RESUMO
Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.
Assuntos
Lipídeos , Mitose , Animais , Ciclo Celular , Fase G1 , Fosforilação , MamíferosRESUMO
With age, skeletal muscle stem cells (MuSCs) activate out of quiescence more slowly and with increased death, leading to defective muscle repair. To explore the molecular underpinnings of these defects, we combined multiomics, single-cell measurements, and functional testing of MuSCs from young and old mice. The multiomics approach allowed us to assess which changes are causal, which are compensatory, and which are simply correlative. We identified glutathione (GSH) metabolism as perturbed in old MuSCs, with both causal and compensatory components. Contrary to young MuSCs, old MuSCs exhibit a population dichotomy composed of GSHhigh cells (comparable with young MuSCs) and GSHlow cells with impaired functionality. Mechanistically, we show that antagonism between NRF2 and NF-κB maintains this bimodality. Experimental manipulation of GSH levels altered the functional dichotomy of aged MuSCs. These findings identify a novel mechanism of stem cell aging and highlight glutathione metabolism as an accessible target for reversing MuSC aging.
Assuntos
Multiômica , Músculo Esquelético , Camundongos , Animais , Músculo Esquelético/metabolismo , Células-Tronco/metabolismo , Senescência Celular , Envelhecimento/fisiologiaRESUMO
Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Humanos , Fase S , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismoRESUMO
Skeletal muscle possesses remarkable regenerative ability because of the resident muscle stem cells (MuSCs). A prominent feature of quiescent MuSCs is a high content of heterochromatin. However, little is known about the mechanisms by which heterochromatin is maintained in MuSCs. By comparing gene-expression profiles from quiescent and activated MuSCs, we found that the mammalian Hairless (Hr) gene is expressed in quiescent MuSCs and rapidly down-regulated upon MuSC activation. Using a mouse model in which Hr can be specifically ablated in MuSCs, we demonstrate that Hr expression is critical for MuSC function and muscle regeneration. In MuSCs, loss of Hr results in reduced trimethylated Histone 3 Lysine 9 (H3K9me3) levels, reduced heterochromatin, increased susceptibility to genotoxic stress, and the accumulation of DNA damage. Deletion of Hr leads to an acceleration of the age-related decline in MuSC numbers. We have also demonstrated that despite the fact that Hr is homologous to a family of histone demethylases and binds to di- and trimethylated H3K9, the expression of Hr does not lead to H3K9 demethylation. In contrast, we show that the expression of Hr leads to the inhibition of the H3K9 demethylase Jmjd1a and an increase in H3K9 methylation. Taking these data together, our study has established that Hr is a H3K9 demethylase antagonist specifically expressed in quiescent MuSCs.
Assuntos
Inativação Gênica , Heterocromatina , Histona Desmetilases/antagonistas & inibidores , Músculo Esquelético/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Animais , Histonas/genética , Histonas/metabolismo , Metilação , Camundongos , Camundongos Pelados , Músculo Esquelético/citologia , Células-Tronco/citologia , Fatores de Transcrição/genéticaRESUMO
Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.
Assuntos
Ciclina D/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Células MCF-7 , Camundongos , Microscopia de Fluorescência , Piperazinas/farmacologia , Ligação Proteica , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismoRESUMO
Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/CCDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this entry mechanism operates remains poorly characterized, and whether cells use this path under normal conditions remains unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with a slowed and fluctuating cyclin E-CDK2 activity increase. Surprisingly, with low CDK4/6 activity, the order of APC/CCDH1 and Rb inactivation is reversed in both cell lines and wild-type mice. Finally, we show that as a consequence of this signaling inversion, Rb inactivation replaces APC/CCDH1 inactivation as the point of no return. Together, we elucidate the molecular steps that enable cell-cycle entry without CDK4/6 activity. Our findings not only have implications in cancer resistance, but also reveal temporal plasticity underlying the G1 regulatory circuit.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fase G1 , Animais , Linhagem Celular , Proliferação de Células , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Feminino , Humanos , Masculino , Camundongos , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de SinaisRESUMO
Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here, we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.
Assuntos
Quinase 2 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Fase G1/genética , Estresse Fisiológico , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Genes Reporter , Humanos , Mitose , Fase S/genética , Análise de Célula Única/métodosRESUMO
Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.
Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Mitógenos/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.
