RESUMEN
Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.
Asunto(s)
Diferenciación Celular , Epigénesis Genética , Colorantes Fluorescentes , Histonas , Células Madre Pluripotentes Inducidas , Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Histonas/metabolismo , Histonas/genética , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Neuronas/metabolismo , Neuronas/citología , Animales , RatonesRESUMEN
CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy and microfluidics analyses we demonstrate that plasmids can persist for multiple generations in some Escherichia coli cell lineages at conditions of continuous targeting by the type I-E CRISPR-Cas system. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to continuous maintenance of mobile genetic elements in some cells, which leads to diversification of phenotypes in the entire community and allows rapid changes in the population structure to meet the demands of a changing environment.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli , Secuencias Repetitivas Esparcidas , Plásmidos , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Escherichia coli/genética , Interacción Gen-Ambiente , Secuencias Repetitivas Esparcidas/genética , Modelos Genéticos , Plásmidos/genéticaRESUMEN
Papain-like protease PLpro, a domain within a large polyfunctional protein, nsp3, plays key roles in the life cycle of SARS-CoV-2, being responsible for the first events of cleavage of a polyprotein into individual proteins (nsp1-4) as well as for the suppression of cellular immunity. Here, we developed a new genetically encoded fluorescent sensor, named PLpro-ERNuc, for detection of PLpro activity in living cells using a translocation-based readout. The sensor was designed as follows. A fragment of nsp3 protein was used to direct the sensor on the cytoplasmic surface of the endoplasmic reticulum (ER) membrane, thus closely mimicking the natural target of PLpro. The fluorescent part included two bright fluorescent proteins-red mScarlet I and green mNeonGreen-separated by a linker with the PLpro cleavage site. A nuclear localization signal (NLS) was attached to ensure accumulation of mNeonGreen into the nucleus upon cleavage. We tested PLpro-ERNuc in a model of recombinant PLpro expressed in HeLa cells. The sensor demonstrated the expected cytoplasmic reticular network in the red and green channels in the absence of protease, and efficient translocation of the green signal into nuclei in the PLpro-expressing cells (14-fold increase in the nucleus/cytoplasm ratio). Then, we used PLpro-ERNuc in a model of Huh7.5 cells infected with the SARS-CoV-2 virus, where it showed robust ER-to-nucleus translocation of the green signal in the infected cells 24 h post infection. We believe that PLpro-ERNuc represents a useful tool for screening PLpro inhibitors as well as for monitoring virus spread in a culture.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Células HeLa , COVID-19/virología , COVID-19/diagnóstico , COVID-19/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Proteasas 3C de Coronavirus/metabolismo , Transporte de Proteínas , Técnicas Biosensibles/métodosRESUMEN
Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.
Asunto(s)
Histonas , Optogenética , Humanos , Histonas/genética , Histonas/metabolismo , Epigénesis Genética , Cromatina , Procesamiento Proteico-Postraduccional , ColorantesRESUMEN
Proteins of the Green Fluorescent Protein (GFP) family, being used as genetically encoded labels, have brought fluorescence molecular imaging into the dynamic world of living cells and organisms. Flavin-, bilirubin-, and biliverdin-binding fluorescent proteins further enriched the palette of genetic markers with novel spectral and physico-chemical properties. What are the possible next steps in the development of this methodology?
Asunto(s)
Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , Proteínas Luminiscentes/metabolismoRESUMEN
Epigenome contains a lot of information about cell state. Epigenetic analysis includes primarily sequence-based methods, which provide detailed data on distribution of modifications along the genome, but are poorly applicable for screenings. Specific fluorescence labeling and imaging of epigenetic modifications is an attractive complementary approach. It is currently based mainly on histone modifications study. We expect that inclusion of DNA modifications into imaging-based study would empower the method. In this review we discuss methods for fluorescence imaging of DNA modifications (mainly 5-methylcytosine). It opens an easy way to single cell analysis and high-throughput screening. Moreover, tracking epigenome changes in live cells becomes possible with genetically encoded probes.
Asunto(s)
Epigénesis Genética , Genoma , ADN/genética , Metilación de ADN , Código de Histonas , Imagen ÓpticaRESUMEN
The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Compuestos de Boro/química , Biología Computacional , Cristalografía por Rayos X , Diseño de Fármacos , Fluorescencia , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Ingeniería de Proteínas/estadística & datos numéricos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Programas InformáticosRESUMEN
Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.
