RESUMO
Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0-1.75 µM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Ouro , Nanopartículas Metálicas , Muramidase , Peptídeos , Ouro/química , Transferência Ressonante de Energia de Fluorescência/métodos , Muramidase/análise , Muramidase/química , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , Aptâmeros de Nucleotídeos/química , Peptídeos/química , Limite de Detecção , Corantes Fluorescentes/química , RodaminasRESUMO
Cells execute specific responses to diverse environmental cues by encoding information in distinctly compartmentalized biochemical signalling reactions. Genetically encoded fluorescent biosensors enable the spatial and temporal monitoring of signalling events in live cells. Temporal and spatiotemporal computational models can be used to interpret biosensor experiments in complex biochemical networks and to explore hypotheses that are difficult to test experimentally. In this review, we first provide brief discussions of the experimental toolkit of fluorescent biosensors as well as computational basics with a focus on temporal and spatiotemporal deterministic models. We then describe how we used this combined approach to identify and investigate a protein kinase A (PKA) - cAMP - Ca2+ oscillatory circuit in MIN6 ß cells, a mouse pancreatic ß cell system. We describe the application of this combined approach to interrogate how this oscillatory circuit is differentially regulated in a nano-compartment formed at the plasma membrane by the scaffolding protein A kinase anchoring protein 79/150. We leveraged both temporal and spatiotemporal deterministic models to identify the key regulators of this oscillatory circuit, which we confirmed with further experiments. The powerful approach of combining live-cell biosensor imaging with quantitative modelling, as discussed here, should find widespread use in the investigation of spatiotemporal regulation of cell signalling.
Assuntos
Técnicas Biossensoriais , Transdução de Sinais , Animais , Camundongos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diagnóstico por Imagem , Membrana Celular/metabolismo , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodosRESUMO
Budding yeast uses the TORC1-Sch9p and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these cascades will improve our understanding of the cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9p and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV measures the Sch9p- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose, and fructose, but heterogeneous for mannose. Cells that start to grow after a transition to mannose correspond to higher normalized Förster resonance energy transfer (FRET) levels, in line with the involvement of Sch9p and PKA pathways to stimulate growth-related processes. The Sch9p and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose-derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that Sch9p- and PKA-dependent phosphorylation activities are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.
Assuntos
Técnicas Biossensoriais , Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Animais , Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Manose/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Mamíferos/metabolismoRESUMO
The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.
Assuntos
Canabidiol , Canabinoides , Humanos , Canabidiol/farmacologia , Cálcio/metabolismo , Proteínas Quinases Ativadas por AMP , Linhagem Celular , Canabinoides/farmacologia , Homeostase , RNA Mensageiro/metabolismo , ColesterolRESUMO
Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system.
Assuntos
Condrócitos/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Osteogênese/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , GMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Lâmina de Crescimento/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.
Assuntos
Adenilil Ciclases , Canal de Liberação de Cálcio do Receptor de Rianodina , Adenilil Ciclases/metabolismo , Adrenérgicos , Cálcio/metabolismo , Sinalização do Cálcio , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
3',5'-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3',5'-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions.
RESUMO
In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Glicólise/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Células Ependimogliais/metabolismo , Luz , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação Oxidativa , Fótons , Retina/metabolismoRESUMO
Activating mutations in RAS are present in ~ 30% of human tumors, and the resulting aberrations in ERK/MAPK signaling play a central role in oncogenesis. However, the form of these signaling changes is uncertain, with activating RAS mutants linked to both increased and decreased ERK activation in vivo. Rationally targeting the kinase activity of this pathway requires clarification of the quantitative effects of RAS mutations. Here, we use live-cell imaging in cells expressing only one RAS isoform to quantify ERK activity with a new level of accuracy. We find that despite large differences in their biochemical activity, mutant KRAS isoforms within cells have similar ranges of ERK output. We identify roles for pathway-level effects, including variation in feedback strength and feedforward modulation of phosphatase activity, that act to rescale pathway sensitivity, ultimately resisting changes in the dynamic range of ERK activity while preserving responsiveness to growth factor stimuli. Our results reconcile seemingly inconsistent reports within the literature and imply that the signaling changes induced by RAS mutations early in oncogenesis are subtle.
Assuntos
Carcinogênese/genética , Genes ras/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Processamento de Imagem Assistida por Computador , Cinética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Mutação , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Isoformas de Proteínas , Análise de Célula ÚnicaRESUMO
Light-induced extrasynaptic dopamine release in the retina reduces adenosine 3',5'-cyclic monophosphate (cAMP) in rod photoreceptor cells, which is thought to mediate light-dependent desensitization. However, the fine time course of the cAMP dynamics in rods remains elusive due to technical difficulty. Here, we visualized the spatiotemporal regulation of cAMP-dependent protein kinase (PKA) in mouse rods by two-photon live imaging of retinal explants of PKAchu mice, which express a fluorescent biosensor for PKA. Unexpectedly, in addition to the light-on-induced suppression, we observed prominent light-off-induced PKA activation. This activation required photopic light intensity and was confined to the illuminated rods. The estimated maximum spectral sensitivity of 489 nm and loss of the light-off-induced PKA activation in rod-transducin-knockout retinas strongly suggest the involvement of rhodopsin. In support of this notion, rhodopsin-deficient retinal explants showed only the light-on-induced PKA suppression. Taken together, these results suggest that, upon photopic light stimulation, rhodopsin and dopamine signals are integrated to shape the light-off-induced cAMP production and following PKA activation. This may support the dark adaptation of rods.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dopamina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Rodopsina/metabolismo , Animais , Ativação Enzimática , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Transducina/metabolismoRESUMO
Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with light-inducible nuclear localization signal. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ.
