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1.
Cell ; 144(6): 897-909, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21414482

RESUMO

Our understanding of the plasma membrane, once viewed simply as a static barrier, has been revolutionized to encompass a complex, dynamic organelle that integrates the cell with its extracellular environment. Here, we discuss how bidirectional signaling across the plasma membrane is achieved by striking a delicate balance between restriction and propagation of information over different scales of time and space and how underlying dynamic mechanisms give rise to rich, context-dependent signaling responses. In this Review, we show how computer simulations can generate counterintuitive predictions about the spatial organization of these complex processes.


Assuntos
Membrana Celular/metabolismo , Transdução de Sinais , Animais , Membrana Celular/química , Simulação por Computador , Humanos
2.
Bioinformatics ; 40(8)2024 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-39078116

RESUMO

MOTIVATION: Chemical reaction networks (CRNs) play a pivotal role in diverse fields such as systems biology, biochemistry, chemical engineering, and epidemiology. High-level definitions of CRNs enables to use various simulation approaches, including deterministic and stochastic methods, from the same model. However, existing Python tools for simulation of CRN typically wrap external C/C++ libraries for model definition, translation into equations and/or numerically solving them, limiting their extensibility and integration with the broader Python ecosystem. RESULTS: In response, we developed Poincaré and SimBio, two novel Python packages for simulation of dynamical systems and CRNs. Poincaré serves as a foundation for dynamical systems modeling, while SimBio extends this functionality to CRNs, including support for the Systems Biology Markup Language (SBML). Poincaré and SimBio are developed as pure Python packages enabling users to easily extend their simulation capabilities by writing new or leveraging other Python packages. Moreover, this does not compromise the performance, as code can be just-in-time compiled with Numba. Our benchmark tests using curated models from the BioModels repository demonstrate that these tools may provide a potentially superior performance advantage compared to other existing tools. In addition, to ensure a user-friendly experience, our packages use standard typed modern Python syntax that provides a seamless integration with integrated development environments. Our Python-centric approach significantly enhances code analysis, error detection, and refactoring capabilities, positioning Poincaré and SimBio as valuable tools for the modeling community. AVAILABILITY AND IMPLEMENTATION: Poincaré and SimBio are released under the MIT license. Their source code is available on GitHub (https://github.com/maurosilber/poincare and https://github.com/hgrecco/simbio) and can be installed from PyPI or conda-forge.


Assuntos
Linguagens de Programação , Software , Biologia de Sistemas , Biologia de Sistemas/métodos , Simulação por Computador , Modelos Biológicos
3.
J Cell Sci ; 135(1)2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34859817

RESUMO

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.


Assuntos
Proteínas Quinases Ativadas por AMP , Mitocôndrias , Proteínas Quinases Ativadas por AMP/genética , Núcleo Celular , Humanos , Hibridização in Situ Fluorescente , Mitocôndrias/genética , Serina-Treonina Quinases TOR/genética
4.
J Microsc ; 294(3): 420-439, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38747464

RESUMO

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

5.
BMC Bioinformatics ; 24(1): 230, 2023 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-37270479

RESUMO

BACKGROUND: In tissues and organisms, the coordination of neighboring cells is essential to maintain their properties and functions. Therefore, knowing which cells are adjacent is crucial to understand biological processes that involve physical interactions among them, e.g. cell migration and proliferation. In addition, some signaling pathways, such as Notch or extrinsic apoptosis, are highly dependent on cell-cell communication. While this is straightforward to obtain from membrane images, nuclei labelling is much more ubiquitous for technical reasons. However, there are no automatic and robust methods to find neighboring cells based only on nuclear markers. RESULTS: In this work, we describe Nfinder, a method to assess the cell's local neighborhood from images with nuclei labeling. To achieve this goal, we approximate the cell-cell interaction graph by the Delaunay triangulation of nuclei centroids. Then, links are filtered by automatic thresholding in cell-cell distance (pairwise interaction) and the maximum angle that a pair of cells subtends with shared neighbors (non-pairwise interaction). We systematically characterized the detection performance by applying Nfinder to publicly available datasets from Drosophila melanogaster, Tribolium castaneum, Arabidopsis thaliana and C. elegans. In each case, the result of the algorithm was compared to a cell neighbor graph generated by manually annotating the original dataset. On average, our method detected 95% of true neighbors, with only 6% of false discoveries. Remarkably, our findings indicate that taking into account non-pairwise interactions might increase the Positive Predictive Value up to + 11.5%. CONCLUSION: Nfinder is the first robust and automatic method for estimating neighboring cells in 2D and 3D based only on nuclear markers and without any free parameters. Using this tool, we found that taking non-pairwise interactions into account improves the detection performance significantly. We believe that using our method might improve the effectiveness of other workflows to study cell-cell interactions from microscopy images. Finally, we also provide a reference implementation in Python and an easy-to-use napari plugin.