Asunto(s)
Aptitud Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Animales , Epistasis Genética , Evolución Molecular , Fluorescencia , Estudios de Asociación Genética , Genotipo , Hidrozoos/química , Hidrozoos/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación/genética , FenotipoRESUMEN
The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.
Asunto(s)
Ciclo Celular , Neoplasias del Colon/patología , Colorantes Fluorescentes/química , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Imagen Individual de Molécula/métodos , Animales , Proliferación Celular , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Fluorescente RojaRESUMEN
In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target-green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker-were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.
Asunto(s)
COVID-19 , SARS-CoV-2 , Proteasas Similares a la Papaína de Coronavirus , Células HeLa , Humanos , Papaína/metabolismo , SARS-CoV-2/genéticaRESUMEN
Epigenetic modifications of histones (methylation, acetylation, phosphorylation, etc.) are of great importance in determining the functional state of chromatin. Changes in epigenome underlay all basic biological processes, such as cell division, differentiation, aging, and cancerous transformation. Post-translational histone modifications are mainly studied by immunoprecipitation with high-throughput sequencing (ChIP-Seq). It enables an accurate profiling of target modifications along the genome, but suffers from the high cost of analysis and the inability to work with living cells. Fluorescence microscopy represents an attractive complementary approach to characterize epigenetics. It can be applied to both live and fixed cells, easily compatible with high-throughput screening, and provide access to rich spatial information down to the single cell level. In this review, we discuss various fluorescent probes for histone modification detection. Various types of live-cell imaging epigenetic sensors suitable for conventional as well as super-resolution fluorescence microscopy are described. We also focus on problems and future perspectives in the development of fluorescent probes for epigenetics.
Asunto(s)
Cromatina , Colorantes Fluorescentes , Cromatina/genética , Epigénesis Genética , Epigenómica/métodos , Microscopía Fluorescente , Procesamiento Proteico-PostraduccionalRESUMEN
The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore's pKa and demonstrate that, in the pH range of 7.5-9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range.
Asunto(s)
Colorantes , Colorantes Fluorescentes , Animales , Humanos , Fluorescencia , Células HEK293 , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente/métodos , Concentración de Iones de Hidrógeno , MamíferosRESUMEN
Oxygen concentrations vary between tissues of multicellular organisms and change under certain physiological or pathological conditions. Multiple methods have been developed for measuring oxygenation of biological samples in vitro and in vivo However, most require complex equipment, are laborious and have significant limitations. Here we report that oxygen concentration determines the choice between two maturation pathways of DsRed FT (Timer). At high oxygen levels, this DsRed derivate matures predominantly into a red fluorescent isoform. By contrast, a green fluorescent isoform is favored by low oxygen levels. Ratiometric analysis of green and red fluorescence after a pulse of Timer expression in Drosophila larvae provides a record of the history of tissue oxygenation during a subsequent chase period, for the whole animal with single-cell precision. Tissue spreads revealed fine differences in oxygen exposure among different cells of the same organ. We expect that the simplicity and robustness of our approach will greatly impact hypoxia research, especially in small animal models.
Asunto(s)
Drosophila melanogaster/metabolismo , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Oxígeno/análisis , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/genética , Microscopía Fluorescente/métodos , Isoformas de Proteínas/genéticaRESUMEN
Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.
Asunto(s)
Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Línea Celular , Humanos , Microscopía Intravital/métodos , Cinética , Luz , Microscopía Fluorescente/métodos , Nanotecnología , Procesos Fotoquímicos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Espectrofotometría , Proteína Fluorescente RojaRESUMEN
mKate red-to-green photoconversion is a non-canonical type of phototransformation in fluorescent proteins, with a poorly understood mechanism. We have hypothesized that the daughter mKate2 protein may also be photoconvertible, and that this phenomenon would be connected with mKate(2) chromophore photoreduction. Indeed, upon the intense irradiation of the protein sample supplemented by sodium dithionite, the accumulation of green as well as blue spectral forms is enhanced. The reaction was shown to be reversible upon the reductant's removal. However, an analysis of the fluorescence microscopy data, absorption spectra, kinetics and time-resolved fluorescence spectroscopy revealed that the short-wavelength spectral forms of mKate(2) exist before photoactivation, that their fractions increase light-independently after dithionite addition, and that the conversion is facilitated by the photobleaching of the red chromophore form.
