scholarly journals Knockdown of Dinoflagellate Condensin CcSMC4 Subunit Leads to S-Phase Impediment and Decompaction of Liquid Crystalline Chromosomes

2020 ◽  
Vol 8 (4) ◽  
pp. 565
Author(s):  
Ting Hin Kosmo Yan ◽  
Zhihao Wu ◽  
Alvin Chun Man Kwok ◽  
Joseph Tin Yum Wong
Keyword(s):  
Liquid Crystalline ◽  
Protein Complexes ◽  
Early Stage ◽  
S Phase ◽  
Packaging System ◽  
Local Rigidity ◽  
M Phase ◽  
G2 Phase ◽  
Phase Progression ◽  
Structural Maintenance Of Chromosomes

Dinoflagellates have some of the largest genomes, and their liquid-crystalline chromosomes (LCCs) have high degrees of non-nucleosomal superhelicity with cation-mediated DNA condensation. It is currently unknown if condensins, pentameric protein complexes containing structural maintenance of chromosomes 2/4, commonly involved in eukaryotic chromosomes condensation in preparation for M phase, may be involved in the LCC structure. We find that CcSMC4p (dinoflagellate SMC4 homolog) level peaked at S/G2 phase, even though LCCs do not undergo global-decondensation for replication. Despite the differences in the chromosomal packaging system, heterologous CcSMC4p expression suppressed conditional lethality of the corresponding fission yeast mutant, suggesting conservation of some canonical condensin functions. CcSMC4p-knockdown led to sustained expression of the S-phase marker PCNAp, S-phase impediment, and distorted nuclei in the early stage of CcSMC4p depletion. Prolonged CcSMC4p-knockdown resulted in aneuploidal cells and nuclear swelling with increasing LCC decompaction–decondensation. Cumulatively, our data suggested CcSMC4p function was required for dinoflagellate S-phase progression, and we propose that condensin-mediated higher-order compaction provisioning is involved in the provision of local rigidity for the replisome.

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

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.

scholarly journals Cyclin A2/Cdk1 is Essential for the in vivo S Phase Entry by Phosphorylating Top2a

2019 ◽  
Author(s):  
Miaomiao Jin ◽  
Ruikun Hu ◽  
Baijie Xu ◽  
Weilai Huang ◽  
Hong Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin B1 ◽  
S Phase ◽  
Downstream Target ◽  
Early Embryonic Lethality ◽  
M Phase ◽  
Phase Progression ◽  
Cyclin A2 ◽  
Phase Entry

AbstractCyclin-dependent kinase 1 (CDK1) plays essential roles in cell cycle regulation. However, due to the early embryonic lethality of mouse Cdk1 mutants, the in vivo role of CDK1 in regulating cell cycle and embryonic development remains unclear. Here, by generating zebrafish cdk1 mutants using CRISPR/Cas9 system, we show that cdk1−/− embryos exhibit severe microphthalmia accompanied with multiple defects in polarized cell division, S phase entry and M phase progression, cell apoptosis and cell differentiation, but not in interkinetic nuclear migration (IKNM). By informatics analysis, we identified Top2a as a potential downstream target, and Cyclin A2 and Cyclin B1 as partners of Cdk1 in cell cycle. Depletion of either Cyclin A2 or Top2a leads to decreased S phase entry and increased DNA damage response in zebrafish retinal cells, and depletion of Cyclin B1 leads to M phase arrest. Immunoprecipitation shows that Cdk1 and Cyclin A2 physically interact in vivo. Moreover, phosphorylation of Top2a on Serine 1213 (S1213) site is almost absent in either cdk1 or ccna2 mutants, but in not ccnb1 mutants. Furthermore, overexpression of TOP2AS1213, the phosphomimetic form of human TOP2A, rescues S phase entry and microphthalmia defects in cdk1−/− and ccna2−/− embryos. Taken together, our data suggests that Cdk1 interacts with Cyclin A2 to regulate S phase entry through phosphorylating Top2a, and with Cyclin B1 to regulate M phase progression in vivo.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

