Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.; Gilbert, David M.

doi:10.1083/jcb.201007111

Cited by 79 publications

(92 citation statements)

References 68 publications

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“…We conclude that MCM proteins maintain long-lived interactions within the nucleus in contrast to Cdt1. This is consistent with findings in CHO cells (48) and indicates that the FIGURE 2. GFP-MCM2 and GFP-MCM4 bind to chromatin during G 1 phase, similar to endogenous MCM2 and MCM4 proteins.…”

Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 81%

“…These findings show that the presence of an immobile MCM pool, as measured by FRAP, closely mirrors MCM-chromatin association during licensing, consistent with earlier work (48). Our assay can therefore be used to assess licensing in vivo.…”

Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 73%

“…GFP-MCM2 localized to the nucleus and was excluded from the nucleoli, similar to endogenous MCM2 (data not shown). GFP-MCM4, transiently expressed, showed both nuclear and occasional cytoplasmic localization (data not shown), consistent with previous results showing that MCM4 lacks a nuclear localization signal and is transported to the nucleus in complex with other subunits (48,57). Several MCF7 cell lines stably expressing GFP-MCM2, GFP-MCM4 or a nuclear localized construct of GFP (GFP-NLS) were generated and analyzed by GFP fluorescence and Western blot analysis (data not shown).…”

Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 64%

“…A similar dynamic behavior was also reported for ORC components (45). In contrast, a recent study showed stable binding of MCM proteins to chromatin in CHO cells (48). To investigate the behavior of MCM proteins in live human cells, real-time in vivo confocal fluorescence microscopy was used.…”

Section: A System For Studying MCM Protein Dynamics Within Livementioning

confidence: 95%

“…Live cell imaging studies revealed both the ORC (45) and Cdt1 (46,47) to be highly mobile within the cell nucleus, whereas a recent study showed stable binding of MCM proteins throughout G 1 and S phases in CHO cells (48). Here, we use live-cell imaging to assess MCM chromatin loading at different cell cycle stages of human cancer cells.…”

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confidence: 99%

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Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Symeonidou

Kotsantis

Roukos

et al. 2013

Journal of Biological Chemistry

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Background: MCM2-7 loading onto chromatin licenses origins for replication. Results: MCMs exhibit transient interactions with chromatin in late mitosis, stable binding in G 1 phase and increased loading in late G 1 phase. Conclusion: Multilevel regulation of MCM2-7 loading to chromatin occurs during mitosis and preceding the G 1 /S phase transition. Significance: The dynamics of the DNA licensing system within live human cells reveal multiple control points.

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Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 81%

Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 73%

Section: A System For Studying MCM Protein Dynamics Within Livesupporting

confidence: 64%

Section: A System For Studying MCM Protein Dynamics Within Livementioning

confidence: 95%

mentioning

confidence: 99%

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Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Symeonidou

Kotsantis

Roukos

et al. 2013

Journal of Biological Chemistry

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Quantitative Colocalization Analysis of Fluorescence Microscopy Images

Zinchuk

Grossenbacher-Zinchuk

2014

CP Cell Biology

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Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of fluorescence microscopy images with colocalization based on calculation of a number of specialized coefficients. Images of double-stained sections are first subjected to background correction, and then various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of the results of calculations based on the use of linguistic variables are given. Success in colocalization studies depends on the quality of images analyzed, proper preparation of the images for coefficients calculations, and correct interpretation of results obtained. This protocol helps ensure reliability of colocalization coefficient calculations. Curr.Keywords: quantitative colocalization r fluorescence microscopy r image analysis r data interpretation

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High‐content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics

Harkness

McNulty

Prestil

et al. 2015

Biotechnology Journal

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Understanding the mechanisms underpinning cellular responses to microenvironmental cues requires tight control not only of the complex milieu of soluble signaling factors, extracellular matrix (ECM) connections and cell-cell contacts within cell culture, but also of the biophysics of human cells. Advances in biomaterial fabrication technologies have recently facilitated detailed examination of cellular biophysics and revealed that constraints on cell geometry arising from the cellular microenvironment influence a wide variety of human cell behaviors. Here, we create an in vitro platform capable of precise and independent control of biochemical and biophysical microenvironmental cues by adapting microcontact printing technology into the format of standard six- to 96-well plates to create MicroContact Printed Well Plates (μCP Well Plates). Automated high-content imaging of human cells seeded on μCP Well Plates revealed tight, highly consistent control of single-cell geometry, cytoskeletal organization, and nuclear elongation. Detailed subcellular imaging of the actin cytoskeleton and chromatin within live human fibroblasts on μCP Well Plates was then used to describe a new relationship between cellular geometry and chromatin dynamics. In summary, the μCP Well Plate platform is an enabling high-content screening technology for human cell biology and cellular engineering efforts that seek to identify key biochemical and biophysical cues in the cellular microenvironment.

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Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

Abstract: Components of the minichromosome maintenance complex (Mcm2-7) remain indefinitely bound to chromatin during G1 phase and replication arrest.

Cited by 79 publications

References 68 publications

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Quantitative Colocalization Analysis of Fluorescence Microscopy Images

High‐content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics

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