Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Chem Biol ; 17(9): 989-997, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34341587

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the ß-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Desdobramento de Proteína
2.
Nanoscale ; 13(24): 10837-10848, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34114594

RESUMO

Gold nanorods (GNRs) are a promising platform for nanoplasmonic biosensing. The localised surface plasmon resonance (LSPR) peak of GNRs is located in the near-infrared optical window and is sensitive to local binding events, enabling label-free detection of biomarkers in complex biological fluids. A key challenge in the development of such sensors is achieving target affinity and selectivity, while both minimizing non-specific binding and maintaining colloidal stability. Herein, we reveal how GNRs decorated with galactosamine-terminated polymer ligands display significantly different binding responses in buffer compared to serum, due to biocorona formation, and how biocorona displacement due to lectin binding plays a key role in their optical responses. GNRs were coated with either poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) or poly(N-hydroxyethyl acrylamide) (PHEA) prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerisation and end-functionalised with galactosamine (Gal) as the lectin-targeting unit. In buffer Gal-PHEA-coated GNRs aggregated upon soybean agglutinin (SBA) addition, whereas Gal-PHPMA-coated GNRs exhibited a red-shift of the LSPR spectrum without aggregation. In contrast, when incubated in serum Gal-PHPMA-coated nanorods showed no binding response, while Gal-PHEA GNRs exhibited a dose-dependent blue-shift of the LSPR peak, which is the opposite direction (red-shift) to what was observed in buffer. This differential behaviour was attributed to biocorona formation onto both polymer-coated GNRs, shown by differential centrifugal sedimentation and nanoparticle tracking analysis. Upon addition of SBA to the Gal-PHEA coated nanorods, signal was generated due to displacement of weakly-bound biocorona components by lectin binding. However, in the case of Gal-PHPMA which had a thicker corona, attributed to lower polymer grafting densities, addition of SBA did not lead to biocorona displacement and there was no signal output. These results show that plasmonic optical responses in complex biological media can be significantly affected by biocorona formation, and that biocorona formation itself does not prevent sensing so long as its exact nature (e.g. 'hard versus soft') is tuned.


Assuntos
Técnicas Biossensoriais , Nanotubos , Ouro , Polímeros , Ressonância de Plasmônio de Superfície
3.
Nat Commun ; 12(1): 2541, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953187

RESUMO

Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Óptica/métodos , Anisotropia , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos
4.
Elife ; 102021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33779550

RESUMO

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Biologia Molecular/métodos , Imagem Individual de Molécula/métodos , Biologia Molecular/instrumentação , Imagem Individual de Molécula/instrumentação
5.
Small ; 17(5): e2006786, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33448084

RESUMO

Extracellular vesicles (EV) are biological nanoparticles that play an important role in cell-to-cell communication. The phenotypic profile of EV populations is a promising reporter of disease, with direct clinical diagnostic relevance. Yet, robust methods for quantifying the biomarker content of EV have been critically lacking, and require a single-particle approach due to their inherent heterogeneous nature. Here, multicolor single-molecule burst analysis microscopy is used to detect multiple biomarkers present on single EV. The authors classify the recorded signals and apply the machine learning-based t-distributed stochastic neighbor embedding algorithm to cluster the resulting multidimensional data. As a proof of principle, the authors use the method to assess both the purity and the inflammatory status of EV, and compare cell culture and plasma-derived EV isolated via different purification methods. This methodology is then applied to identify intercellular adhesion molecule-1 specific EV subgroups released by inflamed endothelial cells, and to prove that apolipoprotein-a1 is an excellent marker to identify the typical lipoprotein contamination in plasma. This methodology can be widely applied on standard confocal microscopes, thereby allowing both standardized quality assessment of patient plasma EV preparations, and diagnostic profiling of multiple EV biomarkers in health and disease.


Assuntos
Células Endoteliais , Vesículas Extracelulares , Análise por Conglomerados , Humanos , Plasma , Aprendizado de Máquina não Supervisionado
7.
ACS Nano ; 14(9): 10775-10783, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32820634

RESUMO

The molecular composition of viral particles indicates that a single virion is capable of initiating an infection. However, the majority of viruses that come into contact with cells fails to infect them. Understanding what makes one viral particle more successful than others requires visualizing the infection process directly in living cells, one virion at a time. In this Perspective, we explain how single-virus imaging using fluorescence microscopy can provide answers to unsolved questions in virology. We discuss fluorescent labeling of virus particles, resolution at the subviral and molecular levels, tracking in living cells, and imaging of interactions between viral and host proteins. We end this Perspective with a set of remaining questions in understanding the life cycle of retroviruses and how imaging a single virus can help researchers address these questions. Although we use examples from the HIV field, these methods are of value for the study of other viruses as well.


