scholarly journals Single Cell Analysis Reveals Multiple Requirements for Zinc in the Mammalian Cell Cycle

2019 ◽  
Author(s):  
Maria N. Lo ◽  
Leah J. Damon ◽  
Jian Wei Tay ◽  
Amy E. Palmer
Keyword(s):  
Cell Cycle ◽  
Mammalian Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Genome Integrity ◽  
Cell Analysis ◽  
Fluorescent Reporters ◽  
Cell Cycle Reentry ◽  
Mammalian Cell Cycle

AbstractDespite recognition of the fundamental role of zinc (Zn2+) for growth and proliferation, mechanisms of how Zn2+ deficiency arrests these processes remain enigmatic. We induced subtle intracellular Zn2+ perturbations and tracked asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. We found that Zn2+ deficiency induces quiescence and Zn2+ resupply stimulates cell-cycle reentry. By monitoring single cells after Zn2+ deprivation, we found that depending on where cells were in the cell cycle, they either went quiescent or entered the cell cycle but stalled in S phase. Stalled cells were defective in DNA synthesis and had increased DNA damage levels, suggesting a role for Zn2+ in maintaining genome integrity. Finally, we found that Zn2+ deficiency-induced quiescence does not require the cell-cycle inhibitor p21. Overall, our study provides new insights into when Zn2+ is required during the mammalian cell cycle and the consequences Zn2+ deficiency.

scholarly journals Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Maria N Lo ◽  
Leah J Damon ◽  
Jian Wei Tay ◽  
Shang Jia ◽  
Amy E Palmer
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Zinc Deficiency ◽  
Single Cell Analysis ◽  
S Phase ◽  
Fluorescent Reporters ◽  
Cell Cycle Reentry ◽  
Mammalian Cell Cycle ◽  
Before And After ◽  
Cycle Regulation

Zinc is widely recognized as essential for growth and proliferation, yet the mechanisms of how zinc deficiency arrests these processes remain enigmatic. Here we induce subtle zinc perturbations and track asynchronously cycling cells throughout division using fluorescent reporters, high throughput microscopy, and quantitative analysis. Zinc deficiency induces quiescence and resupply stimulates synchronized cell-cycle reentry. Monitoring cells before and after zinc deprivation we found the position of cells within the cell cycle determined whether they either went quiescent or entered another cell cycle but stalled in S-phase. Stalled cells exhibited prolonged S-phase, were defective in DNA synthesis and had increased DNA damage levels, suggesting a role for zinc in maintaining genome integrity. Finally, we demonstrate zinc deficiency-induced quiescence occurs independently of DNA-damage response pathways, and is distinct from mitogen removal and spontaneous quiescence. This suggests a novel pathway to quiescence and reveals essential micronutrients play a role in cell cycle regulation.

scholarly journals Methods and sensors for functional genomic studies of cell-cycle transitions in single cells

2020 ◽  
Vol 52 (10) ◽  
pp. 468-477
Author(s):  
Alexander C. Zambon ◽  
Tom Hsu ◽  
Seunghee Erin Kim ◽  
Miranda Klinck ◽  
Jennifer Stowe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Imaging System ◽  
Single Cells ◽  
Cell Types ◽  
Cell Analysis ◽  
Mcf7 Cells ◽  
Genomic Studies ◽  
Cell Subpopulations

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1–5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

The role of E2F in the mammalian cell cycle

1993 ◽  
Vol 1155 (2) ◽  
pp. 125-131 ◽  
Author(s):  
Peggy J. Farnham ◽  
Jill E. Slansky ◽  
Richard Kollamar
Keyword(s):  
Cell Cycle ◽  
Mammalian Cell ◽  
Mammalian Cell Cycle

Statistical single-cell analysis of cell cycle-dependent quantum dot cytotoxicity and cellular uptake using a microfluidic system

RSC Advances ◽  
2014 ◽  
Vol 4 (47) ◽  
pp. 24929-24934 ◽  
Author(s):  
Jing Wu ◽  
Haifang Li ◽  
Qiushui Chen ◽  
Xuexia Lin ◽  
Wu Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Quantum Dot ◽  
Single Cell ◽  
Cellular Uptake ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Microfluidic System ◽  
Cell Analysis ◽  
System P ◽  
Cycle Dependent

The response of single cells in different cell cycle phases to QD cytotoxicity studied on a microfluidic device.

scholarly journals The Expression and Prognostic Value of SUMO1-Activating Enzyme Subunit 1 and Its Potential Mechanism in Triple-Negative Breast Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Qingshui Wang ◽  
Wenting Zhong ◽  
Lin Deng ◽  
Qili Lin ◽  
Youyu Lin ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Protein Expression ◽  
Single Cell ◽  
Prognostic Model ◽  
Triple Negative ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Mrna And Protein Expression

Background: Triple-negative breast cancer (TNBC) is the most invasive and metastatic subtype of breast cancer. SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme, is indispensable for protein SUMOylation. SAE1 has been found to be a relevant biomarker for progression and prognosis in several tumor types. However, the role of SAE1 in TNBC remains to be elucidated.Methods: In the research, the mRNA expression of SAE1 was analyzed via the cancer genome atlas (TCGA) and gene expression omnibus (GEO) database. Cistrome DB Toolkit was used to predict which transcription factors (TFs) are most likely to increase SAE1 expression in TNBC. The correlation between the expression of SAE1 and the methylation of SAE1 or quantity of tumor-infiltrating immune cells was further invested. Single-cell analysis, using CancerSEA, was performed to query which functional states are associated with SAE1 in different cancers in breast cancer at the single-cell level. Next, weighted gene coexpression network (WGCNA) was applied to reveal the highly correlated genes and coexpression networks of SAE1 in TNBC patients, and a prognostic model containing SAE1 and correlated genes was constructed. Finally, we also examined SAE1 protein expression of 207 TNBC tissues using immunohistochemical (IHC) staining.Results: The mRNA and protein expression of SAE1 were increased in TNBC tissues compared with adjacent normal tissues, and the protein expression of SAE1 was significantly associated with overall survival (OS) and disease-free survival (DFS). Correlation analyses revealed that SAE1 expression was positively correlated with forkhead box M1 (FOXM1) TFs and negatively correlated with SAE1 methylation site (cg14042711) level. WGCNA indicated that the genes coexpressed with SAE1 belonged to the green module containing 1,176 genes. Through pathway enrichment analysis of the module, 1,176 genes were found enriched in cell cycle and DNA repair. Single-cell analysis indicated that SAE1 and its coexpression genes were associated with cell cycle, DNA damage, DNA repair, and cell proliferation. Using the LASSO COX regression, a prognostic model including SAE1 and polo-like kinase 1 (PLK1) was built to accurately predict the likelihood of DFS in TNBC patients.Conclusion: In conclusion, we comprehensively analyzed the mRNA and protein expression, prognosis, and interaction genes of SAE1 in TNBC and constructed a prognostic model including SAE1 and PLK1. These results might be important for better understanding of the role of SAE1 in TNBC. In addition, DNA methyltransferase and TFs inhibitor treatments targeting SAE1 might improve the survival of TNBC patients.

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