RESUMEN
Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit. We introduce as well super-resolved re-scan two-photon excited fluorescence microscopy, an imaging modality not explored to date.
Asunto(s)
Microscopía de Generación del Segundo Armónico , Microscopía de Generación del Segundo Armónico/métodos , Microscopía Fluorescente/métodos , Colágeno , Fotones , CintigrafíaRESUMEN
Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.
Asunto(s)
Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Hemidesmosomas/metabolismo , Integrina beta4/metabolismo , Queratinocitos/metabolismo , Queratinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Colágenos no Fibrilares/metabolismo , Piel/metabolismo , Autoantígenos/genética , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Distonina , Hemidesmosomas/genética , Hemidesmosomas/ultraestructura , Humanos , Integrina beta4/genética , Queratinocitos/ultraestructura , Queratinas/genética , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Colágenos no Fibrilares/genética , Piel/ultraestructura , Colágeno Tipo XVIIRESUMEN
Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE: This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.
Asunto(s)
Bacillus subtilis/fisiología , Citoplasma/metabolismo , Ácido Sórbico/farmacología , Estrés Fisiológico , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Bacillus subtilis/ultraestructura , Citoplasma/química , Citoplasma/efectos de los fármacos , Dermatitis Fototóxica , Conservación de Alimentos , Proteínas Fluorescentes Verdes/genética , Concentración de Iones de Hidrógeno , Acetato de Potasio/farmacología , Análisis de la Célula Individual , Imagen de Lapso de TiempoRESUMEN
Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F. oxysporum is subject to a developmental transition from a uninucleate to a multinucleate state after completion of colony initiation. We observed a special type of hypha that exhibits a higher growth rate, possibly acting as a nutrient scout. The higher growth rate is associated with a higher nuclear count and mitotic waves involving 2 to 6 nuclei in the apical compartment. Further, we found that dormant nuclei of intercalary compartments can reenter the mitotic cycle, resulting in multinucleate compartments with up to 18 nuclei in a single compartment.
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Núcleo Celular/fisiología , Fusarium/citología , Hifa/citología , Fusarium/crecimiento & desarrollo , Fusarium/fisiología , Hifa/crecimiento & desarrollo , MitosisRESUMEN
The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced Ih density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous Ih, and enhances the rebound-response ("voltage-sag") of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.
Asunto(s)
Dinaminas/metabolismo , Filaminas/metabolismo , Hipocampo/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Animales , Dinaminas/genética , Filaminas/genética , Hipocampo/citología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Potenciales de la Membrana/fisiología , Ratones , Neuronas/citología , Canales de Potasio/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type-specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb) folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH). Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010) Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.). We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Apoptosis , Factor de Unión a CCCTC , Proteínas de Ciclo Celular , Células Cultivadas , Cromatina/genética , Proteínas Cromosómicas no Histona , Biología Computacional , Simulación por Computador , Proteínas de Unión al ADN , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polímeros , Proteínas Represoras/genética , CohesinasRESUMEN
Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3'-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis. To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool "SporeTracker". In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3'-digallate, clearly affected the growth of emerging vegetative cells.
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Bacillus subtilis/efectos de los fármacos , Biflavonoides/farmacología , Catequina/farmacología , Ácido Gálico/farmacología , Té/química , Bacillus subtilis/citología , Bacillus subtilis/crecimiento & desarrollo , Polifenoles/farmacología , Esporas Bacterianas , Factores de Tiempo , Imagen de Lapso de TiempoRESUMEN
Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry. Its effect on spore germination and outgrowth in a combined, 'hurdle', preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 °C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new 'burst' time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Ácido Sórbico/farmacología , Esporas Bacterianas/citología , Bacillus subtilis/química , Bacillus subtilis/citología , Microbiología de Alimentos , Calor , Concentración de Iones de Hidrógeno , Esporas Bacterianas/química , Esporas Bacterianas/efectos de los fármacosRESUMEN
The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.