Assuntos
Arabidopsis , Drosophila melanogaster , Animais , Caenorhabditis elegans , Microscopia/métodos , Núcleo Celular/metabolismo , Algoritmos , Processamento de Imagem Assistida por Computador/métodos
6.
J Opt Soc Am A Opt Image Sci Vis ; 40(4): C8-C15, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-37132946

RESUMO

Background estimation is the first step in quantitative analysis of images. It has an impact on all subsequent analyses, in particular for segmentation and calculation of ratiometric quantities. Most methods recover only a single value such as the median or yield a biased estimation in non-trivial cases. We introduce, to our knowledge, the first method to recover an unbiased estimation of background distribution. It leverages the lack of local spatial correlation in background pixels to robustly select a subset that accurately represents the background. The resulting background distribution can be used to test for foreground membership of individual pixels or estimate confidence intervals in derived quantities.

7.
Mol Cell ; 47(5): 797-809, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22819327

RESUMO

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.


Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Ubiquitina/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , NF-kappa B/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Infecções por Salmonella/metabolismo , Transdução de Sinais , Ubiquitina/química
8.
Appl Opt ; 59(13): D89-D94, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32400629

RESUMO

The development of light-sheet fluorescence microscopy has been a revolution for developmental biology as it allows long-term imaging during embryonic development. An important reason behind the quick adoption has been the availability of open hardware alternatives. In this work, we present a robust and compact version of a light-sheet fluorescence microscope that is easy to assemble and requires little to no maintenance. An important aspect of the design is that the illumination unit consists of reflective elements, thereby reducing chromatic aberrations an order of magnitude as compared to refractive counterparts.


Assuntos
Drosophila/embriologia , Embrião de Mamíferos/embriologia , Microscopia de Fluorescência/instrumentação , Imagem Óptica/instrumentação , Imagem com Lapso de Tempo/instrumentação , Animais , Biologia do Desenvolvimento , Desenvolvimento Embrionário , Fatores de Tempo
9.
Arch Toxicol ; 93(5): 1401-1415, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30778631

RESUMO

Age-related macular degeneration (AMD) is a multifactorial retinal disease characterized by a progressive loss of central vision. Retinal pigment epithelium (RPE) degeneration is a critical event in AMD. It has been associated to A2E accumulation, which sensitizes RPE to blue light photodamage. Mitochondrial quality control mechanisms have evolved to ensure mitochondrial integrity and preserve cellular homeostasis. Particularly, mitochondrial dynamics involve the regulation of mitochondrial fission and fusion to preserve a healthy mitochondrial network. The present study aims to clarify the cellular and molecular mechanisms underlying photodamage-induced RPE cell death with particular focus on the involvement of defective mitochondrial dynamics. Light-emitting diodes irradiation (445 ± 18 nm; 4.43 mW/cm2) significantly reduced the viability of both unloaded and A2E-loaded human ARPE-19 cells and increased reactive oxygen species production. A2E along with blue light, triggered apoptosis measured by MC540/PI-flow cytometry and activated caspase-3. Blue light induced mitochondrial fusion/fission imbalance towards mitochondrial fragmentation in both non-loaded and A2E-loaded cells which correlated with the deregulation of mitochondria-shaping proteins level (OPA1, DRP1 and OMA1). To our knowledge, this is the first work reporting that photodamage causes mitochondrial dynamics deregulation in RPE cells. This process could possibly contribute to AMD pathology. Our findings suggest that the regulation of mitochondrial dynamics may be a valuable strategy for treating retinal degeneration diseases, such as AMD.


