenesis and function of silencing small RNAs (sRNAs) enrich in nuage, and it is often assumed that nuage is the cellular site where sRNAs are synthesized and encounter target transcripts for silencing. Using C. elegans as a model, we examine the complex multicondensate architecture of nuage and review evidence for compartmentalization of silencing pathways. We consider the possibility that nuage condensates balance the activity of competing sRNA pathways and serve to limit, rather than enhance, sRNA amplification to protect transcripts from dangerous runaway silencing.


Assuntos
Condensados Biomoleculares/química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , Interferência de RNA , RNA de Helmintos/química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Embrião não Mamífero , Grânulos de Ribonucleoproteínas de Células Germinativas/metabolismo , Grânulos de Ribonucleoproteínas de Células Germinativas/ultraestrutura , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , RNA de Helmintos/metabolismo , RNA Interferente Pequeno/metabolismo
9.
STAR Protoc ; 2(4): 101013, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34917984

RESUMO

Myristoylation is a type of lipidation with important functions. Owing to the lack of high-quality antibodies against myristoylation, developing alternative methods for profiling myristoylated proteins is important. Here, we provide a protocol for metabolic labeling using click chemistry to profile myristoylated proteins in C. elegans. Our approach improves the signal/noise ratio by covalently linking the myristoylated proteins to the beads. This protocol provides a highly specific and reproducible way for enriching myristoylated proteins, which could be modified to analyze other types of lipidations. For complete details on the use and execution of this protocol, please refer to Tang et al. (2021).


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Ácido Mirístico , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Química Click/métodos , Ácido Mirístico/análise , Ácido Mirístico/química , Ácido Mirístico/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/análise , Proteoma/química , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
10.
Elife ; 102021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34787570

RESUMO

Functional requirements constrain protein evolution, commonly manifesting in a conserved amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) is a ~100-nm-wide ladder-like meiotic structure present in all eukaryotic clades, where it aligns parental chromosomes and regulates exchanges between them. Despite the conserved ultrastructure and functions of the SC, SC proteins are highly divergent within Caenorhabditis. However, SC proteins have highly conserved length and coiled-coil domain structure. We found the same unconventional conservation signature in Drosophila and mammals, and used it to identify a novel SC protein in Pristionchus pacificus, Ppa-SYP-1. Our work suggests that coiled-coils play wide-ranging roles in the structure and function of the SC, and more broadly, that expanding sequence analysis beyond measures of per-site similarity can enhance our understanding of protein evolution and function.


Assuntos
Caenorhabditis elegans/química , Drosophila melanogaster/química , Complexo Sinaptonêmico/química , Animais , Rabditídios/química , Especificidade da Espécie , Relação Estrutura-Atividade
11.
Sci Rep ; 11(1): 21346, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725424

RESUMO

The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Animais , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Ciclo Celular/química , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP90/química , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Mapas de Interação de Proteínas
12.
mBio ; 12(5): e0257921, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34634942

RESUMO

A variety of effector proteins contribute to host defense in Caenorhabditis elegans. However, beyond lytic enzymes and antimicrobial peptides and proteins, little is known about the exact function of these infection-related effectors. This study set out to identify pathogen-dependent cytokine-like molecules, focusing on C-type lectin domain-containing proteins (CLECs). In total, 38 CLECs that are differentially regulated in response to bacterial infections have been previously identified by microarray and transcriptome sequencing (RNA-seq) analyses in C. elegans. We successfully cloned 18 of these 38 CLECs and chose to focus on CLEC-47 because, among these 18 cloned CLECs, it was the smallest protein and was recombinantly expressed at the highest levels in prokaryotic cells examined by SDS-PAGE. Quantitative real-time PCR (qRT-PCR/qPCR) showed that the expression of clec-47 was induced by a variety of Gram-positive bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, and Cutibacterium acnes, but was suppressed by the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. By expressing CLEC-47 in HEK 293 cells, we showed that CLEC-47 is released into the culture media, which the Golgi apparatus inhibitors (brefeldin A [BFA] and GolgiStop) could block. Purified recombinant CLEC-47 (maltose binding protein [MBP]-CLEC-47-His) did not display antimicrobial activity against ESKAPE pathogen isolates but bound directly to murine macrophage J774A.1 cells. Recombinant CLEC-47 attracted and recruited J774A.1 cells in a chemotaxis assay. In addition, qPCR studies and enzyme-linked immunosorbent assays (ELISAs) showed that CLEC-47 activates J774A.1 cells in a dose- and time-dependent manner to express the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, and Macrophage Inflammatory Protein 2 (MIP-2). Moreover, C. elegans, fed with CLEC-47-expressing Escherichia coli, demonstrated enhanced expression of several antimicrobial proteins (CNC-1, CNC-2, CPR-1, and CPR-2) as well as the detoxification protein MTL-1. These data suggest that CLEC-47 functions as a novel cytokine-like signaling molecule and exemplify how the study of infection-related effectors in C. elegans can help elucidate the evolution of immune responses. IMPORTANCE A variety of effector proteins contribute to host defense in the nematode Caenorhabditis elegans. However, little is known about the exact function of these infection-related effectors beyond lytic enzymes and antimicrobial peptides and proteins. This study set out to identify pathogen-dependent cytokine-like molecules, and we focus on the C-type lectin domain-containing proteins (CLECs). Our data suggest that CLEC-47 functions as a novel cytokine-like signaling molecule and exemplify how the study of infection-related effectors in nematodes can help elucidate the evolution of immune responses.