Asunto(s)
Compartimento Celular , Laminas/metabolismo , Membrana Nuclear/patología , Animales , Proteínas Bacterianas/metabolismo , División Celular , Dextranos/metabolismo , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/metabolismo , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares/metabolismo , Ratones , Peso Molecular , Membrana Nuclear/ultraestructura , Señales de Localización Nuclear , Transportador 1 de Catión Orgánico/metabolismo , Transporte de ProteínasRESUMEN
Genome function in higher eukaryotes involves major changes in the spatial organization of the chromatin fiber. Nevertheless, our understanding of chromatin folding is remarkably limited. Polymer models have been used to describe chromatin folding. However, none of the proposed models gives a satisfactory explanation of experimental data. In particularly, they ignore that each chromosome occupies a confined space, i.e., the chromosome territory. Here, we present a polymer model that is able to describe key properties of chromatin over length scales ranging from 0.5 to 75 Mb. This random loop (RL) model assumes a self-avoiding random walk folding of the polymer backbone and defines a probability P for 2 monomers to interact, creating loops of a broad size range. Model predictions are compared with systematic measurements of chromatin folding of the q-arms of chromosomes 1 and 11. The RL model can explain our observed data and suggests that on the tens-of-megabases length scale P is small, i.e., 10-30 loops per 100 Mb. This is sufficient to enforce folding inside the confined space of a chromosome territory. On the 0.5- to 3-Mb length scale chromatin compaction differs in different subchromosomal domains. This aspect of chromatin structure is incorporated in the RL model by introducing heterogeneity along the fiber contour length due to different local looping probabilities. The RL model creates a quantitative and predictive framework for the identification of nuclear components that are responsible for chromatin-chromatin interactions and determine the 3-dimensional organization of the chromatin fiber.
Asunto(s)
Cromatina/química , Fibroblastos/citología , Interfase , Conformación de Ácido Nucleico , Células Cultivadas , Femenino , Humanos , Modelos MolecularesRESUMEN
BACKGROUND: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose. RESULTS: In this paper, we describe the development of an improved FRET-based Gq activity sensor that consists of a yellow fluorescent protein (YFP)-tagged Gγ2 subunit and a Gαq subunit with an inserted monomeric Turquoise (mTurquoise), the best cyan fluorescent protein variant currently available. This sensor enabled us to determine, for the first time, the kon (2/s) of Gq activation. In addition, we found that the guanine nucleotide exchange factor p63RhoGEF has a profound effect on the number of Gq proteins that become active upon stimulation of endogenous histamine H1 receptors. The sensor was also used to measure ligand-independent activation of the histamine H1 receptor (H1R) upon addition of a hypotonic stimulus. CONCLUSIONS: Our observations reveal that the application of a truncated mTurquoise as donor and a YFP-tagged Gγ2 as acceptor in FRET-based Gq activity sensors substantially improves their dynamic range. This optimization enables the real-time single cell quantification of Gq signalling dynamics, the influence of accessory proteins and allows future drug screening applications by virtue of its sensitivity.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Luminiscentes/metabolismo , Receptores Histamínicos H1/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Humanos , RatonesRESUMEN
The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca2+-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores. Notably, the SpoVAEa-SGFP2 proteins were present in a single spot in spores, similar to the IM complex formed by all GRs termed the germinosome. However, while the GRs' spot remains in one location, the SpoVAEa-SGFP2 spot in the IM moved randomly with high frequency. It seems possible that this movement may be a means of communicating germination signals from the germinosome to the IM SpoVA channel, thus stimulating CaDPA release in germination. The dynamics of the SpoVAEa-SGFP2 and its surrounding IM region as stained by fluorescent dyes were also tracked during spore germination, as the dormant spore IM appeared to have an immobile germination related functional microdomain. This microdomain disappeared around the time of appearance of a germinated spore, and the loss of fluorescence of the IM with fluorescent dyes, as well as the appearance of peak SpoVAEa-SGFP2 fluorescent intensity occurred in parallel. These observed events were highly related to spores' rapid phase darkening, which is considered as due to rapid Ca2+DPA release. We also tested the response of SpoVAEa and the IM to thermal treatments at 40-80 °C. Heat treatment triggered an increase of green autofluorescence, which is speculated to be due to coat protein denaturation, and 80 °C treatments induce the appearance of phase-grey-like spores. These spores presumably have a similar intracellular physical state as the phase grey spores detected in the germination but lack the functional proteins for further germination events.