Assuntos
Luz/efeitos adversos , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/patologia , Retinoides/metabolismo , Apoptose/fisiologia , Linhagem Celular , Humanos , Degeneração Macular/etiologia , Dinâmica Mitocondrial/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia
10.
Cytometry A ; 89(8): 761-75, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27183498

RESUMO

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.


Assuntos
Citoplasma/química , Proteínas de Fluorescência Verde/química , Imagem Molecular/métodos , Mapas de Interação de Proteínas , Citoplasma/genética , Microscopia de Fluorescência
11.
Biophys J ; 106(1): 93-105, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24411241

RESUMO

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.


Assuntos
Processamento de Proteína Pós-Traducional , Tioléster Hidrolases/metabolismo , Acilação , Animais , Citoplasma/metabolismo , Cães , Complexo de Golgi/metabolismo , Lipoilação , Células Madin Darby de Rim Canino , Transporte Proteico
12.
J Biol Chem ; 288(50): 35852-67, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24158436

RESUMO

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2-7 complex onto chromatin during G1 phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G1 phase. The immobile fraction of MCM2 and MCM4 increases during G1 phase, suggestive of reiterative licensing. In late G1 phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G1 phase progresses and do not colocalize with sites of DNA synthesis in S phase.


Assuntos
Proteínas de Manutenção de Minicromossomo/metabolismo , Ciclo Celular , Sobrevivência Celular , Cromatina/metabolismo , Humanos , Células MCF-7 , Imagem Molecular , Transporte Proteico
13.
Biochem Pharmacol ; 229: 116480, 2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-39128587

RESUMO

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1-7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1ß were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR-MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1ß secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1ß was prevented by MasR blockade and MasR downregulation, suggesting MasR-MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1ß secretion independently of MasR. MasR-MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR-MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1-7) reduced cellular proliferation in MasR -but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR-MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR-MrgDR interaction. MasR-MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases.

14.
Nat Methods ; 7(4): 295-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20228813

RESUMO

We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Mapeamento de Interação de Proteínas/métodos , Pontos Quânticos , Espectrometria de Fluorescência/métodos , Animais , Células COS , Chlorocebus aethiops , Imunoprecipitação/métodos
15.
Nat Methods ; 7(6): 467-72, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20453867

RESUMO

Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.


Assuntos
Microscopia de Fluorescência/métodos , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Fosfoproteínas/análise , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo
16.
J Mol Biol ; 435(16): 168153, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37210029

RESUMO

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".


Assuntos
Nucleoproteínas , Vírus Sincicial Respiratório Humano , Compartimentos de Replicação Viral , Proteínas Estruturais Virais , RNA Polimerases Dirigidas por DNA/metabolismo , Nucleoproteínas/metabolismo , Vírus Sincicial Respiratório Humano/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Compartimentos de Replicação Viral/metabolismo , Replicação Viral , Proteínas Estruturais Virais/metabolismo , Humanos
17.
Opt Express ; 20(18): 20730-41, 2012 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-23037122

RESUMO

Fluorescence Lifetime Imaging Microscopy (FLIM) is a quantitative technique to probe the nanoenvironment of fluorescent molecules. It is the most robust way to quantify Förster Resonance Energy Transfer (FRET) as it allows reliable differentiation between concentration changes and quenching. In this way, molecular interactions can be imaged in single living cells. The most common wide-field implementation is homodyne Frequency Domain (FD) FLIM, which determines the fluorescence lifetime by measuring the phase and modulation changes of the fluorescence in each pixel upon excitation with a light source modulated at a high frequency. The fluorescence lifetimes are derived from a stack of images acquired at different phase shifts between excitation and detection. In this work we describe a simple method to enhance the dynamic range of FD-FLIM based on precompensating the expected fluorescence modulation by varying the laser power through the phase stack. We show theoretically and experimentally that most of the dynamic range of the camera can be recovered to quantify cells with different intensities. This improvement can be added to any FD-FLIM setup with minimal modifications, enhancing the throughput of information content.