Assuntos
Técnicas Biossensoriais/métodos , Núcleo Celular/metabolismo , Citosol/metabolismo , Optogenética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Frações Subcelulares/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Sinais de Localização Nuclear , Transporte ProteicoRESUMO
Genetically encoded Förster resonance energy transfer (FRET)-based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Förster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.
Assuntos
Técnicas Biossensoriais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Adenilil Ciclases , Animais , Proteínas de Bactérias , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cães , Feminino , Proteínas de Fluorescência Verde , Células HEK293 , Células HeLa , Humanos , Luz , Proteínas Luminescentes , Células Madin Darby de Rim Canino , Masculino , Camundongos Transgênicos , Optogenética , Espectrometria de FluorescênciaRESUMO
The EphA4 receptor tyrosine kinase is well-known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensor based on Förster resonance energy transfer (FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensor can produce a robust and specific FRET response upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRET responses for the non-raft targeting biosensor than for the raft targeting biosensor, suggesting that stronger EphA4 activation may occur in non-raft regions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensor could serve as a powerful tool to visualize and investigate EphA4 activation and signaling in specific subcellular compartments of single live cells.
Assuntos
Técnicas Biossensoriais/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Receptor EphA4/metabolismo , Citoesqueleto de Actina/metabolismo , Células HEK293 , Células HeLa , HumanosRESUMO
The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP-PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP-PKA activity in this pathogen, we here present the usage of two FRET-based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time-resolved manner, as we exemplify by glucose-induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP-PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.
Assuntos
Técnicas Biossensoriais/métodos , Candida glabrata/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Candida glabrata/patogenicidade , Glucose/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Transdução de SinaisRESUMO
Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells.
Assuntos
Anticorpos/química , Técnicas Biossensoriais/métodos , Corantes/química , Transferência Ressonante de Energia de Fluorescência/métodos , Metaloproteinase 14 da Matriz/análise , Ficoeritrina/química , Anticorpos/genética , Evolução Molecular Direcionada , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Imagem Óptica/métodos , Peptídeos/química , Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Quinase CDC2/metabolismo , Ciclina A2/metabolismo , Ciclina B1/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinase 1 Polo-LikeRESUMO
Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Biologia de Sistemas/métodos , Animais , Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Células PC12 , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca(2+) signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca(2+), ER Ca(2+) release is the source of intracellular Ca(2+) oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca(2+) release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca(2+) permeable channels on the plasma membrane, specifically TRPM7. However, Ca(2+) influx at the plasma membrane via mechanosensitive Ca(2+) permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células-Tronco Mesenquimais/metabolismo , Estresse Mecânico , Actomiosina/metabolismo , Animais , Técnicas Biossensoriais , Cálcio/metabolismo , Bovinos , Citoesqueleto/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Espaço Intracelular/metabolismo , Células-Tronco Mesenquimais/citologia , TransfecçãoRESUMO
The mechanical microenvironment surrounding cells has a significant impact on cellular function. One prominent example is that the stiffness of the substrate directs stem cell differentiation. However, the underlying mechanisms of how mechanical cues affect stem cell functions are largely elusive. Here, we report that in human mesenchymal stem cells (HMSCs), substrate stiffness can regulate cellular responses to a ß-adrenergic receptor (ß-AR) agonist, Isoproterenol (ISO). Fluorescence resonance energy transfer-based A-Kinase Activity Reporter revealed that HMSCs displayed low activity of ISO-induced protein kinase A (PKA) signal on soft substrate, whereas a significantly higher activity can be observed on hard substrate. Meanwhile, there is an increasing ISO-induced internalization of ß2-AR with increasing substrate stiffness. Further experiments revealed that the effects of substrate stiffness on both events were disrupted by interfering the polymerization of microtubules, but not actin filaments. Mechanistic investigation revealed that inhibiting ISO-induced PKA activation abolished ß2-AR internalization and vice versa, forming a feedback loop. Thus, our results suggest that the cellular sensing mechanism of its mechanical environment, such as substrate stiffness, affects its response to chemical stimulation of ß-AR signaling and PKA activation through the coordination of microtubules, which may contribute to how mechanical cues direct stem cell differentiation.
Assuntos
Materiais Biocompatíveis/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microtúbulos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Alicerces Teciduais/química , Acrilamida/química , Agonistas Adrenérgicos beta/farmacologia , Fenômenos Biomecânicos , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Dureza , Humanos , Isoproterenol/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Microtúbulos/ultraestruturaRESUMO
Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin ß1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.