Bimodal Degradation of MLL Protein by the Cell Cycle SCFSkp2 and APCCdc20 E3 Lilgases Assures Cell Cycle Execution: A Critical Regulatory Circuit Lost in Leukemogenic MLL-Fusions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 828-828 ◽  
Author(s):  
Han Liu ◽  
David Chen ◽  
Todd Westergard ◽  
Shugaku Takeda ◽  
Satoru Sasagawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hox Genes ◽  
S Phase ◽  
Polycomb Group ◽  
Cell Cycle Gene ◽  
E3 Ligases ◽  
M Phase ◽  
Phase Progression

Abstract The MLL (mixed lineage leukemia) gene encodes a highly conserved 3,969 aa histone H3 K4 methyl transferase, of which chromosome translocations result in poor prognostic infant and therapy-related leukemias. Mysteriously, the pathognomonic MLL leukemia fusions consist of a common amino(N)-terminal ∼1400 aa of MLL fused in frame with more than 60 different partners with no shared characteristics. The most well known genetic function of MLL is to antagonize polycomb group of proteins for proper Hox gene expression. Consequently deregulated Hox genes caused by MLL translocations contribute to the MLL leukemogenesis. Besides Hox genes, it remains largely undetermined whether MLL participates in other biological processes. The 500kD MLL precursor undergoes evolutionarily conserved proteolytic maturation mediated by Taspase1 (Hsieh et al. 2003, MCB, 23, 186–194; Hsieh et al. 2003, Cell, 115, 293–303). Our recent studies on Taspase1 knockout cells established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins and possibly Cdk inhibitors (Takeda et al. 2006, Genes & Development, 20, 2397–2409). As MLL actively participates in the cell cycle regulation, we investigated the regulation of MLL through cell cycle transition. We uncovered a unique biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M phase progression. Conversely, over-expression of MLL blocks S phase progression. Remarkably, MLL degradation initiates at its N-terminal ∼1400 aa that is retained in all MLL leukemia fusions. We examined prevalent MLL-fusions, including MLL-AF4, MLL-AF9, MLL-ELL and MLL-ELL, and observed their increased resistance to degradation. Furthermore, the same resistance was observed with the leukemogenic MLL-lacZ but not the non-leukemogenic MLL-Myc tag fusion. Thus, non-oscillating expression of MLL-fusions through the cell cycle, resulted from impaired degradation, likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) to assure the temporal necessity of MLL in coordinating cell cycle progression. Future studies aim at providing a comprehensive analysis on the cell cycle consequences associated with MLL-fusions using genetically modified cells derived from mice carrying various MLL-fusion knockin alleles, including MLL-AF4, MLL-AF9, and MLL-CBP.

scholarly journals ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin

2012 ◽  
Vol 200 (1) ◽  
pp. 31-44 ◽  
Author(s):  
Kyoo-young Lee ◽  
Haiqing Fu ◽  
Mirit I. Aladjem ◽  
Kyungjae Myung
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Nuclear Antigen ◽  
Spatial Regulation ◽  
Replication Factory ◽  
G2 Phase ◽  
Phase Progression ◽  
Temporal And Spatial ◽  
Proliferating Cell

Temporal and spatial regulation of the replication factory is important for efficient DNA replication. However, the underlying molecular mechanisms are not well understood. Here, we report that ATAD5 regulates the lifespan of replication factories. Reduced expression of ATAD5 extended the lifespan of replication factories by retaining proliferating cell nuclear antigen (PCNA) and other replisome proteins on the chromatin during and even after DNA synthesis. This led to an increase of inactive replication factories with an accumulation of replisome proteins. Consequently, the overall replication rate was decreased, which resulted in the delay of S-phase progression. Prevalent detection of PCNA foci in G2 phase cells after ATAD5 depletion suggests that defects in the disassembly of replication factories persist after S phase is complete. ATAD5-mediated regulation of the replication factory and PCNA required an intact ATAD5 ATPase domain. Taken together, our data imply that ATAD5 regulates the cycle of DNA replication factories, probably through its PCNA-unloading activity.

