RESUMEN
A lipid droplet (LD) core of a cell consists mainly of neutral lipids, triacylglycerols and/or steryl esters (SEs). The structuration of these lipids inside the core is still under debate. Lipid segregation inside LDs has been observed but is sometimes suggested to be an artefact of LD isolation and chemical fixation. LD imaging in their native state and in unaltered cellular environments appears essential to overcome these possible technical pitfalls. Here, imaging techniques for ultrastructural study of native LDs in cellulo are provided and it is shown that LDs are organized structures. Cryo soft X-ray tomography and deep-ultraviolet (DUV) transmittance imaging are showing a partitioning of SEs at the periphery of the LD core. Furthermore, DUV transmittance and tryptophan/tyrosine auto-fluorescence imaging on living cells are combined to obtain complementary information on cell chemical contents. This multimodal approach paves the way for a new label-free organelle imaging technique in living cells.
Asunto(s)
Gotas Lipídicas/química , Gotas Lipídicas/ultraestructura , Imagen Multimodal , Microscopía por Crioelectrón , Saccharomyces cerevisiae , Sincrotrones , Triglicéridos/químicaRESUMEN
Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.
Asunto(s)
Anemia de Células Falciformes/enzimología , Eritrocitos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Tomografía por Rayos X/métodos , Animales , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/metabolismo , Eritrocitos/efectos de los fármacos , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.
Asunto(s)
Algoritmos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Modelos Teóricos , Actinas/química , Actinas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Línea Celular , Línea Celular Tumoral , Exocitosis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Polimerizacion , Reproducibilidad de los Resultados , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína Fluorescente RojaRESUMEN
A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.
Asunto(s)
Antígenos CD/metabolismo , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Melanoma/metabolismo , Microscopía Confocal/métodos , Transporte de Proteínas , Factores de TiempoRESUMEN
Each class of microscope is limited to imaging specific aspects of cell structure and/or molecular organization. However, imaging the specimen by complementary microscopes and correlating the data can overcome this limitation. Whilst not a new approach, the field of correlative imaging is currently benefitting from the emergence of new microscope techniques. Here we describe the correlation of cryogenic fluorescence tomography (CFT) with soft X-ray tomography (SXT). This amalgamation of techniques integrates 3D molecular localization data (CFT) with a high-resolution, 3D cell reconstruction of the cell (SXT). Cells are imaged in both modalities in a near-native, cryopreserved state. Here we describe the current state of the art in correlative CFT-SXT, and discuss the future outlook for this method.
Asunto(s)
Imagenología Tridimensional , Microscopía Fluorescente/métodos , Tomografía por Rayos X/métodos , Levaduras/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/tendencias , Estadística como Asunto , Tomografía por Rayos X/tendenciasRESUMEN
Beamline 2.1 (XM-2) is a transmission soft X-ray microscope in sector 2 of the Advanced Light Source at Lawrence Berkeley National Laboratory. XM-2 was designed, built and is now operated by the National Center for X-ray Tomography as a National Institutes of Health Biomedical Technology Research Resource. XM-2 is equipped with a cryogenic rotation stage to enable tomographic data collection from cryo-preserved cells, including large mammalian cells. During data collection the specimen is illuminated with `water window' X-rays (284-543â eV). Illuminating photons are attenuated an order of magnitude more strongly by biomolecules than by water. Consequently, differences in molecular composition generate quantitative contrast in images of the specimen. Soft X-ray tomography is an information-rich three-dimensional imaging method that can be applied either as a standalone technique or as a component modality in correlative imaging studies.
Asunto(s)
Criopreservación , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Animales , Biología Celular/instrumentación , Células Cultivadas , Criopreservación/instrumentación , Criopreservación/métodos , Diseño de Equipo , Luz , Manejo de Especímenes , Tomografía Computarizada por Rayos X/métodosRESUMEN
Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.
Asunto(s)
Microscopía Fluorescente/métodos , Tomografía por Rayos X/métodos , Criopreservación/métodosRESUMEN
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.
RESUMEN
In the present work, droplet-based microfluidics and sol-gel techniques were combined to synthesize highly monodisperse zinc oxide (ZnO) microspheres, which can be doped easily and precisely with dyes, such as rhodamine B (RhB), and whose size can be finely tuned in the 10-30 µm range. The as-synthesized microparticles were analyzed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. The results reveal that the microspheres exhibit an excellent size monodispersity, hollow feature, and a porous shell with a thickness of about 0.6 µm, in good agreement with our calculations. We show in particular by means of fluorescence recovery after photobleaching (FRAP) analysis that the electric charges carried by ZnO nanoparticles primary units play a crucial role not just in the formation and structure of the synthesized ZnO microcapsules, but also in the confinement of dye molecules inside the microcapsules despite a demonstrated porosity of their shell in regards to the solvent (oil). Our results enable also the measurement of the diffusion coefficient of RhB molecules inside the microcapsules (DRhB=3.8×10-8 cm2/s), which is found two order of magnitude smaller than the literature value. We attribute such feature to a strong interaction between dye molecules and the electrical charges carried by ZnO nanoparticles. These results are important for potential applications in micro-thermometry (as shown recently in our previous study), photovoltaics, or photonics such as whispering gallery mode resonances.
