Quantification of tissue-specific protein translation in whole C. elegans using O-propargyl-puromycin labeling and fluorescence microscopy

Cell Reports Methods, Год журнала: 2022, Номер 2(4), С. 100203 - 100203

Опубликована: Апрель 1, 2022

The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify in C. elegans, they limited using lysate from whole worms. To achieve tissue-specific quantification ribosome activity intact we report the application O-propargyl-puromycin a cuticle defective mutant followed by conjugation an azide fluorophore detection fluorescent confocal microscopy. We apply this technique response heat shock, cycloheximide, or knockdown factors. Furthermore, demonstrate that can be between tissues within tissue like germline. This expected broad range applications determining how altered different knockdowns with age.

Язык: Английский

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Non-autonomous insulin signaling regulates the duration of mitosis inC. elegansgermline stem and progenitor cells

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 2, 2024

Abstract Stem and progenitor cell mitosis is essential for tissue development homeostasis. How these cells ensure proper chromosome segregation, thereby maintain mitotic fidelity, in the complex physiological environment of a living animal poorly understood. Here we use situ live-cell imaging C. elegans germline stem (GSPCs) to ask how signaling influences vivo . Through candidate screen identify new role insulin/IGF receptor (IGFR), daf-2, during GSPC mitosis. Mitosis delayed daf-2/ IGFR mutants, delays require canonical, DAF-2/IGFR DAF-16/FoxO insulin signaling, here acting non-autonomously from soma. Interestingly, daf-2 /IGFR mutants depend on spindle assembly checkpoint but are not accompanied by loss fidelity. Correspondingly, show that caloric restriction, which compromises does act via canonical pathway, instead requires AMP-activated kinase (AMPK). Together this work demonstrates influenced at least two genetically separable pathways highlights importance networks Author Summary drive sustain adult turnover producing daughter division, success depends segregation perform animal, yet relatively little known about events context. In particular, whether coordinate other aspects behavior with physiology also play took advantage model nematode address question. mitosis, uncover pathway. We find reducing but, surprisingly, addition, somatic tissues sufficient delay indicating pathway acts non-autonomously. Finally, while link division nutritional status many species, found it did mediate effects restriction Instead, conserved energy-sensing regulator AMPK. These results regulators emphasize

Язык: Английский

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Actomyosin-mediated apical constriction promotes physiological germ cell death in C. elegans

PLoS Biology, Год журнала: 2024, Номер 22(8), С. e3002775 - e3002775

Опубликована: Авг. 23, 2024

Germ cell apoptosis in Caenorhabditis elegans hermaphrodites is a physiological process eliminating around 60% of all cells meiotic prophase to maintain tissue homeostasis. In contrast programmed death the C . soma, selection germ undergoing stochastic. By live-tracking individual at pachytene stage, we found that smaller than their neighbors are selectively eliminated through before differentiating into oocytes. Thus, size strong predictor death. The RAS/MAPK and ECT/RHO/ROCK pathways together regulate by controlling actomyosin constriction apical rachis bridges, which cellular openings connecting syncytial shared cytoplasmic core. Enhancing reduces increases rate while inhibiting network prevents We propose contractility bridges amplifies intrinsic disparities size. Through this mechanism, animals can adjust balance between oocyte differentiation.

Язык: Английский

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Non-autonomous insulin signaling delays mitotic progression in C. elegans germline stem and progenitor cells

PLoS Genetics, Год журнала: 2024, Номер 20(12), С. e1011351 - e1011351

Опубликована: Дек. 23, 2024

Stem and progenitor cell mitosis is essential for tissue development homeostasis. How these cells ensure proper chromosome segregation, thereby maintain mitotic fidelity, in the complex physiological environment of a living animal poorly understood. Here we use situ live-cell imaging C . elegans germline stem (GSPCs) to ask how signaling influences vivo Through candidate screen identify new role insulin/IGF receptor (IGFR), daf-2 , during GSPC mitosis. Mitosis delayed daf-2/ IGFR mutants, delays require canonical, DAF-2/IGFR DAF-16/FoxO insulin signaling, here acting non-autonomously from soma. Interestingly, /IGFR mutants depend on spindle assembly checkpoint but are not accompanied by loss fidelity. Correspondingly, show that caloric restriction, which compromises does act via canonical pathway, instead requires AMP-activated kinase (AMPK). Together this work demonstrates influenced at least two genetically separable pathways highlights importance networks

Язык: Английский

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EGFR signal transduction is downregulated in C. elegans vulval precursor cells during dauer diapause

Development, Год журнала: 2022, Номер 149(21)

Опубликована: Окт. 13, 2022

Caenorhabditis elegans larvae display developmental plasticity in response to environmental conditions: adverse conditions, second-stage enter a reversible, long-lived dauer stage instead of proceeding reproductive adulthood. Dauer entry interrupts vulval induction and is associated with reprogramming-like event that preserves the multipotency precursor cells (VPCs), allowing development reinitiate if conditions improve. Vulval requires LIN-3/EGF-like signal from gonad, which activates EGFR-Ras-ERK transduction nearest VPC, P6.p. Here, using biosensor live imaging we show activity downregulated P6.p dauers. We investigated this process gene mutations or transgenes manipulate different steps pathway, by analyzing LET-23/EGFR subcellular localization during life history. found EGF attenuated at upstream Ras activation, discuss potential membrane-associated mechanisms could achieve this. also describe other findings pertaining maintenance VPC competence quiescence larvae. Our analysis indicates VPCs have L2-like unique features rather than L3 continuous development.