scholarly journals Transforming growth factor β decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase

1994 ◽  
Vol 299 (1) ◽  
pp. 227-235 ◽  
Author(s):  
D J Grainger ◽  
P R Kemp ◽  
C M Witchell ◽  
P L Weissberg ◽  
J C Metcalfe
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Cyclic Amp ◽  
Vascular Smooth Muscle Cells ◽  
Transforming Growth Factor ◽  
S Phase ◽  
Tgf Beta ◽  
M Phase ◽  
G2 Phase

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.

scholarly journals Assessing the ubiquity and origins of structural maintenance of chromosomes (SMC) proteins in eukaryotes

2021 ◽  
Author(s):  
Mari Yoshinaga ◽  
Yuji Inagaki
Keyword(s):  
Protein Complexes ◽  
Early Stage ◽  
Functional Divergence ◽  
Protein S ◽  
Single Type ◽  
Eukaryotic Evolution ◽  
Taxonomic Groups ◽  
Related Proteins ◽  
Structural Maintenance Of Chromosomes ◽  
Smc Proteins

Structural maintenance of chromosomes (SMC) protein complexes are common in Bacteria, Archaea, and Eukaryota. SMC proteins, together with the proteins related to SMC (SMC-related proteins), constitute a superfamily of ATPases. Bacteria/Archaea and Eukaryotes are distinctive from one another in terms of the repertory of SMC proteins. A single type of SMC protein is dimerized in the bacterial and archaeal complexes, whereas eukaryotes possess six distinct SMC subfamilies (SMC1-6), constituting three heterodimeric complexes, namely cohesin, condensin, and SMC5/6 complex. Thus, to bridge the homodimeric SMC complexes in Bacteria and Archaea to the heterodimeric SMC complexes in Eukaryota, we need to invoke multiple duplications of a SMC gene followed by functional divergence. However, to our knowledge, the evolution of the SMC proteins in Eukaryota had not been examined for more than a decade. In this study, we reexamined the ubiquity of SMC1-6 in phylogenetically diverse eukaryotes that cover the major eukaryotic taxonomic groups recognized to date (101 species in total) and provide two novel insights into the SMC evolution in eukaryotes. First, multiple secondary losses of SMC5 and SMC6 occurred in the eukaryotic evolution. Second, the SMC proteins constituting cohesin and condensin (i.e., SMC1-4), and SMC5 and SMC6 were derived from closely related but distinct ancestral proteins. Finally, we discuss how SMC1-4 were evolved from the ancestral SMC protein(s) in the very early stage of eukaryotic evolution.

scholarly journals An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells

2007 ◽  
Vol 178 (2) ◽  
pp. 257-268 ◽  
Author(s):  
Helfrid Hochegger ◽  
Donniphat Dejsuphong ◽  
Eiichiro Sonoda ◽  
Alihossein Saberi ◽  
Eeson Rajendra ◽  
...  
Keyword(s):  
Chemical Genetics ◽  
Phase Control ◽  
S Phase ◽  
Replication Initiation ◽  
Cyclin Dependent Kinases ◽  
Dna Replication Initiation ◽  
Atp Analogs ◽  
G2 Phase ◽  
Phase Progression ◽  
Rapid Activation

In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences

2021 ◽  
Author(s):  
P Stamatiadis ◽  
A Boel ◽  
G Cosemans ◽  
M Popovic ◽  
B Bekaert ◽  
...  
Keyword(s):  
Blastocyst Stage ◽  
S Phase ◽  
Preimplantation Development ◽  
Human Embryos ◽  
Immunofluorescence Analysis ◽  
Media Control ◽  
Cas9 Protein ◽  
M Phase ◽  
Developmental Capacity

Abstract STUDY QUESTION What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY Clustered regularly interspaced short palindromic repeats—CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain—B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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