RESUMEN
Current research findings clearly reveal the role of the microalga's cell wall as a key obstacle to an efficient and optimal compound extraction. Such extraction process is therefore closely related to the microalga species used. Effects of electrical or mechanical constraints on C. reinhardtii's structure and particularly its cell wall and membrane, is therefore investigated in this paper using a combination of microscopic tools. Membrane pores with a radius between 0.77 and 1.59 nm were determined for both reversible (5 kVâcm-1) and irreversible (7 kVâcm-1) electroporation with a 5 µs pulse duration. Irreversible electroporation with longer pulses (10 µs) lead to the entry of large molecules (at least 5.11 nm). Additionally, for the first time, the effect of pulsed electric fields on the cell wall was observed. The combined electrical and mechanical treatment showed a significant impact on the cell wall structure as observed under Transmission Electron Microscopy. This treatment permits the penetration of larger molecules (at least 5.11 nm) within the cell, shown by tracking the penetration of dextran molecules. For the first time, the size of pores on the cell membrane and the structural changes on the microalgae cell wall induced by electrical and mechanical treatments is reported.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de la radiación , Chlamydomonas reinhardtii/ultraestructura , Radiación Electromagnética , Estrés Mecánico , Membrana Celular/efectos de la radiación , Membrana Celular/ultraestructura , Chlamydomonas reinhardtii/efectos de la radiación , Electroporación , Fenómenos FísicosRESUMEN
Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.
Asunto(s)
Aminas/toxicidad , Endocitosis/efectos de los fármacos , Endocitosis/efectos de la radiación , Espacio Intracelular/metabolismo , Luz , Aminas/química , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de la radiación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de FluorescenciaRESUMEN
Confocal laser scanning microscopy (CLSM) is one of the most relevant technologies for studying biofilms in situ. Several tools have been developed to investigate and quantify the architecture of biofilms. However, an approach to accurately quantify the intensity of a fluorescent signal over biofilm depth is still lacking. Here we present a tool developed in the ImageJ open-source software that can be used to extract both structure and fluorescence intensity from CLSM data: BIAM (Biofilm Intensity and Architecture Measurement). This is of utmost significance when studying the fundamental mechanisms of biofilm development, differentiation, and in situ gene expression or when aiming to understand the effect of external molecules on biofilm phenotypes.
Asunto(s)
Biopelículas , Procesamiento de Imagen Asistido por Computador/métodos , Conjuntos de Datos como Asunto , Fluorescencia , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Programas InformáticosRESUMEN
BACKGROUND: Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. METHODS: The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively. RESULTS: We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. CONCLUSIONS: We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells. GENERAL SIGNIFICANCE: Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.
Asunto(s)
Aparato de Golgi/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Luz , NADP , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Humanos , NADP/análogos & derivados , NADP/farmacologíaRESUMEN
We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.
Asunto(s)
Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Animales , Línea Celular , Proteínas Fluorescentes Verdes/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Conformación Proteica , Espectrometría de Fluorescencia , Temperatura , TermodinámicaRESUMEN
The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.
Asunto(s)
Antibacterianos/análisis , Bacterias/química , Espectrometría de Fluorescencia/métodos , Antibacterianos/farmacocinética , Membranas/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
Confocal laser scanning microscopy (CLSM) is one of the most relevant technologies for studying biofilms in situ. Several tools have been developed to investigate and quantify the architecture of biofilms. However, an approach to quantify correctly the evolution of intensity of a fluorescent signal as a function of the structural parameters of a biofilm is still lacking. Here we present a tool developed in the ImageJ open source software that can be used to extract both structural and fluorescence intensity from CLSM data: BIAM (Biofilm Intensity and Architecture Measurement). This is of utmost significance when studying the fundamental mechanisms of biofilm growth, differentiation and development or when aiming to understand the effect of external molecules on biofilm phenotypes. In order to provide an example of the potential of such a tool in this study we focused on biofilm dispersion. cis-2-Decenoic acid (CDA) is a molecule known to induce biofilm dispersion of multiple bacterial species. The mechanisms by which CDA induces dispersion are still poorly understood. To investigate the effects of CDA on biofilms, we used a reporter strain of Escherichia coli (E. coli) that expresses the GFPmut2 protein under control of the rrnBP1 promoter. Experiments were done in flow cells and image acquisition was made with CLSM. Analysis carried out using the new tool, BIAM, indicates that CDA affects the fluorescence intensity of the biofilm structures as well as biofilm architectures. Indeed, our results demonstrate that CDA removes more than 35% of biofilm biovolume and suggest that it results in an increase of the biofilm's mean fluorescence intensity (MFI) by more than 26% compared to the control biofilm in the absence of CDA.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Microscopía Confocal/métodos , Programas Informáticos , Técnicas Bacteriológicas/instrumentación , Técnicas Bacteriológicas/métodos , Biopelículas/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Ácidos Grasos Monoinsaturados/farmacología , Fluorescencia , Regiones Promotoras GenéticasRESUMEN
Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed "Structure Intracellular Concentration Activity Relationship" (SICAR) concept.
Asunto(s)
Antibacterianos/metabolismo , Bacterias/metabolismo , Membrana Celular/metabolismo , Fluoroquinolonas/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Fluoroquinolonas/química , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interacted with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Metabolismo Energético , Glucosa/metabolismo , Metabolismo de los Lípidos , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Quinasas de la Proteína-Quinasa Activada por el AMP , Viabilidad Microbiana , Saccharomyces cerevisiae/metabolismoRESUMEN
A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining "Structure Intracellular Accumulation Activity Relationship" for the development of new therapeutic candidates.
Asunto(s)
Antibacterianos/farmacocinética , Ceftazidima/farmacocinética , Bacterias Gramnegativas/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Ceftazidima/síntesis química , Ceftazidima/química , Membrana Celular/química , Pruebas de Sensibilidad Microbiana , Microespectrofotometría , Estructura Molecular , Periplasma/química , PermeabilidadRESUMEN
Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.