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1.
EMBO J ; 42(7): e108533, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36825437

RESUMO

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.


Assuntos
Mitocôndrias , Proteínas , Humanos , Células HeLa , Substâncias Macromoleculares/metabolismo , Proteínas/metabolismo , Solventes/metabolismo , Mitocôndrias/metabolismo
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.
Int J Mol Sci ; 22(20)2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34681888

RESUMO

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores' extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor's (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)-SGFP2 and GerD-mScarlet-I, but there was no FRET between GerD-mScarlet-I and either GerRA-SGFP2 or GerRC-SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)-SGFP2 and GerD-mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)-SGFP2 and GerD-mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Domínios e Motivos de Interação entre Proteínas , Esporos Bacterianos/metabolismo , Bacillus cereus/genética , Bacillus cereus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento
4.
Nat Methods ; 14(1): 53-56, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869816

RESUMO

We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Engenharia de Proteínas/métodos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Sobrevivência Celular , Células HeLa , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Células Tumorais Cultivadas , Proteína Vermelha Fluorescente
5.
Plant Cell ; 26(10): 4188-99, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25351493

RESUMO

Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.


Assuntos
Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Lipopolissacarídeos/metabolismo , Medicago truncatula/genética , Medicago truncatula/microbiologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Quinases/química , Proteínas Quinases/genética , Multimerização Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/fisiologia , Simbiose
6.
Nucleic Acids Res ; 41(22): 10358-70, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24049073

RESUMO

Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Afidicolina/farmacologia , Proteínas Arqueais/análise , Proteínas Arqueais/genética , Tamanho Celular/efeitos dos fármacos , Dano ao DNA , DNA Helicases/análise , DNA Helicases/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/análise , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Fluorescência , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Resolvases de Junção Holliday/fisiologia , Multimerização Proteica , Proteínas Recombinantes de Fusão/análise
7.
Nat Cell Biol ; 9(11): 1319-26, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17952059

RESUMO

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3(PP)) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3(PP) activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3(PP)-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.


Assuntos
Substâncias Macromoleculares/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Feromônios/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Difusão , Monoéster Fosfórico Hidrolases/metabolismo , Espectrometria de Fluorescência
8.
Biophys J ; 104(9): 1875-85, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23663830

RESUMO

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology.


Assuntos
Anexina A4/metabolismo , Membrana Celular/metabolismo , Multimerização Proteica , Anexina A4/química , Cálcio/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos
9.
Nat Methods ; 7(2): 137-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20081836

RESUMO

Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/classificação , Microscopia de Fluorescência/métodos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Fluorescência Verde/análise , Dados de Sequência Molecular
10.
J Am Chem Soc ; 134(18): 7913-23, 2012 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-22486524

RESUMO

Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.


Assuntos
Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Espectrometria de Fluorescência/métodos , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , Proteínas Luminescentes/genética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Transfecção
11.
Elife ; 102021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34190040

RESUMO

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals, but the underlying molecular mechanism remains incompletely understood. Specifically, quantitative insight into endogenous protein behavior is missing. Here, we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure the dynamics, diffusion characteristics and absolute concentrations of fluorescently tagged, endogenous CTNNB1 in human cells under both physiological and oncogenic conditions. State-of-the-art imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing cytoplasmic complexes, irrespective of the activation status of the pathway. This cytoplasmic CTNNB1 complex undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. Based on our biophysical measurements, we build a computational model of WNT/CTNNB1 signaling. Our integrated experimental and computational approach reveals that WNT pathway activation regulates the dynamic distribution of free and complexed CTNNB1 across different subcellular compartments through three regulatory nodes: the destruction complex, nucleocytoplasmic shuttling, and nuclear retention.


