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1.
PLoS Genet ; 17(7): e1009635, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34237064

RESUMO

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.


Assuntos
Aldeído Desidrogenase/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Lipase/genética , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise/genética , Mutação , Peroxissomos/genética , Receptores Citoplasmáticos e Nucleares/genética
2.
Bioessays ; 43(1): e2000199, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33169432

RESUMO

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein-protein and protein-lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER-LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage-like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin-positive peri-LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.


Assuntos
Caenorhabditis elegans , Fosfolipídeos , Animais , Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Fosfolipídeos/metabolismo
3.
Genes Dev ; 26(8): 846-56, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22508728

RESUMO

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/química , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
4.
Anal Chem ; 91(3): 2279-2287, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30589537

RESUMO

Quantitative methods to precisely measure cellular states in vivo have become increasingly important and desirable in modern biology. Recently, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool to visualize small biological molecules tagged with alkyne (C≡C) or carbon-deuterium (C-D) bonds in the cell-silent region. In this study, we developed a technique based on SRS microscopy of vibrational tags for quantitative imaging of lipid synthesis and lipolysis in live animals. The technique aims to overcome the major limitations of conventional fluorescent staining and lipid extraction methods that do not provide the capability of in vivo quantitative analysis. Specifically, we used three bioorthogonal lipid molecules (the alkyne-tagged fatty acid 17-ODYA, deuterium-labeled saturated fatty acid PA-D31, and unsaturated fatty acid OA-D34) to investigate the metabolic dynamics of lipid droplets (LDs) in live Caenorhabditis elegans ( C. elegans). Using a hyperspectral SRS (hsSRS) microscope and subtraction method, the interfering non-Raman background was eliminated to improve the accuracy of lipid quantification. A linear relationship between SRS signals and fatty acid molar concentrations was accurately established. With this quantitative analysis tool, we imaged and determined the changes in concentration of the three fatty acids in LDs of fed or starved adult C. elegans. Using the hsSRS imaging mode, we also observed the desaturation of fatty acids in adult C. elegans via spectral analysis on the SRS signals from LDs. The results demonstrated the unique capability of hsSRS microscopy in quantitative analysis of lipid metabolism in vivo.


Assuntos
Caenorhabditis elegans/metabolismo , Ácidos Graxos Insaturados/análise , Lipogênese/fisiologia , Lipólise/fisiologia , Ácido Oleico/análise , Ácido Palmítico/análise , Animais , Deutério/química , Ácidos Graxos Insaturados/metabolismo , Microscopia Óptica não Linear , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Triglicerídeos/biossíntese , Triglicerídeos/metabolismo
5.
Biochim Biophys Acta ; 1853(10 Pt A): 2481-91, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26025681

RESUMO

The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Metabolismo dos Lipídeos/fisiologia , Triglicerídeos/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Especificidade de Órgãos/fisiologia , Triglicerídeos/genética
6.
Nat Genet ; 38(3): 363-8, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16462744

RESUMO

Tubby mice and individuals with Bardet-Biedl syndrome have defects in ciliated neuron function and obesity, suggesting an as-yet unknown metabolic signaling axis from ciliated neurons to fat storage tissues. Here we show coordinate regulation of Caenorhabditis elegans fat storage by orthologues of these genes acting in ciliated neurons and by a 3-ketoacyl-coA thiolase (encoded by kat-1) that acts in fat storage tissue. A genetic screen for markedly enhanced fat storage in tub-1 mutants led to the isolation only of kat-1 alleles, which impair fatty acid beta-oxidation. kat-1 acts in the intestine, the major C. elegans fat storage tissue, and is transcriptionally upregulated in animals with high fat storage. A genetic screen for synergistic increase in fat storage of a kat-1 mutant identified bbs-1. bbs-1 acts in 15 ciliated neurons that are poised to sense external and internal nutrient levels, supporting a model in which bbs-1 and tub-1 in ciliated neurons form part of an ancient, conserved neuroendocrine axis. This pathway also includes genes encoding intraflagellar transport proteins and cyclic nucleotide gated channels, demonstrating that C. elegans fat storage is under polygenic control.


