scholarly journals In vitro cell cycle oscillations exhibit a robust and hysteretic response to changes in cytoplasmic density

2021 ◽  
Author(s):  
Minjun Jin ◽  
Franco Tavella ◽  
Shiyuan Wang ◽  
Qiong Yang
Keyword(s):  
Cell Cycle ◽  
Oscillatory Behavior ◽  
Cycle Number ◽  
Biochemical Processes ◽  
Cellular Processes ◽  
Wide Range ◽  
A Cell ◽  
Xenopus Egg ◽  
Number Of Cycles

Cells control the properties of the cytoplasm to ensure the proper functioning of biochemical processes. Recent studies showed that the density of the cytoplasm varies in both physiological and pathological states of cells undergoing growth, division, differentiation, apoptosis, senescence, and metabolic starvation. Little is known about how cellular processes cope with these cytoplasmic variations. Here we study how a cell cycle oscillator comprising cyclin-dependent kinase (CDK1) responds to cytoplasmic density changes by systematically diluting or concentrating a cycling Xenopus egg cytoplasm in cell-like microfluidic droplets. We found that the cell cycle maintains robust oscillations over a wide range of deviations from the endogenous density by as low as 0.2x to more than 1.22x. A further dilution or concentration from these values will arrest the system in a low or high steady-state of CDK1 activity, respectively. Interestingly, diluting a concentrated arrested cytoplasm recovers its oscillatory behavior but requires a significantly lower concentration than 1.22x. Thus, the cell cycle switches reversibly between oscillatory and stable steady states at distinct thresholds depending on the direction of density tuning, forming a hysteresis loop. We recapitulated these observations by a mathematical model. The model predicted that Wee1 and Cdc25 positive feedback do not contribute to the observed robustness, confirmed by experiments. Nevertheless, modulating these feedback strengths and cytoplasmic density changes the total number of cycles, revealing a new role of Wee1 and Cdc25 in controlling the cycle number of early embryonic extracts. Our system can be applied to study how cytoplasmic density affects other cellular processes.

scholarly journals Conserved Promoter Motif Is Required for Cell Cycle Timing of dnaX Transcription inCaulobacter

2001 ◽  
Vol 183 (16) ◽  
pp. 4860-4865 ◽  
Author(s):  
Kenneth C. Keiler ◽  
Lucy Shapiro
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Caulobacter Crescentus ◽  
Transcriptional Networks ◽  
Cellular Processes ◽  
Genes Encoding ◽  
Constitutive Gene Expression ◽  
A Cell ◽  
Replication Factors

ABSTRACT Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, includingdnaX, dnaA, dnaN,gyrB, and dnaK, are induced at the G1/S-phase transition. By analyzing mutations in thednaX promoter, we identified a motif between the −10 and −35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

scholarly journals Overexpression of 14-3-3σ Modulates Cholangiocarcinoma Cell Survival by PI3K/Akt Signaling

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Qiao Wu ◽  
Hua Fan ◽  
Ren Lang ◽  
Xianliang Li ◽  
Xingmao Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Physiological Function ◽  
Tumor Stage ◽  
Akt Signaling ◽  
Cellular Processes ◽  
Cholangiocarcinoma Cell ◽  
A Cell ◽  
Xenograft Models

The protein 14-3-3σ is involved in numerous cellular processes through its ability to bind phosphorylated serine/threonine residues. It is a key regulator of the cell cycle involving in G2 arrest by p53. Deregulation of 14-3-3σ expression has been associated with a large variety of human cancers. However, its physiological function and therapeutic significance have rarely been investigated in cholangiocarcinoma. Using immunohistochemistry (IHC), we evaluated 14-3-3σ expression in 65 human extrahepatic cholangiocarcinomas. As a result, we found that 14-3-3σ is expressed in the tissue of 56 patients (86.2%), and its expression is positively correlated with tumor size, lymph node metastasis, and tumor stage. We also explored the significance of 14-3-3σ and found that 14-3-3σ exerts cell type-dependent effects on cell proliferation through PI3K/Akt signaling in both in vitro and in vivo xenograft models. These results suggest that 14-3-3σ assumes a constitutive role in tumorigenesis rather than acting as a cell cycle regulator in cholangiocarcinoma, which makes 14-3-3σ a new potential target for therapeutic intervention.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