Assuntos
Simulação por Computador , Modelos Biológicos , Transdução de Sinais/fisiologia , Análise de Célula Única/métodos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Mutação , Proteínas Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genética
12.
ISME J ; 15(2): 435-449, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32989245

RESUMO

Arbuscular mycorrhizal fungi function as conduits for underground nutrient transport. While the fungal partner is dependent on the plant host for its carbon (C) needs, the amount of nutrients that the fungus allocates to hosts can vary with context. Because fungal allocation patterns to hosts can change over time, they have historically been difficult to quantify accurately. We developed a technique to tag rock phosphorus (P) apatite with fluorescent quantum-dot (QD) nanoparticles of three different colors, allowing us to study nutrient transfer in an in vitro fungal network formed between two host roots of different ages and different P demands over a 3-week period. Using confocal microscopy and raster image correlation spectroscopy, we could distinguish between P transfer from the hyphae to the roots and P retention in the hyphae. By tracking QD-apatite from its point of origin, we found that the P demands of the younger root influenced both: (1) how the fungus distributed nutrients among different root hosts and (2) the storage patterns in the fungus itself. Our work highlights that fungal trade strategies are highly dynamic over time to local conditions, and stresses the need for precise measurements of symbiotic nutrient transfer across both space and time.


Assuntos
Micorrizas , Apatitas , Nutrientes , Fósforo , Raízes de Plantas , Simbiose
13.
Am J Physiol Cell Physiol ; 297(6): C1477-89, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19741200

RESUMO

Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, K(D)(eff), values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 (K(D)(eff) 345 nM). VEGF administration affected differentially the K(D)(eff) values of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Homeostase , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Movimento Celular/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Interações Medicamentosas , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Distribuição Tecidual , Transcrição Gênica/fisiologia , Veias Umbilicais/citologia , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Curr Biol ; 29(12): 2043-2050.e8, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31178314

RESUMO

The world's ecosystems are characterized by an unequal distribution of resources [1]. Trade partnerships between organisms of different species-mutualisms-can help individuals cope with such resource inequality [2-4]. Trade allows individuals to exchange commodities they can provide at low cost for resources that are otherwise impossible or more difficult to access [5, 6]. However, as resources become increasingly patchy in time or space, it is unknown how organisms alter their trading strategies [7, 8]. Here, we show how a symbiotic fungus mediates trade with a host root in response to different levels of resource inequality across its network. We developed a quantum-dot-tracking technique to quantify phosphorus-trading strategies of arbuscular mycorrhizal fungi simultaneously exposed to rich and poor resource patches. By following fluorescent nanoparticles of different colors across fungal networks, we determined where phosphorus was hoarded, relocated, and transferred to plant hosts. We found that increasing exposure to inequality stimulated trade. Fungi responded to high resource variation by (1) increasing the total amount of phosphorus distributed to host roots, (2) decreasing allocation to storage, and (3) differentially moving resources within the network from rich to poor patches. Using single-particle tracking and high-resolution video, we show how dynamic resource movement may help the fungus capitalize on value differences across the trade network, physically moving resources to areas of high demand to gain better returns. Such translocation strategies can help symbiotic organisms cope with exposure to resource inequality.


Assuntos
Daucus carota/microbiologia , Glomeromycota/metabolismo , Micorrizas/fisiologia , Fósforo/metabolismo , Raízes de Plantas/microbiologia , Simbiose , Nutrientes , Pontos Quânticos
15.
Biophys J ; 94(3): 1052-62, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17905839

RESUMO

Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/química , Arabidopsis/embriologia , Células Cultivadas , Dimerização , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Espectrometria de Fluorescência/métodos
16.
Biochim Biophys Acta ; 1767(7): 940-7, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17490603

RESUMO

We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYFP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When CI activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Baculoviridae/genética , Células Cultivadas , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/ultraestrutura , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Rotenona/farmacologia , Pele/citologia , Pele/enzimologia , Pele/ultraestrutura , Espectrometria de Fluorescência , Desacopladores/farmacologia
17.
Nucleic Acids Res ; 34(20): e137, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17040895

RESUMO

Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3' end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.