Assuntos
Tecido Adiposo/fisiologia , Caenorhabditis elegans/genética , Acetil-CoA C-Aciltransferase/genética , Tecido Adiposo/enzimologia , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Cílios/fisiologia , Mutação , Neurônios/fisiologia
7.
PLoS Genet ; 7(5): e1002065, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21573134

RESUMO

Elevation of the second messenger cGMP by nitric oxide (NO) activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1-dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/genética , Feminino , Perfilação da Expressão Gênica , Modelos Biológicos , Mutação/genética , Ligação Proteica , Reprodução/genética
8.
Proc Natl Acad Sci U S A ; 107(10): 4640-5, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20176933

RESUMO

Dietary fat accumulates in lipid droplets or endolysosomal compartments that undergo selective expansion under normal or pathophysiological conditions. We find that genetic defects in a peroxisomal beta-oxidation pathway cause size expansion in lipid droplets that are distinct from the lysosome-related organelles in Caenorhabditis elegans. Expansion of lipid droplets is accompanied by an increase in triglycerides (TAG) that are resistant to fasting- or TAG lipase-triggered lipolysis. Nevertheless, in mutant animals, a diet poor in vaccenic acid reduced the TAG level and lipid droplet size. Our results implicate peroxisomal dysfunction in pathologic lipid droplet expansion in animals and illustrate how dietary factors modulate the phenotype of such genetic defects.


Assuntos
Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Animais , Animais Geneticamente Modificados , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipase/genética , Lipase/metabolismo , Lipólise , Lisossomos/metabolismo , Masculino , Microscopia Confocal , Microscopia Eletrônica , Mutação , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/metabolismo , Oxirredução , Peroxissomos/metabolismo , Triglicerídeos/metabolismo
9.
J Lipid Res ; 53(1): 28-33, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22049244

RESUMO

Lipid droplets are evolutionarily conserved organelles where cellular fat storage and mobilization are exquisitely regulated. Recent studies have defined lipid droplets in C. elegans and explored how they are regulated by genetic and dietary factors. C. elegans offers unique opportunities to visualize lipid droplets at single-cell resolution in live animals. The development of novel microscopy techniques and protein markers for lipid droplets will accelerate studies on how nutritional states and subcellular organization are linked in vivo. Together with powerful tools for genetic and biochemical analysis of metabolic pathways, alteration in lipid droplet abundance, size, and distribution in C. elegans can be readily connected to whole-animal energy homeostasis, behavior, and life span. Therefore, further studies on lipid droplets in C. elegans promise to yield valuable insights that complement our knowledge gained from yeast, Drosophila, and mammalian systems on cellular and organismal fat storage.


Assuntos
Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos , Organelas/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Intestinos/citologia , Lipase/metabolismo , Lipídeos , Microscopia Eletrônica
10.
Proc Natl Acad Sci U S A ; 106(6): 1875-9, 2009 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-19174521

RESUMO

To sense its population density and to trigger entry into the stress-resistant dauer larval stage, Caenorhabditis elegans uses the dauer pheromone, which consists of ascaroside derivatives with short, fatty acid-like side chains. Although the dauer pheromone has been studied for 25 years, its biosynthesis is completely uncharacterized. The daf-22 mutant is the only known mutant defective in dauer pheromone production. Here, we show that daf-22 encodes a homolog of human sterol carrier protein SCPx, which catalyzes the final step in peroxisomal fatty acid beta-oxidation. We also show that dhs-28, which encodes a homolog of the human d-bifunctional protein that acts just upstream of SCPx, is also required for pheromone production. Long-term daf-22 and dhs-28 cultures develop dauer-inducing activity by accumulating less active, long-chain fatty acid ascaroside derivatives. Thus, daf-22 and dhs-28 are required for the biosynthesis of the short-chain fatty acid-derived side chains of the dauer pheromone and link dauer pheromone production to metabolic state.


Assuntos
Caenorhabditis elegans/metabolismo , Feromônios/biossíntese , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Transporte , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Humanos , Redes e Vias Metabólicas , Oxirredução , Homologia de Sequência de Aminoácidos
11.
Front Cell Dev Biol ; 10: 856474, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35445028