1995 ◽  
Vol 108 (6) ◽  
pp. 2187-2196 ◽  
Author(s):  
L.J. Wangh ◽  
D. DeGrace ◽  
J.A. Sanchez ◽  
A. Gold ◽  
Y. Yeghiazarians ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Nuclear Transplantation ◽  
Optimal Time ◽  
Genome Replication ◽  
Cell Nuclei ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

scholarly journals RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
G Quadruplex ◽  
Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

scholarly journals Lanthionine synthetase C–like protein 2 (LanCL2) is a novel regulator of Akt

2014 ◽  
Vol 25 (24) ◽  
pp. 3954-3961 ◽  
Author(s):  
Min Zeng ◽  
Wilfred A. van der Donk ◽  
Jie Chen
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptor Substrate ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Cellular Processes ◽  
Human Liver Cells ◽  
Wide Range ◽  
Physical Interactions ◽  
Protein Kinase Akt

The serine/threonine protein kinase Akt controls a wide range of biochemical and cellular processes under the modulation of a variety of regulators. In this study, we identify the lanthionine synthetase C–like 2 (LanCL2) protein as a positive regulator of Akt activation in human liver cells. LanCL2 knockdown dampens serum- and insulin-stimulated Akt phosphorylation, whereas LanCL2 overexpression enhances these processes. Neither insulin receptor phosphorylation nor the interaction between insulin receptor substrate and phosphatidylinositide 3-kinase (PI3K) is affected by LanCL2 knockdown. LanCL2 also does not function through PP2A, a phosphatase of Akt. Instead, LanCL2 directly interacts with Akt, with a preference for inactive Akt. Moreover, we show that LanCL2 also binds to the Akt kinase mTORC2, but not phosphoinositide-dependent kinase 1. Whereas LanCL2 is not required for the Akt-mTORC2 interaction, recombinant LanCL2 enhances Akt phosphorylation by target of rapamycin complex 2 (mTORC2) in vitro. Finally, consistent with a function of Akt in regulating cell survival, LanCL2 knockdown increases the rate of apoptosis, which is reversed by the expression of a constitutively active Akt. Taken together, our findings reveal LanCL2 as a novel regulator of Akt and suggest that LanCL2 facilitates optimal phosphorylation of Akt by mTORC2 via direct physical interactions with both the kinase and the substrate.

scholarly journals Evidence for a dual role for TC4 protein in regulating nuclear structure and cell cycle progression.

1994 ◽  
Vol 125 (4) ◽  
pp. 705-719 ◽  
Author(s):  
S Kornbluth ◽  
M Dasso ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Structure ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
A Cell ◽  
Xenopus Egg Extracts

TC4, a ras-like G protein, has been implicated in the feedback pathway linking the onset of mitosis to the completion of DNA replication. In this report we find distinct roles for TC4 in both nuclear assembly and cell cycle progression. Mutant and wild-type forms of TC4 were added to Xenopus egg extracts capable of assembling nuclei around chromatin templates in vitro. We found that a mutant TC4 protein defective in GTP binding (GDP-bound form) suppressed nuclear growth and prevented DNA replication. Nuclear transport under these conditions approximated normal levels. In a separate set of experiments using a cell-free extract of Xenopus eggs that cycles between S and M phases, the GDP-bound form of TC4 had dramatic effects, blocking entry into mitosis even in the complete absence of nuclei. The effect of this mutant TC4 protein on cell cycle progression is mediated by phosphorylation of p34cdc2 on tyrosine and threonine residues, negatively regulating cdc2 kinase activity. Therefore, we provide direct biochemical evidence for a role of TC4 in both maintaining nuclear structure and in the signaling pathways that regulate entry into mitosis.

scholarly journals Gold Nanoparticles Promote Oxidant-Mediated Activation of NF-κB and 53BP1 Recruitment-Based Adaptive Response in Human Astrocytes