Assuntos
Algoritmos , Aumento da Imagem/instrumentação , Interpretação de Imagem Assistida por Computador/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/instrumentação , Imagem Molecular/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Chemphyschem ; 12(3): 484-90, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21344589

RESUMO

Interest in imaging of Förster resonance energy transfer (FRET) in biological systems has been steadily increasing in the last 30 years. The ability to transduce a near-field interaction into a far-field signal has provided a unique optical tool to assess biological phenomena well below the resolution of standard optical microscopy. In recent years, sub-diffraction microscopy techniques have achieved maturation and are increasingly used in biological applications. As the resolution of these methods increases they will slowly encroach on the domains where FRET is now dominant. Herein we review the major applications in biological FRET imaging and we discuss the possibilities and challenges in the super-resolution era.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Biologia Computacional , Humanos , Microscopia de Fluorescência , Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , Proteoma , RNA Interferente Pequeno/metabolismo
19.
ACS Sens ; 6(7): 2642-2653, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34191492

RESUMO

Understanding signal propagation across biological networks requires to simultaneously monitor the dynamics of several nodes to uncover correlations masked by inherent intercellular variability. To monitor the enzymatic activity of more than two components over short time scales has proven challenging. Exploiting the narrow spectral width of homo-FRET-based biosensors, up to three activities can be imaged through fluorescence polarization anisotropy microscopy. We introduce Caspase Activity Sensor by Polarization Anisotropy Multiplexing (CASPAM) a single-plasmid triple-modality reporter of key nodes of the apoptotic network. Apoptosis provides an ideal molecular framework to study interactions between its three composing pathways (intrinsic, extrinsic, and effector). We characterized the biosensor performance and demonstrated the advantages that equimolar expression has in both simplifying experimental procedure and reducing observable variation, thus enabling robust data-driven modeling. Tools like CASPAM become essential to analyze molecular pathways where multiple nodes need to be simultaneously monitored.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Anisotropia , Caspases/genética , Microscopia de Fluorescência
20.
Phys Rev E ; 104(6-1): 064410, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35030833

RESUMO

Apoptosis is a mechanism of programmed cell death in which cells engage in a controlled demolition and prepare to be digested without damaging their environment. In normal conditions, apoptosis is repressed until it is irreversibly induced by an appropriate signal. In adult organisms, apoptosis is a natural way to dispose of damaged cells and its disruption or excess is associated with cancer and autoimmune diseases. Apoptosis is regulated by a complex signaling network controlled by caspases, specialized enzymes that digest essential cellular components and promote the degradation of genomic DNA. In this work, we propose an effective description of the signaling network focused on caspase-3 as a readout of cell fate. We integrate intermediate network interactions into a nonlinear feedback function acting on caspase-3 and introduce the effect of pro-apoptotic stimuli and regulatory elements as a saturating activation function. We show that activation dynamics in the theory is similar to previously reported experimental results. We compute bifurcation diagrams and obtain cell fate maps describing how stimulus intensity and feedback strength affect cell survival and death fates. These fates overlap within a bistable region that depends on total caspase concentration, regulatory elements, and feedback nonlinearity. We study a strongly nonlinear regime to obtain analytical expressions for bifurcation curves and fate map boundaries. For a broad range of parameters, strong stimuli can induce an irreversible switch to the death fate. We use the theory to explore dynamical stimulation conditions and determine how cell fate depends on stimulation temporal patterns. This analysis predicts a critical relation between transient stimuli intensity and duration to trigger irreversible apoptosis. We derive an analytical expression for this critical relation, valid for short stimuli. Our description provides distinct predictions and offers a framework to study how this signaling network processes different stimuli to make a cell fate decision.


Assuntos
Apoptose , Modelos Biológicos , Transdução de Sinais
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