Asunto(s)
Proteínas Luminiscentes/química , Microscopía Fluorescente , Oxidación-Reducción , Fotoblanqueo , Proteína Fluorescente RojaRESUMEN
Insect odorant receptors (ORs) have been suggested to function as ligand-gated cation channels, with OrX/Orco heteromers combining ionotropic and metabotropic activity. The latter is mediated by different G proteins and results in Orco self-activation by cyclic nucleotide binding. In this contribution, we co-express the odor-specific subunits DmOr49b and DmOr59b with either wild-type Orco or an Orco-PKC mutant lacking cAMP activation heterologously in mammalian cells. We show that the characteristics of heteromers strongly depend on both the OrX type and the coreceptor variant. Thus, methyl acetate-sensitive Or59b/Orco demonstrated 25-fold faster response kinetics over o-cresol-specific Or49b/Orco, while the latter required a 10-100 times lower ligand concentration to evoke a similar electrical response. Compared to wild-type Orco, Orco-PKC decreased odorant sensitivity in both heteromers, and blocked an outward current rectification intrinsic to the Or49b/Orco pair. Our observations thus provide an insight into insect OrX/Orco functioning, highlighting their natural and artificial tuning features and laying the groundwork for their application in chemogenetics, drug screening, and repellent design.
Asunto(s)
Proteínas de Drosophila/genética , Canales Iónicos Activados por Ligandos/genética , Receptores Odorantes/genética , Acetatos/química , Acetatos/farmacología , Animales , Cresoles/química , Cresoles/farmacología , AMP Cíclico/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Proteínas de Unión al GTP/genética , Cinética , Odorantes/análisis , Transducción de Señal/efectos de los fármacosRESUMEN
Fluorescent proteins are commonly used to label target proteins in live cells. However, the conventional approach based on covalent fusion of targeted proteins with fluorescent protein probes is limited by the slow rate of fluorophore maturation and irretrievable loss of fluorescence due to photobleaching. Here, we report a genetically encoded protein labeling system utilizing transient interactions of small, 21-28 residues-long helical protein tags (K/E coils, KEC). In this system, a protein of interest, covalently tagged with a single coil, is visualized through binding to a cytoplasmic fluorescent protein carrying a complementary coil. The reversible heterodimerization of KECs, whose affinity can be tuned in a broad concentration range from nanomolar to micromolar, allows continuous exchange and replenishment of the tag bound to a targeted protein with the entire cytosolic pool of soluble fluorescent coils. We found that, under conditions of partial illumination of living cells, the photostability of labeling with KECs exceeds that of covalently fused fluorescent probes by approximately one order of magnitude. Similarly, single-molecule localization microscopy with KECs provided higher labeling density and allowed a much longer duration of imaging than with conventional fusion to fluorescent proteins. We also demonstrated that this method is well suited for imaging newly synthesized proteins, because the labeling efficiency by KECs is not dependent on the rate of fluorescent protein maturation. In conclusion, KECs can be used to visualize various target proteins which are directly exposed to the cytosol, thereby enabling their advanced characterization in time and space.
Asunto(s)
Colorantes Fluorescentes/química , Proteínas/análisis , Animales , Línea Celular , Supervivencia Celular , Células HEK293 , Células HeLa , Humanos , Proteínas Luminiscentes/análisis , Ratones , Microscopía Fluorescente , Imagen Óptica , Fotólisis , Multimerización de Proteína , Ratas , Coloración y EtiquetadoRESUMEN
The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.
Asunto(s)
Microscopía Fluorescente , SARS-CoV-2/fisiología , COVID-19/patología , COVID-19/virología , Endocitosis , Colorantes Fluorescentes/química , Genes Reporteros , Humanos , ARN Viral/química , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
Fluorescent labeling is an established method for visualizing cellular structures and dynamics. The fundamental diffraction limit in image resolution was recently bypassed with the development of super-resolution microscopy. Notably, both localization microscopy and stimulated emission depletion (STED) microscopy impose tight restrictions on the physico-chemical properties of labels. One of them-the requirement for high photostability-can be satisfied by transiently interacting labels: a constant supply of transient labels from a medium replenishes the loss in the signal caused by photobleaching. Moreover, exchangeable tags are less likely to hinder the intrinsic dynamics and cellular functions of labeled molecules. Low-affinity labels may be used both for fixed and living cells in a range of nanoscopy modalities. Nevertheless, the design of optimal labeling and imaging protocols with these novel tags remains tricky. In this review, we highlight the pros and cons of a wide variety of transiently interacting labels. We further discuss the state of the art and future perspectives of low-affinity labeling methods.
Asunto(s)
Colorantes Fluorescentes/química , Fotoblanqueo , Fluorescencia , Microscopía FluorescenteRESUMEN
Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures.