Advances and enabling technologies for phase-specific cell cycle synchronisation

Bordhan, Pritam; Bazaz, Sajad Razavi; Jin, Dayong; Warkiani, Majid Ebrahimi

doi:10.1039/d1lc00724f

Cited by 7 publications

(3 citation statements)

References 156 publications

(202 reference statements)

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“…PM-localized phosphoinositides (PI4P, PI(4,5)P2, and PI(3,4,5)P3) play several key roles throughout cell division, including cell rounding beginning in prophase, spindle orientation during metaphase, and furrow ingression during cytokinesis 48 . These functions have been investigated largely by genetic, pharmacological, and chemogenetic methods for chronically or acutely manipulating PIP levels that are often employed in conjunction with harsh cell synchronization methods that can perturb physiology [49][50][51] . MARS presents an opportunity to manipulate PM PI (3,4,5)P3 levels in bulk cell populations without synchronization, prompting us to investigate whether MARS-p85 iSH2 might reveal new mitotic roles for PI (3,4,5)P3.…”

Section: Mars-p85 Ish2 Reveals An Inhibitory Effect Of a Pm Pi(345)p3...mentioning

confidence: 99%

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

Cao,

Huang,

Wagner

et al. 2024

Preprint

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Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled Anchor-away/Recruiter System (MARS) comprises an engineered, 15-kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP–GFP nanobody interaction. Applications of MARS include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.

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Section: Mars-p85 Ish2 Reveals An Inhibitory Effect Of a Pm Pi(345)p3...mentioning

confidence: 99%

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

Cao,

Huang,

Wagner

et al. 2024

Preprint

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“…In addition to the assessment of cell viability using the classical clonogenic assay, developed by Puck andMarcus in 1956 (Puck andMarcus 1956), cell cycle synchronization in culture cells is one of the experimental techniques that have supported the significant progress in this field. However, conventional synchronization techniques using chemical blockade or physical fraction have various technical limitations, such as the physiological load on living cells and the contamination of dead cells (Bordhan et al 2022). Furthermore, the accurate detection of cell cycle changes in individual cells under cell cycle-synchronized culture conditions is technically difficult.…”

Section: Introductionmentioning

confidence: 99%

Cell cycle dependence of cell survival following exposure to X-rays in synchronous HeLa cells expressing fluorescent ubiquitination-based cell cycle indicators

SEINO,

UNO,

PRISE

et al. 2024

Biomed. Res.

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The cell cycle dependence of radiosensitivity has yet to be fully determined, as it is technically difficult to achieve a high degree of cell cycle synchronization in cultured cell systems and accurately detect the cell cycle phase of individual cells simultaneously. We used human cervical carcinoma HeLa cells expressing fluorescent ubiquitination-based cell cycle indicators (FUCCI), and employed the mitotic harvesting method that is one of the cell cycle synchronization methods. The imaging analysis confirmed that the cell cycle is highly synchronized after mitotic cell harvesting until 18-20 h of the doubling time has elapsed. Also, flow cytometry analysis revealed that the S and G2 phases peak at approximately 12 and 14-16 h, respectively, after mitotic harvesting. In addition, the clonogenic assay showed the changes in surviving fractions following exposure to X-rays according to the progress through the cell cycle. These results indicate that HeLa-FUCCI cells become radioresistant in the G1 phase, become radiosensitive in the early S phase, rapidly become radioresistant in the late S phase, and become radiosensitive again in the G2 phase. Our findings may contribute to the further development of combinations of radiation and cell cycle-specific anticancer agents.

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“…Among the contributing factors toward cellular heterogeneity, unsynchronized cycle of cells is one of the most important factors that affect the homologous cell phenotypes in the culture and leads to the drug-response, aging, and many other heterogeneous behaviors. , Homologous cells under the mitotic phase (M phase), G 0 phase, G 1 phase, DNA synthesis phase (S phase), or G 2 phase are significantly different from intracellular active compounds to cellular morphology, and information collected from known specific cell cycle stages is undoubtedly more accurate than average results for disease and cell biology research. Protocols based on cell cycle synchronization before analysis have been employed to analyze cell cycle-related pathways, but the synchronization is time-consuming and might affect the biochemical pathways. Ideally, the natural cell cycle stage needs to be identified in real time for each cell with other cellular information during a high-throughput cytometric analysis …”

Section: Introductionmentioning

confidence: 99%

Organic Mass Cytometry Discriminating Cycle Stages of Single Cells with Small Molecular Indicators

Yang

Yan

2023

Anal. Chem.

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Cell cycle is a significant factor toward cellular heterogeneity, so cell cycle discrimination is a precise measurement on the top of single-cell analysis. Single-cell analysis based on organic mass spectrometry has received great attention for its unique ability to profile single-cell metabolome, but the influence of cell cycle on cellular metabolome heterogeneity has been overlooked until now due to the lack of a compatible cell cycle discrimination method. Here, we report a robust protocol based on the combination of three small molecular indicators, consisting of two small molecular labels (Hoechst and docetaxel) and one cellular endogenous compound [phosphocholine (34:1)], to discriminate single cells at different cycle stages in real time by organic mass cytometry. More than 6000 HeLa cells were acquired by an improved organic mass cytometry system to build a cell cycle differentiation model. The model successfully discriminated single HeLa cells, SCC7, and Hep G2 cells, at G0/G1, S, and G2/M stages with larger than 85% sensitivity and larger than 89% specificity. Along with cell cycle discrimination, obvious heterogeneity of amino acids, nucleotides, energy metabolic intermediates, and phospholipids was observed among single cells at different cycle stages by this protocol, further demonstrating the necessity of cell cycle discrimination for cellular metabolome heterogeneity research and the potential of more endogenous small molecular compounds for cell cycle discrimination.

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Advances and enabling technologies for phase-specific cell cycle synchronisation

Abstract: Cell cycle synchronisation is the process of isolating cell populations at specific phases of the cell cycle from heterogeneous, asynchronous cell cultures. The process has important implications in targeted gene-editing...

Cited by 7 publications

References 156 publications

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

A phosphorylation-controlled switch confers cell cycle-dependent protein relocalization

Cell cycle dependence of cell survival following exposure to X-rays in synchronous HeLa cells expressing fluorescent ubiquitination-based cell cycle indicators

Organic Mass Cytometry Discriminating Cycle Stages of Single Cells with Small Molecular Indicators

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