The Consequences of Differential Origin Licensing Dynamics

MCM complexes are loaded onto chromosomes to license DNA replication origins in G1 phase of the cell cycle, but it is not yet known how mammalian MCM complexes are adequately distributed to both euchromatin and heterochromatin. To address this question, we combined time-lapse live-cell imaging with fixed cell immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in heterochromatin and euchromatin at different times within G1. We report here that MCM loading in euchromatin is faster than in heterochromatin in very early G1, but surprisingly, heterochromatin loading accelerates faster than euchromatin in middle and late G1. These different loading dynamics require ORCA-dependent differences in ORC distribution during G1. A consequence of heterochromatin origin licensing dynamics is that cells experiencing a truncated G1 phase from premature Cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.

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Efficiency and equity in origin licensing to ensure complete DNA replication

Biochemical Society Transactions ◽

10.1042/bst20210161 ◽

2021 ◽

Author(s):

Liu Mei ◽

Jeanette Gowen Cook

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Dna Replication ◽

Genome Stability ◽

S Phase ◽

Replication Initiation ◽

G1 Phase ◽

Dna Replication Initiation ◽

Origin Licensing ◽

Dna Replication Origins

The cell division cycle must be strictly regulated during both development and adult maintenance, and efficient and well-controlled DNA replication is a key event in the cell cycle. DNA replication origins are prepared in G1 phase of the cell cycle in a process known as origin licensing which is essential for DNA replication initiation in the subsequent S phase. Appropriate origin licensing includes: (1) Licensing enough origins at adequate origin licensing speed to complete licensing before G1 phase ends; (2) Licensing origins such that they are well-distributed on all chromosomes. Both aspects of licensing are critical for replication efficiency and accuracy. In this minireview, we will discuss recent advances in defining how origin licensing speed and distribution are critical to ensure DNA replication completion and genome stability.

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Time-Lapse, Live-Cell Imaging of Potassium Efflux in Cell Exposed to Nanosecond Pulsed Electric Fields

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1493 ◽

2021 ◽

Vol 120 (3) ◽

pp. 223a

Author(s):

Flavia Mazzarda ◽

Esin B. Sozer ◽

Julia L. Pittaluga ◽

Claudia Muratori ◽

P. Thomas Vernier

Keyword(s):

Electric Fields ◽

Live Cell Imaging ◽

Cell Imaging ◽

Pulsed Electric Fields ◽

Time Lapse ◽

Live Cell ◽

Potassium Efflux ◽

Nanosecond Pulsed Electric Fields

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Dye selection for live cell imaging of intact siRNA

Biological Chemistry ◽

10.1515/bc-2011-256 ◽

2012 ◽

Vol 393 (1-2) ◽

pp. 23-35 ◽

Cited By ~ 10

Author(s):

Markus Hirsch ◽

Dennis Strand ◽

Mark Helm

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Dyes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Lapse ◽

Live Cell ◽

High Yield ◽

Ph Variation ◽

Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

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Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Journal of Visualized Experiments ◽

10.3791/3145 ◽

2011 ◽

Cited By ~ 35

Author(s):

Imke G. de Jong ◽

Katrin Beilharz ◽

Oscar P. Kuipers ◽

Jan- Willem Veening

Keyword(s):

Bacillus Subtilis ◽

Streptococcus Pneumoniae ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Live Cell ◽

Time Lapse Microscopy

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Time-lapse live cell imaging to monitor doxorubicin release from DNA origami nanostructures

Journal of Materials Chemistry B ◽

10.1039/c7tb03223d ◽

2018 ◽

Vol 6 (11) ◽

pp. 1605-1612 ◽

Cited By ~ 19

Author(s):

Yun Zeng ◽

Jiajun Liu ◽

Shuo Yang ◽

Wenyan Liu ◽

Liang Xu ◽

...

Keyword(s):

Drug Delivery ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Dna Origami ◽

Live Cell ◽

Doxorubicin Release

DNA origami nanostructures can serve as a promising carrier for drug delivery due to the outstanding programmability and biocompatibility.