Assuntos
Infecções por HIV , Vírus , Humanos , Microscopia de Fluorescência , Vírion , Replicação Viral
9.
Front Immunol ; 11: 1114, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582194

RESUMO

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.


Assuntos
Doenças da Imunodeficiência Primária/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Linhagem Celular , Sobrevivência Celular , Mutação com Ganho de Função , Humanos , Fenótipo
10.
Nat Chem Biol ; 16(8): 834-840, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393900

RESUMO

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.


Assuntos
Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/genética , Genes rel/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Ligases/fisiologia , Nucleotídeos/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
11.
Data Brief ; 29: 105348, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32181308

RESUMO

The data provided with this paper are image series of slowly diffusing GlyRa3 molecules, linked to either eGFP or mCherry fluorescent proteins, at the membrane of HEK cells, acquired on a Zeiss LSM880 confocal laser scanning microscope. Raster spectral image cross-correlation spectroscopy (RSICS) is applied to the data, a technique that exploits intensity fluctuations in confocal image series recorded using a spectral detector to study the diffusion and concentration of molecules, and interactions between them. First, spectral filters are created from reference image series containing GlyRa3 labeled with a single fluorophore. Once experimental data containing GlyRa3 labeled with both fluorophores is acquired, single images are either autocorrelated, or the cross-correlation is calculated between two images, each one containing the data for eGFP or mCherry labeled GyRa 3. Data is then fit with a one-component model assuming a two-dimensional Gaussian point spread function to obtain the diffusion coefficient, D, and average number of molecules in the focus, N. The software package PAM is used to analyze all the acquired data. The data can be used as a reference for artifact-free two-color ccRICS that contains slowly diffusing interacting molecules. Additionally, the analysis workflow described in this paper helps researchers avoid common errors during a RICS experiment.

12.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941774

RESUMO

The HIV-1 capsid protein performs multiple roles in virus replication both during assembly and particle release and during virus trafficking into the nucleus. In order to decipher the roles of capsid protein during early replication, a reliable method to follow its intracellular distribution is required. To complement existing approaches to track HIV-1 capsid during early infection, we developed an HIV-1 imaging strategy, relying on viruses incorporating enhanced green fluorescent protein (eGFP)-tagged capsid (CA-eGFP) protein and mCherry-tagged integrase (IN-mCherry). Wild-type infectivity and sensitivity to inhibition by PF74 point to the functionality of CA-eGFP-containing complexes. Low numbers of CA-eGFP molecules were located inside the viral core and imported into the nucleus without significant loss in intensity. Less than 5% of particles carrying both CA-eGFP and IN-mCherry retained both labelled proteins after nuclear entry, implying a major uncoating event at the nuclear envelope dissociating IN and CA. Still, 20% of all CA-eGFP-containing complexes were detected in the nucleus. Unlike for IN-mCherry complexes, addition of the integrase inhibitor raltegravir had no effect on CA-eGFP-containing complexes, suggesting that these may be not (yet) competent for integration. Our imaging strategy offers alternative visualization of viral capsid trafficking and helps clarify its potential role during integration.IMPORTANCE HIV-1 capsid protein (CA) builds a conical shell protecting viral genomic RNA inside the virus particles. Upon entry into host cells, this shell disassembles in a process of uncoating, which is coordinated with reverse transcription of viral RNA into DNA. After uncoating, a portion of CA remains associated with the viral DNA and mediates its nuclear import and, potentially, integration into host DNA. In this study, we tagged CA with eGFP to follow its trafficking in host cells and address potential CA roles in the nucleus. We found that while functional viruses import the tagged CA into the nucleus, this capsid protein is not part of integration-competent complexes. The roles of nuclear CA thus remain to be established.