Assuntos
Infecções Bacterianas/imunologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/imunologia , Caenorhabditis elegans/química , Caenorhabditis elegans/imunologia , Citocinas/imunologia , Imunidade Inata , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Linhagem Celular , Citocinas/classificação , Citocinas/genética , Células HEK293 , Humanos , Camundongos , Domínios Proteicos
13.
STAR Protoc ; 2(2): 100547, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34095861

RESUMO

Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/química , Proteoma , Proteômica/métodos , Animais , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/isolamento & purificação , Nitrosação , Proteoma/análise , Proteoma/química , Proteoma/isolamento & purificação , S-Nitrosotióis
14.
Elife ; 102021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33950834

RESUMO

Neural circuits develop through a plastic phase orchestrated by genetic programs and environmental signals. We have identified a leucine-rich-repeat domain transmembrane protein PAN-1 as a factor required for synaptic rewiring in C. elegans. PAN-1 localizes on cell membrane and binds with MYRF, a membrane-bound transcription factor indispensable for promoting synaptic rewiring. Full-length MYRF was known to undergo self-cleavage on ER membrane and release its transcriptional N-terminal fragment in cultured cells. We surprisingly find that MYRF trafficking to cell membrane before cleavage is pivotal for C. elegans development and the timing of N-MYRF release coincides with the onset of synaptic rewiring. On cell membrane PAN-1 and MYRF interact with each other via their extracellular regions. Loss of PAN-1 abolishes MYRF cell membrane localization, consequently blocking myrf-dependent neuronal rewiring process. Thus, through interactions with a cooperating factor on the cell membrane, MYRF may link cell surface activities to transcriptional cascades required for development.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Fatores de Transcrição/metabolismo , Animais , Sistemas CRISPR-Cas , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Transporte Proteico , Sinapses/fisiologia , Fatores de Transcrição/genética
15.
J Vis Exp ; (170)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33999031

RESUMO

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.


Assuntos
Cálcio/análise , Animais , Comportamento Animal , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Interneurônios/fisiologia , Vibração
16.
STAR Protoc ; 2(2): 100411, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33870220

RESUMO

RNA tomography or tomo-seq combines mRNA sequencing and cryo-sectioning to spatially resolve gene expression. We have adapted this method for the nematode Caenorhabditis elegans to generate anteroposterior gene expression maps at near-cellular resolution. Here, we provide a detailed overview of the method and present two approaches: one that includes RNA isolation for maximum sensitivity and one that is suitable for partial automatization and is therefore less time-consuming. For complete details on the use and execution of this protocol, please refer to Ebbing et al. (2018).


Assuntos
Caenorhabditis elegans , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Tomografia/métodos , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genética
17.
Methods Mol Biol ; 2252: 151-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33765274

RESUMO

Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.