RESUMO

In addition to coordinating the storage and mobilization of neutral fat, lipid droplets (LDs) are conserved organelles that can accommodate additional cargos in order to support animal development. However, it is unclear if each type of cargo is matched with a specific subset of LDs. Here, we report that SEIP-1/seipin defines a subset of oocyte LDs that are required for proper eggshell formation in C. elegans. Using a photoconvertible fluorescent protein-based imaging assay, we found that SEIP-1 positive LDs were selectively depleted after fertilization, coincident of the formation of a lipid-rich permeability barrier of the eggshell. Loss of SEIP-1 function caused impenetrant embryonic arrest, which could be worsened by FAT-3/fatty acyl-CoA desaturase deficiency or suppressed by PLIN-1/Perilipin deficiency. The embryonic development of seip-1; plin-1 mutant in turn depended on the recruitment of RAB-18/Rab18 to LDs, which was not observed in wild type embryos. We propose that SEIP-1 dependent and independent mechanisms act in parallel to ensure the packaging and export of lipid-rich permeability barrier constituents, which involve LDs. The identity of these LDs, as defined by their associated proteins, exhibits unexpected plasticity that ultimately ensures the survival of embryos ex utero.

12.
Materials (Basel) ; 15(3)2022 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-35160799

RESUMO

Soft polymeric gels are susceptible to buckling-induced instabilities due to their great compliance to surface deformations. The instability patterns at soft interfaces have great potential in engineering functional materials with unique surface properties. In this work, we systematically investigated how swelling-induced instability patterns effectively improved the adhesive properties of soft polydimethylsiloxane (PDMS) gels. We directly imaged the formations of the surface instability features during the relaxation process of a swollen gel substrate. The features were found to greatly increase the adhesion energy of soft gels across multiple length scales, and the adhesion enhancement was associated with the variations of contact lines both inside the contact region and along the contact periphery. We expect that these studies of instability patterns due to swelling will further benefit the design of functional interfaces in various engineering applications.

13.
Gut Microbes ; 14(1): 2013762, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35112996

RESUMO

Dietary and symbiotic bacteria can exert powerful influence on metazoan lipid metabolism. Recent studies have emerged that microbiota have a role in animal obesity and related health disorders, but the mechanisms by which bacteria influence lipid storage in their host are unknown. To reduce the complexity of the relationship between gut microbiota and the host, Caenorhabditis elegans (C. elegans) has been chosen as a model organism to study interspecies interaction. Here, we demonstrate that feeding C. elegans with an opportunistic pathogenic bacterium Stenotrophomonas maltophilia (S. maltophilia) retards growth and promotes excessive neutral lipid storage. Gene expression analysis reveals that dietary S. maltophilia induces a lipogenic transcriptional response that includes the SREBP ortholog SBP-1, and fatty acid desaturases FAT-6 and FAT-7. Live imaging and ultrastructural analysis suggest that excess neutral lipid is stored in greatly expanded lipid droplets (LDs), as a result of enhanced endoplasmic reticulum (ER)-LD interaction. We also report that loss of function mutations in dpy-9 in C. elegans confers resistance to S. maltophilia. Dietary S. maltophilia induces supersized LDs by enhancing lipogenesis and ER-LD contacts in C. elegans. This work delineates a new model for understanding microbial regulation of metazoan physiology.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Gotículas Lipídicas/metabolismo , Lipogênese , Stenotrophomonas maltophilia/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Microbioma Gastrointestinal , Masculino , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
BMC Cell Biol ; 11: 96, 2010 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-21143850

RESUMO

BACKGROUND: Lipid droplets are a class of eukaryotic cell organelles for storage of neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE). We and others have recently reported that lysosome-related organelles (LROs) are not fat storage structures in the nematode C. elegans. We also reported the formation of enlarged lipid droplets in a class of peroxisomal fatty acid ß-oxidation mutants. In the present study, we seek to provide further evidence on the organelle nature and biophysical properties of fat storage structures in wild-type and mutant C. elegans. RESULTS: In this study, we provide biochemical, histological and ultrastructural evidence of lipid droplets in wild-type and mutant C. elegans that lack lysosome related organelles (LROs). The formation of lipid droplets and the targeting of BODIPY fatty acid analogs to lipid droplets in live animals are not dependent on lysosomal trafficking or peroxisome dysfunction. However, the targeting of Nile Red to lipid droplets in live animals occurs only in mutants with defective peroxisomes. Nile Red labelled-lipid droplets are characterized by a fluorescence emission spectrum distinct from that of Nile Red labelled-LROs. Moreover, we show that the recently developed post-fix Nile Red staining method labels lipid droplets exclusively. CONCLUSIONS: Our results demonstrate lipid droplets as ubiquitous fat storage organelles and provide a unified explanation for previous studies on fat labelling methods in C. elegans. These results have important applications to the studies of fat storage and lipid droplet regulation in the powerful genetic system, C. elegans.