Assuntos
Fase G2/genética , Mitose/genética , Fase S/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Ciclina B1/antagonistas & inibidores , Ciclina B1/metabolismo , Dano ao DNA/genética , Replicação do DNA/genética , Proteína Forkhead Box M1/metabolismo , Redes Reguladoras de Genes , Células HCT116 , Humanos , Fosforilação , TelomeraseRESUMO
Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , DNA/biossíntese , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Divisão Celular , Linhagem Celular , Quinase 2 Dependente de Ciclina/fisiologia , Quinases Ciclina-Dependentes/genética , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Humanos , Células MCF-7 , Fase S/fisiologia , Análise de Célula Única/métodosRESUMO
Glucocorticoid and other adipogenic hormones are secreted in mammals in circadian oscillations. Loss of this circadian oscillation pattern correlates with obesity in humans, raising the intriguing question of how hormone secretion dynamics affect adipocyte differentiation. Using live, single-cell imaging of the key adipogenic transcription factors CEBPB and PPARG, endogenously tagged with fluorescent proteins, we show that pulsatile circadian hormone stimuli are rejected by the adipocyte differentiation control system. In striking contrast, equally strong persistent signals trigger maximal differentiation. We identify the mechanism of how hormone oscillations are filtered as a combination of slow and fast positive feedback centered on PPARG. Furthermore, we confirm in mice that flattening of daily glucocorticoid oscillations significantly increases the mass of subcutaneous and visceral fat pads. Together, our study provides a molecular mechanism for why stress, Cushing's disease, and other conditions for which glucocorticoid secretion loses its pulsatility may lead to obesity.
Assuntos
Adipócitos/citologia , Adipogenia/genética , Ritmo Circadiano/genética , Glucocorticoides/metabolismo , PPAR gama/metabolismo , Células Estromais/metabolismo , Transcrição Gênica , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Célula Única , Células Estromais/citologiaRESUMO
To achieve guide RNA (gRNA) multiplexing and an efficient delivery of tens of distinct gRNAs into single cells, we developed a molecular assembly strategy termed chimeric array of gRNA oligonucleotides (CARGO). We coupled CARGO with dCas9 (catalytically dead Cas9) imaging to quantitatively measure the movement of enhancers and promoters that undergo differentiation-associated activity changes in live embryonic stem cells. Whereas all examined functional elements exhibited subdiffusive behavior, their relative mobility increased concurrently with transcriptional activation. Furthermore, acute perturbation of RNA polymerase II activity can reverse these activity-linked increases in loci mobility. Through quantitative CARGO-dCas9 imaging, we provide direct measurements of cis-regulatory element dynamics in living cells and distinct cellular and activity states and uncover an intrinsic connection between cis-regulatory element mobility and transcription.
Assuntos
RNA Guia de Cinetoplastídeos/genética , Sequências Reguladoras de Ácido Nucleico , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Transcrição Gênica , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Linhagem Celular , Núcleo Celular/genética , Endonucleases , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/metabolismo , Ativação TranscricionalRESUMO
The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence.
Assuntos
Ciclo Celular , Quinase 2 Dependente de Ciclina/metabolismo , Linhagem Celular , Inibição de Contato , Ciclina D1/metabolismo , Humanos , Análise de Célula ÚnicaRESUMO
Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1-CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation-quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.
Assuntos
Ciclo Celular/fisiologia , Mitógenos/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Fatores de Transcrição E2F/metabolismo , Humanos , Mitose , Retinoblastoma/metabolismo , Retinoblastoma/patologiaRESUMO
The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, 'cadherin fingers', which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular , Polaridade Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Fagocitose , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actomiosina/metabolismo , Cateninas/metabolismo , Contagem de Células , Membrana Celular/metabolismo , Células HEK293 , Humanos , Imageamento Tridimensional , Junções Intercelulares/metabolismo , Mitose , Modelos Biológicos , Optogenética , Polimerização , Pseudópodes/metabolismoRESUMO
A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.
Assuntos
Ciclo Celular/fisiologia , Proteínas Luminescentes/química , Imagem Molecular/métodos , Proteínas Recombinantes de Fusão/química , Imagem com Lapso de Tempo/métodos , Animais , Técnicas de Cultura de Células , Cromatina/metabolismo , Fase G2/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Mitose , Modelos Moleculares , Células NIH 3T3 , Proteínas Recombinantes de Fusão/genética , Fase S/fisiologia , Proteína Vermelha FluorescenteRESUMO
Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.