scholarly journals Live cell imaging of metabolic heterogeneity by quantitative fluorescent ATP indicator protein, QUEEN-37C.

2021 ◽  
Author(s):  
Hideyuki Yaginuma ◽  
Yasushi Okada
Keyword(s):  
Single Cell ◽  
Mammalian Cells ◽  
Cell Sheet ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Luciferase Assay ◽  
Atp Concentration ◽  
Cell Measurement ◽  
Positive Correlations ◽  
Metabolic Heterogeneity

Adenosine triphosphate (ATP) is often referred as the energy currency of the cell. Yet, non-invasive, real-time, and quantitative measurement of its concentration in living mammalian cells has been difficult. Here we report an improved fluorescent ATP indicator protein, QUEEN-37C, which is optimized for measuring ATP concentration in living mammalian cells. Absolute value of the ATP concentration can be estimated from the ratiometric fluorescence imaging, and its accuracy was verified by the luciferase assay. Since QUEEN-37C enables the single-cell measurement of ATP concentration, we can not only measure its mean but its distribution in the cell population, which revealed that the ATP concentration is tightly regulated in most cells. We also noted the positive correlations in the ATP concentration among adjacent cells in epithelial cell sheet and mouse embryonic stem cell colonies. Thus, QUEEN-37C would serve as a new tool for the investigation of the single cell heterogeneity of metabolic states.

scholarly journals Extensive Loss of Heterozygosity Is Suppressed during Homologous Repair of Chromosomal Breaks

2003 ◽  
Vol 23 (2) ◽  
pp. 733-743 ◽  
Author(s):  
Jeremy M. Stark ◽  
Maria Jasin
Keyword(s):  
Loss Of Heterozygosity ◽  
Meiotic Recombination ◽  
Mammalian Cells ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Human Populations ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Homologous Chromosomes

ABSTRACT Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 × 10−5; however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.

scholarly journals Coupled Homologous and Nonhomologous Repair of a Double-Strand Break Preserves Genomic Integrity in Mammalian Cells

2000 ◽  
Vol 20 (23) ◽  
pp. 9068-9075 ◽  
Author(s):  
Christine Richardson ◽  
Maria Jasin
Keyword(s):  
Mammalian Cells ◽  
Chromosomal Rearrangements ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Homologous Sequence ◽  
Chromosome Loss ◽  
Genomic Integrity ◽  
Gene Sequences ◽  
Exogenous Dna ◽  
Repair Pathways

ABSTRACT DNA double-strand breaks (DSBs) may be caused by normal metabolic processes or exogenous DNA damaging agents and can promote chromosomal rearrangements, including translocations, deletions, or chromosome loss. In mammalian cells, both homologous recombination and nonhomologous end joining (NHEJ) are important DSB repair pathways for the maintenance of genomic stability. Using a mouse embryonic stem cell system, we previously demonstrated that a DSB in one chromosome can be repaired by recombination with a homologous sequence on a heterologous chromosome, without any evidence of genome rearrangements (C. Richardson, M. E. Moynahan, and M. Jasin, Genes Dev., 12:3831–3842, 1998). To determine if genomic integrity would be compromised if homology were constrained, we have now examined interchromosomal recombination between truncated but overlapping gene sequences. Despite these constraints, recombinants were readily recovered when a DSB was introduced into one of the sequences. The overwhelming majority of recombinants showed no evidence of chromosomal rearrangements. Instead, events were initiated by homologous invasion of one chromosome end and completed by NHEJ to the other chromosome end, which remained highly preserved throughout the process. Thus, genomic integrity was maintained by a coupling of homologous and nonhomologous repair pathways. Interestingly, the recombination frequency, although not the structure of the recombinant repair products, was sensitive to the relative orientation of the gene sequences on the interacting chromosomes.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Guillaume Harmange ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

scholarly journals Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

2017 ◽  
Author(s):  
Bogdan Budnik ◽  
Ezra Levy ◽  
Nikolai Slavov
Keyword(s):  
Mass Spectrometry ◽  
Single Cell ◽  
Mammalian Cells ◽  
Human Cancer ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cellular Heterogeneity ◽  
Protein Levels ◽  
Transcriptional Regulatory Mechanisms

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

Sevoflurane regulating LncRNA Rik-203 contributes to neural differentiation via microRNA-466l-3p /BDNF pathway

2019 ◽  
Author(s):  
Lei Zhang ◽  
Qidong Liu ◽  
Zhenyu Xue ◽  
Yan Jia ◽  
Hong Jiang
Keyword(s):  
Neural Differentiation ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Luciferase Assay ◽  
Over Expression ◽  
Mice Brain ◽  
Underlying Mechanisms ◽  
Induced Neuron ◽  
Bdnf Pathway

Abstract Background: The anesthetics inhibit neural differentiation, induced neuron loss and cognitive impairment in young animals. However, the underlying mechanisms of anesthesia on neural differentiation and are unknown. Methods: Embryonic stem cells (ESCs) and mice received sevoflurane anesthesia. RNA sequencing; gene expression of mRNAs, LncRNAs and miRNAs; over-expression and RNA interference of genes; flow cytometry; real-time quantity PCR and Western blot were used in the studies. RNA pull-down assay and PCR were employed to detect any miRNA that attached to Rik-203. The binding of miRNA with mRNA of BDNF was presented by the luciferase assay. Results: Here we found that LncRNA Rik-203 was higher expressed in mice brain than other tissues and increased during neural differentiation. Sevoflurane decreased the level of Rik-203 in mice brain. Knockdown of Rik-203 repressed the neural differentiation derived from mouse embryonic stem cell with the downregulation of the neural progenitor cells markers Sox1 and Nestin. RNA pull-down showed that miR-466l-3p was highly bound to Rik-203 and mediated the function of Rik-203. Inhibition of miR-466l-3p restored the neural differentiation repressed by Rik-203 knockdown. BDNF was directly targeted by miR-466l-3p and also downregulated by sevoflurane. Overexpression of BDNF restored the neural differentiation repressed by miR-466l-3p and Rik-203 knockdown. Conclusion: Our study suggested that sevoflurane related LncRNARik-203 facilitates neural differentiation by inhibiting miR-466l-3p’s ability to reduce BDNF levels. Keywords: Anesthesia; sevoflurane; Rik-203; miR-466l-3p; BDNF; neural differentiation.

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