Язык: Английский

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Genetic and Chemical Controls of Sperm Fate and Spermatocyte Dedifferentiation via PUF-8 and MPK-1 in Caenorhabditis elegans

Cells, Год журнала: 2023, Номер 12(3), С. 434 - 434

Опубликована: Янв. 28, 2023

Using the nematode C. elegans germline as a model system, we previously reported that PUF-8 (a PUF RNA-binding protein) and LIP-1 dual-specificity phosphatase) repress sperm fate at 20 °C dedifferentiation of spermatocytes into mitotic cells (termed "spermatocyte dedifferentiation") 25 °C. Thus, double mutants lacking both produce excess °C, their return to mitotically dividing via resulting in tumors. To gain insight molecular competence for spermatocyte dedifferentiation, compared phenotypes three mutant strains sperm-fem-3(q20gf), puf-8(q725); fem-3(q20gf), lip-1(zh15). Spermatocyte was not observed fem-3(q20gf) mutants, but it more severe lip-1(zh15) than mutants. These results suggest MPK-1 (the ERK1/2 MAPK ortholog) activation absence is required promote dedifferentiation. This idea confirmed using Resveratrol (RSV), potential activator human cells, respectively. Notably, significantly enhanced by RSV treatment PUF-8, its effect blocked mpk-1 RNAi. We, therefore, conclude are essential regulators tumorigenesis. Since these broadly conserved, similar regulatory circuitry may control cellular tumorigenesis other organisms, including humans.

Язык: Английский

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Formation of benign tumors by stem cell deregulation

PLoS Genetics, Год журнала: 2022, Номер 18(10), С. e1010434 - e1010434

Опубликована: Окт. 27, 2022

Within living organisms, stem cells respond to various cues, including niche signals and growth factors. Niche originate from the cell's microenvironment promote undifferentiated state by preventing differentiation, allowing for cell self-renewal. On other hand, factors proliferation, while their sources comprise of a systemic input reflecting animal's nutritional metabolic status, localized, homeostatic feedback signal tissue that serve. That prevents unnecessary proliferation when corresponding differentiated tissues already have optimal contents. Here, we recapitulate progresses made in our understanding vivo regulation, largely using simple models, draw conclusion 2 types deregulations can provoke formation benign tumors. Namely, constitutive signaling promotes "stem cell" tumors, defective leads Finally, provide evidence these general principles may be conserved mammals as such, underlie tumor humans, tumors evolve into cancer.

Язык: Английский

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Simplified Quantification of Progenitor Zone Size, an Indicator of Germ Stem Cell Niche Activity, in the Nematode Caenorhabditis elegans

Methods in molecular biology, Год журнала: 2024, Номер unknown

Опубликована: Янв. 1, 2024

Язык: Английский

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Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Сен. 18, 2024

Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin modulates cell fate decisions controlled by conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich increases vulval induction, germ apoptosis oocyte differentiation. These effects are mediated different metabolites a tissue-specific manner. enhances via choline/phosphatidylcholine metabolism induction down-regulating fat biosynthesis genes increasing H3K4 tri-methylation, results increased expression target genes. Furthermore, nucleoside tri-methylation positively regulate production. Using mammalian cells carrying activated KRAS BRAF alleles, on RAS/MAPK-regulated phenotype mammals. Our findings suggest B12-dependent limiting factor diverse RAS/MAPK-induced cellular responses.

Язык: Английский

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The L1CAM SAX-7 is an antagonistic modulator of Erk Signaling

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Сен. 16, 2024

Abstract L1CAMs are immunoglobulin superfamily cell adhesion molecules that ensure proper nervous system development and function. In addition to being associated with the autism schizophrenia spectrum disorders, mutations in L1CAM family of genes also underlie distinct developmental syndromes neurological conditions, such as intellectual disability, spastic paraplegia, hypotonia congenital hydrocephalus. Studies both vertebrate invertebrate model organisms have established conserved neurodevelopmental roles for L1CAMs; these include axon guidance, dendrite morphogenesis, synaptogenesis, maintenance neural architecture, among others. Caenorhabditis elegans , L1CAMs, encoded by sax-7 gene, required coordinated locomotion. We previously uncovered a genetic interaction between components synaptic vesicle cycle, revealing non-developmental role regulating activity. More recently, we determined genetically interacts extracellular signal-related kinase (ERK) signaling controlling C. ERK, mpk-1 is serine/threonine protein belonging mitogen-activated (MAPK) governs multiple aspects animal cellular homeostasis. Here, show this occurs not only cholinergic neurons locomotion, but extends outside system, novel SAX-7/L1CAM non-neuronal processes, including vulval development. Our findings developing vulva consistent acting an antagonistic modulator ERK signaling.

Язык: Английский

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