Optimization of Cell Cycle Measurement by Time-Lapse Microscopy

ABSTRACT Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 μm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

10.1101/309914 ◽

2018 ◽

Author(s):

Zeynep Kabakci ◽

Simon Käppeli ◽

Giorgio Cozza ◽

Claudio Cantù ◽

Christiane König ◽

...

Keyword(s):

Cell Cycle ◽

Mixed Type ◽

Molecular Level ◽

Time Lapse ◽

Guided Discovery ◽

Enzymatic Assays ◽

Intestinal Crypt ◽

Time Lapse Microscopy ◽

Cycle Arrest ◽

Mechanism Of Inhibition

ABSTRACTCDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

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Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

eLife ◽

10.7554/elife.48063 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Guillaume Witz ◽

Erik van Nimwegen ◽

Thomas Julou

Keyword(s):

Cell Cycle ◽

Time Lapse ◽

Growth Conditions ◽

Replication Initiation ◽

Stochastic Simulations ◽

E Coli ◽

Time Lapse Microscopy ◽

Statistical Framework ◽

Wide Range ◽

Cell Cycle Models

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

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Time‐Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single‐Cell Level

Current Protocols in Cytometry ◽

10.1002/0471142956.cy1204s64 ◽

2013 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Rachel J. Errington ◽

Sally C. Chappell ◽

Imtiaz A. Khan ◽

Nuria Marquez ◽

Marie Wiltshire ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Time Lapse ◽

Single Cell Level ◽

Cell Level ◽

Time Lapse Microscopy

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The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation

Blood ◽

10.1182/blood.v126.23.1188.1188 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1188-1188

Author(s):

Susanne Wingert ◽

Frederic B Thalheimer ◽

Nadine Haetscher ◽

Maike Rehage ◽

Hubert Serve ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Stem Cell ◽

Stem Cell Differentiation ◽

Genotoxic Stress ◽

Time Lapse ◽

Cellular Systems ◽

Hematopoietic Stem ◽

Time Lapse Microscopy ◽

Differentiation Induction

Abstract The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level. (1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014) Disclosures No relevant conflicts of interest to declare.

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Time‐Lapse Microscopy Approaches to Track Cell Cycle Progression at the Single‐Cell Level

Current Protocols in Cytometry ◽

10.1002/0471142956.cy1204s31 ◽

2005 ◽

Vol 31 (1) ◽

Cited By ~ 7

Author(s):

Rachel J. Errington ◽

Nuria Marquez ◽

Sally C. Chappell ◽

Marie Wiltshire ◽

Paul J. Smith

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Cell Cycle Progression ◽

Time Lapse ◽

Single Cell Level ◽

Cell Level ◽

Cycle Progression ◽

Time Lapse Microscopy

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Recombination occurs within minutes of replication blockage by RTS1 producing restarted forks that are prone to collapse

eLife ◽

10.7554/elife.04539 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 29

Author(s):

Michael O Nguyen ◽

Manisha Jalan ◽

Carl A Morrow ◽

Fekret Osman ◽

Matthew C Whitby

Keyword(s):

Cell Cycle ◽

Replication Fork ◽

Genome Duplication ◽

Time Lapse ◽

Genetic Change ◽

Replication Proteins ◽

Site Specific ◽

Time Lapse Microscopy ◽

A Site ◽

Stalled Fork

The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-lapse microscopy and genetic assays to show that recombination is initiated within ∼10 min of replication fork blockage at a site-specific barrier in fission yeast, leading to a restarted fork within ∼60 min, which is only prevented/curtailed by the arrival of the opposing replication fork. The restarted fork is susceptible to further collapse causing hyper-recombination downstream of the barrier. Surprisingly, in our system fork restart is unnecessary for maintaining cell viability. Seemingly, the risk of failing to complete replication prior to mitosis is sufficient to warrant the induction of recombination even though it can cause deleterious genetic change.

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Two-Step Assembly Dynamics of the Bacillussubtilis Divisome

Journal of Bacteriology ◽

10.1128/jb.01758-08 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4186-4194 ◽

Cited By ~ 116

Author(s):

Pamela Gamba ◽

Jan-Willem Veening ◽

Nigel J. Saunders ◽

Leendert W. Hamoen ◽

Richard A. Daniel

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Fluorescent Protein ◽

Time Lapse ◽

Localization Pattern ◽

Ftsz Ring ◽

Time Lapse Microscopy ◽

Green Fluorescent ◽

Posttranscriptional Level

ABSTRACT Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

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Bacillus subtilis SMC Is Required for Proper Arrangement of the Chromosome and for Efficient Segregation of Replication Termini but Not for Bipolar Movement of Newly Duplicated Origin Regions

Journal of Bacteriology ◽

10.1128/jb.182.22.6463-6471.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6463-6471 ◽

Cited By ~ 30

Author(s):

Peter L. Graumann

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Chromosome Segregation ◽

Late Stage ◽

Time Lapse ◽

Chromosome Condensation ◽

Time Lapse Microscopy ◽

Rapid Movement ◽

Segregation Process ◽

Mutant Cells

ABSTRACT SMC protein is required for chromosome condensation and for the faithful segregation of daughter chromosomes in Bacillus subtilis. The visualization of specific sites on the chromosome showed that newly duplicated origin regions in growing cells of ansmc mutant were able to segregate from each other but that the location of origin regions was frequently aberrant. In contrast, the segregation of replication termini was impaired in smcmutant cells. This analysis was extended to germinating spores of ansmc mutant. The results showed that during germination, newly duplicated origins, but not termini, were able to separate from each other in the absence of SMC. Also, DAPI (4′,6′-diamidino-2-phenylindole) staining revealed that chromosomes in germinating spores were able to undergo partial or complete replication but that the daughter chromosomes were blocked at a late stage in the segregation process. These findings were confirmed by time-lapse microscopy, which showed that after duplication in growing cells the origin regions underwent rapid movement toward opposite poles of the cell in the absence of SMC. This indicates that SMC is not a required component of the mitotic motor that initially drives origins apart after their duplication. It is also concluded that SMC is needed to maintain the proper layout of the chromosome in the cell and that it functions in the cell cycle after origin separation but prior to complete segregation or replication of daughter chromosomes. It is proposed here that chromosome segregation takes place in at least two steps: an SMC-independent step in which origins move apart and a subsequent SMC-dependent step in which newly duplicated chromosomes condense and are thereby drawn apart.

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