Assuntos
Proteínas de Bactérias/genética , Bacteriófago T7/genética , Proteínas do Capsídeo/genética , Corantes Fluorescentes , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas Luminescentes/genética , Biblioteca de Peptídeos , Mapeamento de Interação de Proteínas/métodos , Proteínas de Bactérias/análise , Proteínas do Capsídeo/análise , Escherichia coli/genética , Corantes Fluorescentes/análise , Vetores Genéticos , Proteínas Luminescentes/análise , Microscopia de Fluorescência , Plasmídeos/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Temperatura , Vírion/química
18.
ACS Photonics ; 5(6): 2129-2136, 2018 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-29963583

RESUMO

Light-emitting silicon nanoparticles (Si-NPs) are interesting for lighting applications due to their nontoxicity, chemical robustness, and photostability; however, they are not practically considered due to their low emission efficiencies. While large Si-NPs emitting in the red to infrared spectral region show ensemble emission quantum efficiencies up to 60%, the emission efficiencies of smaller Si-NPs, emitting in the visible spectral range, are far lower, typically below 10-20%. In this work, we test this efficiency limit by measuring for the first time the internal quantum efficiency (IQE), i.e., the higher bound of the emission quantum efficiency, considering only the emissive NPs within the ensemble, of Si-NPs emitting in the visible spectral range between 350 and 650 nm. On the basis of photoluminescence decay measurements in a Drexhage geometry, we show that Si-NPs with organic passivation (C:Si-NPs) can have high direct-bandgap-like radiative rates, which enable a high IQE over ∼50%. In this way, we demonstrate that Si-NPs can in principle be considered a competitive candidate as a phosphor in lighting applications and medical imaging also in the visible spectral range. Moreover, our findings show that the reason for the much lower ensemble emission efficiency is due to the fact that the ensemble consists of a low fraction of emissive NPs, most likely due to a low PL "blinking" duty cycle.

19.
Biophys Chem ; 125(2-3): 298-305, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17007994

RESUMO

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.


Assuntos
Difusão , Proteínas/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Modelos Biológicos , Modelos Químicos , Movimento (Física) , Mioglobina/química , Mioglobina/metabolismo , Proteínas/química , Rotação , Espectrometria de Fluorescência , Viscosidade
20.
mBio ; 8(5)2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28900026

RESUMO

One of the mechanisms of ß-lactam antibiotic resistance requires the activity of d,d-carboxypeptidases (d,d-CPases) involved in peptidoglycan (PG) synthesis, making them putative targets for new antibiotic development. The activity of PG-synthesizing enzymes is often correlated with their association with other proteins. The PG layer is maintained in the periplasm between the two membranes of the Gram-negative cell envelope. Because no methods existed to detect in vivo interactions in this compartment, we have developed and validated a Förster resonance energy transfer assay. Using the fluorescent-protein donor-acceptor pair mNeonGreen-mCherry, periplasmic protein interactions were detected in fixed and in living bacteria, in single samples or in plate reader 96-well format. We show that the d,d-CPases PBP5, PBP6a, and PBP6b of Escherichia coli change dimer conformation between resting and active states. Complementation studies and changes in localization suggest that these d,d-CPases are not redundant but that their balanced activity is required for robust PG synthesis.IMPORTANCE The periplasmic space between the outer and the inner membrane of Gram-negative bacteria contains many essential regulatory, transport, and cell wall-synthesizing and -hydrolyzing proteins. To date, no assay is available to determine protein interactions in this compartment. We have developed a periplasmic protein interaction assay for living and fixed bacteria in single samples or 96-well-plate format. Using this assay, we were able to demonstrate conformation changes related to the activity of proteins that could not have been detected by any other living-cell method available. The assay uniquely expands our toolbox for antibiotic screening and mode-of-action studies.


Assuntos
Carboxipeptidases/química , Carboxipeptidases/metabolismo , Escherichia coli/enzimologia , Periplasma/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes , Peptidoglicano/química , Peptidoglicano/metabolismo , Periplasma/química , Periplasma/metabolismo , Conformação Proteica , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Proteína Vermelha Fluorescente
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