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Intracellular mechanisms of fungal space searching in microenvironments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816423116 ◽

2019 ◽

Vol 116 (27) ◽

pp. 13543-13552 ◽

Cited By ~ 2

Author(s):

Marie Held ◽

Ondřej Kašpar ◽

Clive Edwards ◽

Dan V. Nicolau

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Turgor Pressure ◽

Fungal Growth ◽

Time Lapse ◽

Growth Direction ◽

Medical Applications ◽

Initial Growth ◽

Space Partitioning ◽

Constrained Environments

Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

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Live cell imaging of macrophage/bacterium interaction demonstrates cell lysis induced by Corynebacterium diphtheriae and Corynebacterium ulcerans

BMC Research Notes ◽

10.1186/s13104-019-4733-y ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

Dulanthi Weerasekera ◽

Jonas Hahn ◽

Martin Herrmann ◽

Andreas Burkovski

Keyword(s):

Fluorescence Microscopy ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Corynebacterium Diphtheriae ◽

Live Cell ◽

Human Macrophage ◽

Data Description ◽

Corynebacterium Ulcerans ◽

Microscopy Data

Abstract Objectives In frame of a study to characterize the interaction of human macrophage-like cells with pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, live cell imaging experiments were carried out and time lapse fluorescence microscopy videos were generated, which are presented here. Data description The time lapse fluorescence microscopy data revealed new insights in the interaction of corynebacteria with human macrophage-like THP-1 cells. In contrast to uninfected cells and infections with non-pathogenic C. glutamicum used as a control, pathogenic C. diphtheriae and C. ulcerans showed highly detrimental effects towards human cells and induction of cell death of macrophages.

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Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

Scientific Reports ◽

10.1038/s41598-020-77969-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hiroaki Shimono ◽

Atsushi Kaida ◽

Hisao Homma ◽

Hitomi Nojima ◽

Yusuke Onozato ◽

...

Keyword(s):

Cell Cycle ◽

Hela Cells ◽

Time Lapse ◽

S Phase ◽

G1 Phase ◽

M Phase ◽

G2 Phase ◽

The Difference ◽

Time Lapse Imaging ◽

First Time

AbstractIn this study, we examined the fluctuation in radioresponse of HeLa cells during the cell cycle. For this purpose, we used HeLa cells expressing two types of fluorescent ubiquitination-based cell cycle indicators (Fucci), HeLa-Fucci (CA)2 and HeLa-Fucci (SA), and combined this approach with the micronucleus (MN) assay to assess radioresponse. The Fucci system distinguishes cell cycle phases based on the colour of fluorescence and cell morphology under live conditions. Time-lapse imaging allowed us to further identify sub-positions within the G1 and S phases at the time of irradiation by two independent means, and to quantitate the number of MNs by following each cell through M phase until the next G1 phase. Notably, we found that radioresponse was low in late G1 phase, but rapidly increased in early S phase. It then decreased until late S phase and increased in G2 phase. For the first time, we demonstrated the unique fluctuation of radioresponse by the MN assay during the cell cycle in HeLa cells. We discuss the difference between previous clonogenic experiments using M phase-synchronised cell populations and ours, as well as the clinical implications of the present findings.

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Keeping Life in Focus New Systems Prevent Z-axis Drift in Time Lapse Studies

Microscopy Today ◽

10.1017/s1551929500050276 ◽

2006 ◽

Vol 14 (4) ◽

pp. 42-46 ◽

Cited By ~ 1

Author(s):

Edward Lachica

Keyword(s):

Fluorescent Probes ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Living Cells ◽

Dynamic Processes ◽

Biological Structure ◽

Camera Lucida ◽

Developmental Anatomy ◽

Organ Level

Just two decades ago, life scientists studied biological structure, developmental anatomy and intracellular processes by describing individual snapshots of kinetic events. Today, with so much bioscience research focusing on dynamic processes that occur on the molecular, cellular and whole organ level, it is important to record events as they happen, over seconds, minutes or hours, in living cells. Photographs and camera lucida drawings of fixed, stained cells have given way to live cell imaging using fluorescent probes, warming trays to promote cell viability and cinemicrography as a method of recording events.

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