Assuntos
Caenorhabditis elegans/metabolismo , Metabolismo dos Lipídeos/fisiologia , Animais , Caenorhabditis elegans/ultraestrutura , Ésteres do Colesterol/metabolismo , Corantes Fluorescentes/química , Lisossomos/metabolismo , Oxazinas/química , Peroxissomos/metabolismo , Triglicerídeos/metabolismo
15.
Nat Commun ; 10(1): 2902, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263173

RESUMO

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolite-modulated targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduces the size of a subset of LDs while over-expression of SEIP-1 has the opposite effect. Ultrastructural analysis reveals SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purifies with LDs. Analyses of C. elegans and bacterial genetic mutants indicate a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologously expressed SEIP-1 engages nascent lipid droplets and promotes their subsequent expansion in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by promoting LD diversity.


Assuntos
Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Transporte Proteico
16.
J Biomed Opt ; 20(11): 110501, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26580697

RESUMO

The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.


Assuntos
Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Análise Espectral Raman/instrumentação , Vesículas Transportadoras/diagnóstico por imagem , Vesículas Transportadoras/metabolismo , Animais , Caenorhabditis elegans , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Metabolismo dos Lipídeos/fisiologia , Imagem Molecular/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Integração de Sistemas , Distribuição Tecidual , Ultrassonografia
17.
Curr Biol ; 24(8): 832-8, 2014 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-24684930

RESUMO

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , RNA Helicases DEAD-box/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/biossíntese , Animais , Sequência de Bases , Western Blotting , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , RNA Helicases DEAD-box/genética , Primers do DNA/genética , Componentes do Gene , Imunoprecipitação , Microscopia Confocal , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
18.
Methods Cell Biol ; 116: 39-51, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24099286

RESUMO

The powerful forward and reverse genetic tools, and emerging sets of biochemical assays for fat metabolites, make Caenorhabditis elegans an attractive model organism for elucidating conserved mechanisms in fat storage. The ability to observe lipid droplets in live animals at single cell resolution offers a unique advantage for studying cellular fat storage in vivo. In this chapter, we describe transgenic technologies for expressing fluorescent lipid droplet marker proteins at near-physiological levels. Methods to visualize these markers using sensitive confocal microscopy systems are detailed. Additional methods for visualizing lipid droplets by transmission electron microscopy and detection of lipid droplet associated proteins by immunoelectron microscopy are described.


Assuntos
Caenorhabditis elegans/ultraestrutura , Corpos de Inclusão/ultraestrutura , Metabolismo dos Lipídeos , Lipídeos/isolamento & purificação , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/química , Proteínas de Fluorescência Verde , Corpos de Inclusão/química , Lipídeos/química , Microscopia Confocal , Microscopia Eletrônica , Triglicerídeos/química , Triglicerídeos/metabolismo
19.
Lab Chip ; 13(15): 2963-71, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23708469

RESUMO

Direct observation of developmental and physiological changes in certain model organisms over time has been technically challenging. In the model organism Caenorhabditis elegans, these studies require frequent or continuous imaging at physiologically benign conditions. However, standard methods use anaesthetics, glue, or microbeads, which prevent animals from feeding during the experiment. Thus, the animals' normal physiological function may be affected over time. Here we present a platform designed for dynamic studies of C. elegans. The system is capable of immobilizing only the animals' bodies under benign conditions and without physical deformation. Simultaneously, the animals' heads remain free to move and feed for the duration of the experiment. This allows for high-resolution and high-magnification fluorescent imaging of immobilized and feeding animals. The system is very easy to fabricate, set up, and operate, and should be widely applicable to many problems in developmental and physiological studies.


Assuntos
Caenorhabditis elegans/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Fenômenos Fisiológicos da Nutrição Animal , Animais , Caenorhabditis elegans/anatomia & histologia , Desenho de Equipamento , Imagem Óptica/instrumentação
20.
Cell Rep ; 3(5): 1465-75, 2013 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-23684613

RESUMO

Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulated is unknown. Using genetic screens in C. elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion in vitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Vesículas Citoplasmáticas/química , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células COS , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Chlorocebus aethiops , Vesículas Citoplasmáticas/metabolismo , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Interferência de RNA , RNA Interferente Pequeno/metabolismo
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