The Analysis of Cell Cycle, Proliferation, and Asymmetric Cell Division by Imaging Flow Cytometry

Rationale: Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates the analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox. Objective: We used a novel technique-imaging flow cytometry-to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart. Methods and Results: Cardiomyocytes were isolated from 8 subjects undergoing primary heart transplantation and 15 subjects following unloading with left ventricular assist device (LVAD, mean unloading time 13.7 ± 9.1 months). Myocyte size, nuclear number and size, DNA content (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Myocyte size and nuclear morphology was not significantly different between the groups. However, DNA content per nucleus was significantly decreased (P < 0.01) and the correlation between nuclear size and DNA content lost. The frequency of the cell cycle markers, Ki67 and phospho-histone3 (H3P) were not increased after hemodynamic unloading. Conclusions: Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.

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Slit scanning of Saccharomyces cerevisiae cells: quantification of asymmetric cell division and cell cycle progression in asynchronous culture

Biotechnology Progress ◽

10.1021/bp00006a015 ◽

1990 ◽

Vol 6 (6) ◽

pp. 504-512 ◽

Cited By ~ 26

Author(s):

David E. Block ◽

Philip D. Eitzman ◽

Jeffrey D. Wangensteen ◽

Friedrich Srienc

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Cell Division ◽

Cell Cycle Progression ◽

Asymmetric Cell Division ◽

Cycle Progression ◽

Slit Scanning

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The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid

Journal of Bacteriology ◽

10.1128/jb.00900-09 ◽

2009 ◽

Vol 192 (4) ◽

pp. 1058-1065 ◽

Cited By ~ 15

Author(s):

Ole Michelsen ◽

Flemming G. Hansen ◽

Bjarne Albrechtsen ◽

Peter Ruhdal Jensen

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Division ◽

Dna Replication ◽

Cell Size ◽

Lactococcus Lactis ◽

Uv Light ◽

Circular Chromosome ◽

Cellular Content ◽

Slow Growing

ABSTRACT Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.

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Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00524-06 ◽

2006 ◽

Vol 188 (16) ◽

pp. 5958-5965 ◽

Cited By ~ 23

Author(s):

Samer Sakr ◽

Melilotus Thyssen ◽

Michel Denis ◽

Cheng-Cai Zhang

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Division ◽

Cell Growth ◽

Cell Size ◽

Dna Content ◽

Small Cells ◽

Filamentous Cyanobacterium ◽

Pcc 7120 ◽

Heterocyst Development

ABSTRACT When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N2. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.

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Quantitative time-stretch imaging flow cytometry for high-throughput cell-cycle analysis

10.3390/optofluidics2017-04540 ◽

2017 ◽

Author(s):

Tsun Yin Mok ◽

Kevin K.M. Tsia ◽

Chak Man Lee ◽

Kenneth K.Y. Wong

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

High Throughput ◽

Cell Cycle Analysis ◽

Imaging Flow Cytometry

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Virus-induced cell gigantism and asymmetric cell division in archaea

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2022578118 ◽

2021 ◽

Vol 118 (15) ◽

pp. e2022578118 ◽

Cited By ~ 2

Author(s):

Junfeng Liu ◽

Virginija Cvirkaite-Krupovic ◽

Diana P. Baquero ◽

Yunfeng Yang ◽

Qi Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Transcriptional Repression ◽

Asymmetric Cell Division ◽

Cell Cycle Control ◽

Evolutionary Relationship ◽

Giant Cells ◽

Endosomal Sorting ◽

Infected Cells ◽

Cellular Dna

Archaeal viruses represent one of the most mysterious parts of the global virosphere, with many virus groups sharing no evolutionary relationship to viruses of bacteria or eukaryotes. How these viruses interact with their hosts remains largely unexplored. Here we show that nonlytic lemon-shaped virus STSV2 interferes with the cell cycle control of its host, hyperthermophilic and acidophilic archaeon Sulfolobus islandicus, arresting the cell cycle in the S phase. STSV2 infection leads to transcriptional repression of the cell division machinery, which is homologous to the eukaryotic endosomal sorting complexes required for transport (ESCRT) system. The infected cells grow up to 20-fold larger in size, have 8,000-fold larger volume compared to noninfected cells, and accumulate massive amounts of viral and cellular DNA. Whereas noninfected Sulfolobus cells divide symmetrically by binary fission, the STSV2-infected cells undergo asymmetric division, whereby giant cells release normal-sized cells by budding, resembling the division of budding yeast. Reinfection of the normal-sized cells produces a new generation of giant cells. If the CRISPR-Cas system is present, the giant cells acquire virus-derived spacers and terminate the virus spread, whereas in its absence, the cycle continues, suggesting that CRISPR-Cas is the primary defense system in Sulfolobus against STSV2. Collectively, our results show how an archaeal virus manipulates the cell cycle, transforming the cell into a giant virion-producing factory.

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The FACT complex and cell cycle progression are essential to maintain asymmetric transcription factor partitioning during cell division

10.1101/075135 ◽

2016 ◽

Author(s):

Eva Herrero ◽

Sonia Stinus ◽

Eleanor Bellows ◽

Peter H Thorpe

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Cell Division ◽

Cell Cycle Progression ◽

Asymmetric Cell Division ◽

Cycle Progression ◽

Cellular Processes ◽

Daughter Cells ◽

Cycle Arrest ◽

Reverse Genetic Screen

AbstractThe polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. Like most cells, the budding yeast Saccharomyces cerevisiae divides asymmetrically to give two distinct daughter cells. This asymmetry mimics that seen in metazoans and the key regulatory proteins are conserved from yeast to human. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We performed a reverse genetic screen to look for regulators of asymmetry based on the Ace2 localization phenotype. We screened a collection of essential genes in order to analyze the effect of core cellular processes in asymmetric cell division. This identified a large number of mutations that are known to affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model we blocked cells from progressing through mitosis and found that prolonged cell cycle arrest is sufficient to disrupt Ace2 asymmetry after release. We also demonstrate that members of the evolutionary conserved FACT chromatin-remodeling complex are required for both asymmetric and cell cycle-regulated localization of Ace2.

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Label-free cell cycle analysis for high-throughput imaging flow cytometry

Nature Communications ◽

10.1038/ncomms10256 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 128

Author(s):

Thomas Blasi ◽

Holger Hennig ◽

Huw D. Summers ◽

Fabian J. Theis ◽

Joana Cerveira ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

High Throughput ◽

Free Cell ◽

Cell Cycle Analysis ◽

Label Free ◽

Imaging Flow Cytometry ◽

High Throughput Imaging

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Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms?

Open Biology ◽

10.1098/rsob.130083 ◽

2013 ◽

Vol 3 (8) ◽

pp. 130083 ◽

Cited By ~ 31

Author(s):

Anna Noatynska ◽

Nicolas Tavernier ◽

Monica Gotta ◽

Lionel Pintard

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Cell Fate ◽

Cell Polarity ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Asymmetric Cell Division ◽

Control Cell ◽

Model Organisms ◽

Cycle Progression

Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.

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