Assuntos
Caenorhabditis elegans/química , RNA Mensageiro/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Biotina/química , Meios de Cultura/química , Replicação do DNA , Sequenciamento de Nucleotídeos em Larga Escala , Biossíntese de Proteínas , RNA Mensageiro/química , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNA
18.
Nature ; 591(7848): 99-104, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627875

RESUMO

Neuropil is a fundamental form of tissue organization within the brain1, in which densely packed neurons synaptically interconnect into precise circuit architecture2,3. However, the structural and developmental principles that govern this nanoscale precision remain largely unknown4,5. Here we use an iterative data coarse-graining algorithm termed 'diffusion condensation'6 to identify nested circuit structures within the Caenorhabditis elegans neuropil, which is known as the nerve ring. We show that the nerve ring neuropil is largely organized into four strata that are composed of related behavioural circuits. The stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create neural structures that cage the strata within the nerve ring. We use high resolution light-sheet microscopy7,8 coupled with lineage-tracing and cell-tracking algorithms9,10 to resolve the developmental sequence and reveal principles of cell position, migration and outgrowth that guide stratified neuropil organization. Our results uncover conserved structural design principles that underlie the architecture and function of the nerve ring neuropil, and reveal a temporal progression of outgrowth-based on pioneer neurons-that guides the hierarchical development of the layered neuropil. Our findings provide a systematic blueprint for using structural and developmental approaches to understand neuropil organization within the brain.


Assuntos
Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Neurópilo/química , Neurópilo/metabolismo , Algoritmos , Animais , Encéfalo/citologia , Encéfalo/embriologia , Caenorhabditis elegans/química , Caenorhabditis elegans/citologia , Movimento Celular , Difusão , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Neuritos/metabolismo , Neurópilo/citologia , Células Receptoras Sensoriais/metabolismo
19.
Metabolomics ; 17(3): 25, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33594638

RESUMO

INTRODUCTION: Lipidomic profiling allows 100s if not 1000s of lipids in a sample to be detected and quantified. Modern lipidomics techniques are ultra-sensitive assays that enable the discovery of novel biomarkers in a variety of fields and provide new insight in mechanistic investigations. Despite much progress in lipidomics, there remains, as for all high throughput "omics" strategies, the need to develop strategies to standardize and integrate quality control into studies in order to enhance robustness, reproducibility, and usability of studies within specific fields and beyond. OBJECTIVES: We aimed to understand how much results from lipid profiling in the model organism Caenorhabditis elegans are influenced by different culture conditions in different laboratories. METHODS: In this work we have undertaken an inter-laboratory study, comparing the lipid profiles of N2 wild type C. elegans and daf-2(e1370) mutants lacking a functional insulin receptor. Sample were collected from worms grown in four separate laboratories under standardized growth conditions. We used an UPLC-UHR-ToF-MS system allowing chromatographic separation before MS analysis. RESULTS: We found common qualitative changes in several marker lipids in samples from the individual laboratories. On the other hand, even in this controlled experimental system, the exact fold-changes for each marker varied between laboratories. CONCLUSION: Our results thus reveal a serious limitation to the reproducibility of current lipid profiling experiments and reveal challenges to the integration of such data from different laboratories.


Assuntos
Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Lipidômica/métodos , Lipídeos/análise , Animais , Antígenos CD , Biomarcadores , Laboratórios , Receptor de Insulina , Reprodutibilidade dos Testes
20.
Anal Bioanal Chem ; 413(8): 2091-2102, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33575816

RESUMO

Lipid identification is one of the current bottlenecks in lipidomics and lipid profiling, especially for novel lipid classes, and requires multidimensional data for correct annotation. We used the combination of chromatographic and ion mobility separation together with data-independent acquisition (DIA) of tandem mass spectrometric data for the analysis of lipids in the biomedical model organism Caenorhabditis elegans. C. elegans reacts to harsh environmental conditions by interrupting its normal life cycle and entering an alternative developmental stage called dauer stage. Dauer larvae show distinct changes in metabolism and morphology to survive unfavorable environmental conditions and are able to survive for a long time without feeding. Only at this developmental stage, dauer larvae produce a specific class of glycolipids called maradolipids. We performed an analysis of maradolipids using ultrahigh performance liquid chromatography-ion mobility spectrometry-quadrupole-time of flight-mass spectrometry (UHPLC-IM-Q-ToFMS) using drift tube ion mobility to showcase how the integration of retention times, collisional cross sections, and DIA fragmentation data can be used for lipid identification. The obtained results show that combination of UHPLC and IM separation together with DIA represents a valuable tool for initial lipid identification. Using this analytical tool, a total of 45 marado- and lysomaradolipids have been putatively identified and 10 confirmed by authentic standards directly from C. elegans dauer larvae lipid extracts without the further need for further purification of glycolipids. Furthermore, we putatively identified two isomers of a lysomaradolipid not known so far.


Assuntos
Caenorhabditis elegans/química , Glicolipídeos/análise , Animais , Cromatografia Líquida de Alta Pressão , Espectrometria de Mobilidade Iônica , Larva/química , Lipidômica , Espectrometria de Massas
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