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Jennifer Mytych ◽  
Anna Lewinska ◽  
Jacek Zebrowski ◽  
Maciej Wnuk
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Gold Nanoparticles ◽  
Adaptive Response ◽  
Inverse Correlation ◽  
Galactosidase Activity ◽  
Promising Candidate ◽  
Human Astrocytes ◽  
Wide Range

Nanogold-based materials are promising candidate tools for nanobased medicine. Nevertheless, no conclusive information on their cytotoxicity is available. In the present study, we investigated the effects of gold nanoparticles (AuNPs) on human astrocytesin vitro. Nanogold treatment in a wide range of concentrations did not result in cytotoxicity. In contrast, nanogold provoked changes in the astrocyte cell cycle and induced senescence-associatedβ-galactosidase activity. AuNPs promoted oxidative stress and caused activation of NF-κB pathway. After nanogold treatment, an inverse correlation between the formation of 53BP1 foci and micronuclei generation was observed. The robust 53BP1 recruitment resulted in reduced micronuclei production. Thus, nanogold treatment stimulated an adaptive response in a human astrocyte cell.

scholarly journals Reversal of Growth Suppression by p107 via Direct Phosphorylation by Cyclin D1/Cyclin-Dependent Kinase 4

2002 ◽  
Vol 22 (7) ◽  
pp. 2242-2254 ◽  
Author(s):  
Xiaohong Leng ◽  
Martin Noble ◽  
Peter D. Adams ◽  
Jun Qin ◽  
J. Wade Harper
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Control Cell ◽  
Growth Suppression ◽  
Cyclin Dependent Kinase ◽  
Culture Cells ◽  
Cycle Arrest ◽  
A Cell ◽  
Control Cell Division

ABSTRACT p107 functions to control cell division and development through interaction with members of the E2F family of transcription factors. p107 is phosphorylated in a cell cycle-regulated manner, and its phosphorylation leads to its release from E2F. Although it is known that p107 physically associates with E- and A-type cyclin/cyclin-dependent kinase 2 (Cdk2) complexes through a cyclin-binding RXL motif located in the spacer domain, the mechanisms underlying p107 inactivation via phosphorylation remain poorly defined. Recent genetic evidence indicates a requirement for cyclin D1/Cdk4 complexes in p107 inactivation. In this work, we provide direct biochemical evidence for the involvement of cyclin D1/Cdk4 in the inactivation of p107's growth-suppressive function. While coexpression of cyclin D1/Cdk4 can reverse the cell cycle arrest properties of p107 in Saos-2 cells, we find that p107 in which the Lys-Arg-Arg-Leu sequence of the RXL motif is replaced by four alanine residues is largely refractory to inactivation by cyclin D/Cdk4, indicating a role for this motif in p107 inactivation without a requirement for its tight interaction with cyclin D1/Cdk4. We identified four phosphorylation sites in p107 (Thr-369, Ser-640, Ser-964, and Ser-975) that are efficiently phosphorylated by Cdk4 but not by Cdk2 in vitro and are also phosphorylated in tissue culture cells. Growth suppression by p107 containing nonphosphorylatable residues in these four sites is not reversed by coexpression of cyclin D1/Cdk4. In model p107 spacer region peptides, phosphorylation of S640 by cyclin D1/Cdk4 is strictly dependent upon an intact RXL motif, but phosphorylation of this site in the absence of an RXL motif can be partially restored by replacement of S643 by arginine. This suggests that one role for the RXL motif is to facilitate phosphorylation of nonconsensus Cdk substrates. Taken together, these data indicate that p107 is inactivated by cyclin D1/Cdk4 via direct phosphorylation and that the RXL motif of p107 plays a role in its inactivation by Cdk4 in the absence of stable binding.

scholarly journals A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

1972 ◽  
Vol 52 (1) ◽  
pp. 52-65 ◽  
Author(s):  
Michael C. O'Neill ◽  
Frank E. Stockdale
Keyword(s):  
Cell Cycle ◽  
Cell Fusion ◽  
Tritiated Thymidine ◽  
Culture Conditions ◽  
Complete Fusion ◽  
Low Density ◽  
A Cell ◽  
Muscle Cultures ◽  
S Period

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G1 period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G1 period before cell fusion and differentiation during myogenesis.

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