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2.
Nat Methods ; 20(4): 541-545, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36973546

RESUMO

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Humanos , Células HeLa , Proteínas Luminescentes/metabolismo , Proteína Vermelha Fluorescente
3.
J Biol Chem ; 299(2): 102896, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36639026

RESUMO

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.


Assuntos
Receptores de Calcitriol , Receptores do Ácido Retinoico , Receptores X de Retinoides , DNA/metabolismo , Ligantes , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Tretinoína/farmacologia , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo
4.
J Phys Chem B ; 126(40): 7906-7915, 2022 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-36190918

RESUMO

Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (fdark) in the ensemble. To date, fdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine fdark values between 30 and 60% for the different VFPs. The high values for fdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Fótons , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Proteínas de Fluorescência Verde/química , Microscopia de Fluorescência/métodos
6.
J Cell Sci ; 135(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35107584

RESUMO

Kinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters. The kinases were activated with GPCR ligands. We observed ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Receptores Acoplados a Proteínas G , Ativação Enzimática , Histamina/metabolismo , Histamina/farmacologia , Ligantes , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
7.
Nat Commun ; 12(1): 7159, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34887382

RESUMO

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2-9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.


Assuntos
Técnicas Biossensoriais/métodos , Cálcio/análise , Células Endoteliais/metabolismo , Proteínas Luminescentes/química , Técnicas Biossensoriais/instrumentação , Cálcio/metabolismo , Células Endoteliais/química , Fluorescência , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Organoides/química , Organoides/metabolismo
8.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34357388

RESUMO

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development and cell migration. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes in real time with subcellular resolution in living cells and tissue. Until now, relocation Rho biosensors (sensors that relocalize to the native location of active Rho GTPase) seem to have been only useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and Rho-binding peptides on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded fluorescent biosensors for detecting endogenous Rho activity. This enables a broader application of Rho relocation biosensors, which was demonstrated by using the improved biosensor to visualize Rho activity during several cellular processes, such as cell division, migration and G protein-coupled receptor signaling. Owing to the improved avidity of the new biosensors for Rho activity, cellular processes regulated by Rho can be better understood. This article has an associated First Person interview with the first author of the paper.


Assuntos
Técnicas Biossensoriais , Movimento Celular/genética , Humanos , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
9.
Small GTPases ; 12(3): 226-240, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-31469028

RESUMO

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.


Assuntos
Endotélio Vascular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Endotélio Vascular/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Ligação Proteica , Homologia de Sequência de Aminoácidos , Análise de Célula Única/métodos
10.
Sci Rep ; 10(1): 6034, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265472

RESUMO

Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia.


Assuntos
Microtúbulos/genética , Microtúbulos/ultraestrutura , Optogenética , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Microscopia Confocal , Microtúbulos/química , Modelos Moleculares , Imagem Óptica , Tubulina (Proteína)/análise , Tubulina (Proteína)/genética , Tubulina (Proteína)/ultraestrutura
11.
J Phys Chem B ; 124(8): 1383-1391, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32011884

RESUMO

The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.


Assuntos
Proteínas Luminescentes/análise , Fluorescência , Proteínas Luminescentes/isolamento & purificação , Microscopia de Fluorescência , Tamanho da Partícula , Proteína Vermelha Fluorescente
12.
Nat Protoc ; 15(2): 450-478, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31942080

RESUMO

Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using mutagenesis. The optimization process through screening is often based solely on bacterial colony brightness, but multiple parameters ultimately determine the performance of an optimal FP. Instead of characterizing other properties after selection, we developed a multiparameter screening method based on four critical parametersscreened simultaneously: fluorescence lifetime, cellular brightness, maturation efficiency, and photostability. First, a high-throughput primary screen (based on fluorescence lifetime and cellular brightness using a mutated FP library) is performed in bacterial colonies. A secondary multiparameter screen based on all four parameters, using a novel bacterial-mammalian dual-expression vector enables expression of the best FP variants in mammalian cell lines. A newly developed automated multiparameter acquisition and cell-based analysis approach for 96-well plates further increased workflow efficiency. We used this protocol to yield the record-bright mScarlet, a fast-maturating mScarlet-I, and a photostable mScarlet-H. This protocol can also be applied to other FP classes or Förster resonance energy transfer (FRET)-based biosensors with minor adaptations. With an available mutant library of a template FP and a complete and tested laboratory setup, a single round of multiparameter screening (including the primary bacterial screen, secondary mammalian cell screen, sequencing, and data processing) can be performed within 2 weeks.


Assuntos
Proteínas Luminescentes/genética , Mutagênese/genética , Animais , Linhagem Celular , Fluorescência , Proteína Vermelha Fluorescente
13.
Small GTPases ; 11(6): 441-450, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-30182785

RESUMO

Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Luz , Processos Fotoquímicos
14.
Front Plant Sci ; 9: 639, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29868092

RESUMO

Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study. With an effective optimization strategy, we were able to detect the interaction between the stem cell regulators SHORT-ROOT and SCARECROW at endogenous expression levels in the root pole of living Arabidopsis embryos and developing lateral roots by FRET-FLIM. Using this approach we show that the spatial profile of interaction between two transcription factors can be highly modulated in reoccurring and structurally resembling organs, thus providing new information on the dynamic redistribution of nuclear protein complex configurations in different developmental stages. In principle, our optimization procedure for transcription factor complexes is applicable to any biological system.

16.
PLoS One ; 13(3): e0193705, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29505611

RESUMO

Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gß1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/química , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única
17.
Sci Rep ; 7(1): 11999, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931898

RESUMO

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , Proteínas Luminescentes/química , Animais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mutação , Reprodutibilidade dos Testes , Proteína Vermelha Fluorescente
18.
Mol Biol Cell ; 28(23): 3371-3382, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-28954861

RESUMO

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein-coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer-based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway.


Assuntos
Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/fisiologia , Esfingosina/análogos & derivados , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais/genética , Esfingosina/metabolismo , Esfingosina/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Nature ; 548(7665): 97-102, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28746306

RESUMO

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates.


Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Transferência Ressonante de Energia de Fluorescência , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem da Célula , Endoderma/citologia , Endoderma/metabolismo , Células HeLa , Proteínas de Homeodomínio/genética , Humanos , Microscopia de Fluorescência , Mutação , Especificidade de Órgãos , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
20.
Plant Cell Physiol ; 58(7): 1196-1207, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28158855

RESUMO

Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP-C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP-C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo.


Assuntos
Arabidopsis/metabolismo , Diglicerídeos/metabolismo , Arabidopsis/citologia , Técnicas Biossensoriais , Membrana Celular/metabolismo , Células Cultivadas , Citocinese , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Ésteres de Forbol/metabolismo , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Domínios Proteicos , Proteína Quinase C/metabolismo , Nicotiana/citologia , Nicotiana/metabolismo , Rede trans-Golgi/metabolismo
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