Assuntos
Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , HIV-1/fisiologia , Integração Viral , Núcleo Celular/virologia , Citoplasma/metabolismo , DNA Viral/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Membrana Nuclear/metabolismo , RNA Viral/metabolismo , Replicação Viral , Desenvelopamento do Vírus
13.
Biophys J ; 117(10): 1900-1914, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31668746

RESUMO

Raster image correlation spectroscopy (RICS) is a fluorescence image analysis method for extracting the mobility, concentration, and stoichiometry of diffusing fluorescent molecules from confocal image stacks. The method works by calculating a spatial correlation function for each image and analyzing the average of those by model fitting. Rules of thumb exist for RICS image acquisitioning, yet a rigorous theoretical approach to predict the accuracy and precision of the recovered parameters has been lacking. We outline explicit expressions to reveal the dependence of RICS results on experimental parameters. In terms of imaging settings, we observed that a twofold decrease of the pixel size, e.g., from 100 to 50 nm, decreases the error on the translational diffusion constant (D) between three- and fivefold. For D = 1 µm2 s-1, a typical value for intracellular measurements, ∼25-fold lower mean-squared relative error was obtained when the optimal scan speed was used, although more drastic improvements were observed for other values of D. We proposed a slightly modified RICS calculation that allows correcting for the significant bias of the autocorrelation function at small (≪50 × 50 pixels) sizes of the region of interest. In terms of sample properties, at molecular brightness E = 100 kHz and higher, RICS data quality was sufficient using as little as 20 images, whereas the optimal number of frames for lower E scaled pro rata. RICS data quality was constant over the nM-µM concentration range. We developed a bootstrap-based confidence interval of D that outperformed the classical least-squares approach in terms of coverage probability of the true value of D. We validated the theory via in vitro experiments of enhanced green fluorescent protein at different buffer viscosities. Finally, we outline robust practical guidelines and provide free software to simulate the parameter effects on recovery of the diffusion coefficient.


Assuntos
Processamento de Imagem Assistida por Computador , Análise Espectral , Algoritmos , Simulação por Computador , Intervalos de Confiança , Proteínas de Fluorescência Verde/metabolismo , Método de Monte Carlo , Probabilidade
14.
Structure ; 27(1): 90-101.e6, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30471924

RESUMO

SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Förster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.


Assuntos
Proteínas de Escherichia coli/química , Simulação de Dinâmica Molecular , Proteínas SecA/química , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas SecA/metabolismo
15.
Nucleic Acids Res ; 47(3): 1195-1210, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30445610

RESUMO

The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.


Assuntos
Integrases/química , Vírus da Leucemia Murina de Moloney/enzimologia , Vírus da Leucemia Murina de Moloney/genética , Integração Viral , Ciclo Celular , Núcleo Celular/virologia , Citoplasma/virologia , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Mitose , Estrutura Quaternária de Proteína , Proteínas/antagonistas & inibidores , Proteínas/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(48): E11274-E11283, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30429330

RESUMO

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin-cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as "beads on a string," we propose that cohesin-cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Celulossomas/química , Celulossomas/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Clostridium thermocellum/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Celulossomas/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Clostridium thermocellum/química , Clostridium thermocellum/genética , Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica
18.
Nat Methods ; 15(9): 669-676, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30171252

RESUMO

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Laboratórios/normas , Reprodutibilidade dos Testes
19.
Viruses ; 10(5)2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748498

RESUMO

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice.


Assuntos
HIV-1/fisiologia , Microscopia de Fluorescência/instrumentação , Montagem de Vírus , Replicação Viral , Antígeno 2 do Estroma da Médula Óssea/química , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , Humanos , Microscopia Confocal/instrumentação , Estrutura Quaternária de Proteína , Vírus 40 dos Símios
20.
Biophys J ; 114(7): 1518-1528, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29642023

RESUMO

Fluorescence microscopy and spectroscopy data hold a wealth of information on the investigated molecules, structures, or organisms. Nowadays, the same fluorescence data set can be analyzed in many ways to extract different properties of the measured sample. Yet, doing so remains slow and cumbersome, often requiring incompatible software packages. Here, we present PAM (pulsed interleaved excitation analysis with MATLAB), an open-source software package written in MATLAB that offers a simple and efficient workflow through its graphical user interface. PAM is a framework for integrated and robust analysis of fluorescence ensemble, single-molecule, and imaging data. Although it was originally developed for the analysis of pulsed interleaved excitation experiments, PAM has since been extended to support most types of data collection modalities. It combines a multitude of powerful analysis algorithms, ranging from time- and space-correlation analysis, over single-molecule burst analysis, to lifetime imaging microscopy, while offering intrinsic support for multicolor experiments. We illustrate the key concepts and workflow of the software by discussing data handling and sorting and provide step-by-step descriptions for the individual usage cases.


Assuntos
Análise de Dados , Microscopia de Fluorescência , Imagem Individual de Molécula , Software , Espectrometria de Fluorescência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...