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Wide and high resolution tension measurement using FRET in embryo.

ABSTRACT: During embryonic development, physical force plays an important role in morphogenesis and differentiation. Stretch sensitive fluorescence resonance energy transfer (FRET) has the potential to provide non-invasive tension measurements inside living tissue. In this study, we introduced a FRET-based actinin tension sensor into Xenopus laevis embryos and demonstrated that this sensor captures variation of tension across differentiating ectoderm. The actinin tension sensor, containing mCherry and EGFP connected by spider silk protein, was validated in human embryonic kidney (HEK) cells and embryos. It co-localized with actin filaments and changed FRET efficiencies in response to actin filament destruction, myosin deactivation, and osmotic perturbation. Time-lapse FRET analysis showed that the prospective neural ectoderm bears higher tension than the epidermal ectoderm during gastrulation and neurulation, and cells morphogenetic behavior correlated with the tension difference. These data confirmed that the sensor enables us to measure tension across tissues concurrently and with high resolution.

SUBMITTER: Yamashita S

PROVIDER: S-EPMC4917836 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Wide and high resolution tension measurement using FRET in embryo.

Yamashita Satoshi S Tsuboi Takashi T Ishinabe Nanako N Kitaguchi Tetsuya T Michiue Tatsuo T

Scientific reports 20160623

During embryonic development, physical force plays an important role in morphogenesis and differentiation. Stretch sensitive fluorescence resonance energy transfer (FRET) has the potential to provide non-invasive tension measurements inside living tissue. In this study, we introduced a FRET-based actinin tension sensor into Xenopus laevis embryos and demonstrated that this sensor captures variation of tension across differentiating ectoderm. The actinin tension sensor, containing mCherry and EGF ...[more]

PMID: 27335157

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Project description:To persist in their dynamic human host environments, fungal pathogens must sense and adapt by modulating their gene expression to fulfill their cellular needs. Understanding transcriptional regulation on a global scale would uncover cellular processes linked to persistence and virulence mechanisms that could be targeted for antifungal therapeutics. Infections associated with the yeast Candida albicans, a highly prevalent fungal pathogen, and the multiresistant related species Candida auris are becoming a serious public health threat. To define the set of a gene regulated by a transcriptional regulator in C. albicans, chromatin immunoprecipitation (ChIP)-based techniques, including ChIP with microarray technology (ChIP-chip) or ChIP-DNA sequencing (ChIP-seq), have been widely used. Here, we describe a new set of PCR-based micrococcal nuclease (MNase)-tagging plasmids for C. albicans and other Candida spp. to determine the genome-wide location of any transcriptional regulator of interest using chromatin endogenous cleavage (ChEC) coupled to high-throughput sequencing (ChEC-seq). The ChEC procedure does not require protein-DNA cross-linking or sonication, thus avoiding artifacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon. In a proof-of-concept application of ChEC-seq, we provided a high-resolution binding map of the SWI/SNF chromatin remodeling complex, a master regulator of fungal fitness in C. albicans, in addition to the transcription factor Nsi1 that is an ortholog of the DNA-binding protein Reb1 for which genome-wide occupancy was previously established in Saccharomyces cerevisiae The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi.IMPORTANCE Systemic fungal infections caused by Candida albicans and the "superbug" Candida auris are becoming a serious public health threat. The ability of these yeasts to cause disease is linked to their faculty to modulate the expression of genes that mediate their escape from the immune surveillance and their persistence in the different unfavorable niches within the host. Comprehensive knowledge on gene expression control of fungal fitness is consequently an interesting framework for the identification of essential infection processes that could be hindered by chemicals as potential therapeutics. Here, we expanded the use of ChEC-seq, a technique that was initially developed in the yeast model Saccharomyces cerevisiae to identify genes that are modulated by a transcriptional regulator, in pathogenic yeasts from the genus Candida This robust technique will allow a better characterization of key gene expression regulators and their contribution to virulence and antifungal resistance in these pathogenic yeasts.

Project description:BackgroundEpigenetic modifications play important roles in plant and animal development. DNA methylation impacts the transposable element (TE) silencing, gene imprinting and expression regulation.ResultsThrough a genome-wide analysis, DNA methylation peaks were characterized and mapped in maize embryo and endosperm genome, respectively. Distinct methylation level was observed across maize embryo and endosperm. The maize embryo genome contained more DNA methylation than endosperm. Totally, 985,478 CG islands (CGIs) were identified and most of them were unmethylated. More CGI shores were methylated than CGIs in maize suggested that DNA methylation level was not positively correlated with CpG density. The promoter sequence and transcriptional termination region (TTR) were more methylated than the gene body (intron and exon) region based on peak number and methylated depth. Result showed that 99% TEs were methylated in maize embryo, but a large portion of them (34.8%) were not methylated in endosperm. Maize embryo and endosperm exhibit distinct pattern/level of methylation. The most differentially methylated region between embryo and endosperm are CGI shores. Our results indicated that DNA methylation is associated with both gene silencing and gene activation in maize. Many genes involved in embryogenesis and seed development were found differentially methylated in embryo and endosperm. We found 41.5% imprinting genes were similarly methylated and 58.5% imprinting genes were differentially methylated between embryo and endosperm. Methylation level was associated with allelic silencing of only a small number of imprinting genes. The expression of maize DEMETER-like (DME-like) gene and MBD101 gene (MBD4 homolog) were higher in endosperm than in embryo. These two genes may be associated with distinct methylation levels across maize embryo and endosperm.ConclusionsThrough MeDIP-seq we systematically analyzed the methylomes of maize embryo and endosperm and results indicated that the global methylation status of embryo was more than that of the endosperm. Differences could be observed at the total number of methylation peaks, DMRs and specific methylated genes which were tightly associated with development of embryo and endosperm. Our results also revealed that many DNA methylation regions didn't affect transcription of the corresponding genes.

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Wide and high resolution tension measurement using FRET in embryo.

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Wide and high resolution tension measurement using FRET in embryo.

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