Asunto(s)
Bacillus subtilis , Esporas Bacterianas , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Colorantes Fluorescentes/metabolismo , Lípidos de la Membrana/metabolismo , Ácidos Picolínicos/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
Early life stress increases the risk for developing stress-related pathologies later in life. Recent studies in rats suggest that mild early life stress, rather than being overall unfavorable, may program the hippocampus such that it is optimally adapted to a stressful context later in life. Here, we tested whether this principle of "adaptive programming" also holds under severely adverse early life conditions, i.e., 24 h of maternal deprivation (MD), a model for maternal neglect. In young adult male rats subjected to MD on postnatal day 3, we observed reduced levels of adult hippocampal neurogenesis as measured by cell proliferation, cell survival, and neuronal differentiation. Also, mature dentate granule cells showed a change in their dendritic morphology that was most noticeable in the proximal part of the dendritic tree. Lasting structural changes due to MD were paralleled by impaired water maze acquisition but did not affect long-term potentiation in the dentate gyrus. Importantly, in the presence of high levels of the stress hormone corticosterone, even long-term potentiation in the dentate gyrus of MD animals was facilitated. In addition to this, contextual learning in a high-stress environment was enhanced in MD rats. These morphological, electrophysiological, and behavioral observations show that even a severely adverse early life environment does not evolve into overall impaired hippocampal functionality later in life. Rather, adversity early in life can prepare the organism to perform optimally under conditions associated with high corticosteroid levels in adulthood.
Asunto(s)
Hipocampo/fisiopatología , Aprendizaje/fisiología , Neurogénesis/fisiología , Plasticidad Neuronal/fisiología , Estrés Psicológico/fisiopatología , Sinapsis/fisiología , Animales , Corticosterona/sangre , Dendritas/patología , Dendritas/fisiología , Giro Dentado/patología , Giro Dentado/fisiopatología , Emociones/fisiología , Hipocampo/patología , Potenciación a Largo Plazo/fisiología , Masculino , Privación Materna , Aprendizaje por Laberinto/fisiología , Neuronas/patología , Neuronas/fisiología , Distribución Aleatoria , Ratas , Ratas Wistar , Percepción Espacial/fisiología , Estrés Psicológico/sangre , Estrés Psicológico/patología , Sinapsis/patologíaRESUMEN
BACKGROUND: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.
Asunto(s)
Codón sin Sentido , Fibroblastos/fisiología , Lamina Tipo A/genética , Membrana Nuclear/genética , Progeria/genética , Línea Celular , Forma de la Célula , Fibroblastos/patología , Humanos , Inmunohistoquímica , Lamina Tipo A/deficiencia , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Progeria/metabolismo , Progeria/patología , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Valores de Referencia , Piel/citología , Fenómenos Fisiológicos de la PielRESUMEN
The mucosal layers colonized by the pathogenic fungus Candida albicans differ widely in ambient pH. Because the properties and functions of wall proteins are probably pH dependent, we hypothesized that C. albicans adapts its wall proteome to the external pH. We developed an in vitro system that mimics colonization of mucosal surfaces by growing biomats at pH 7 and 4 on semi-solid agarose containing mucin as the sole nitrogen source. The biomats expanded radially for at least 8 days at a rate of ~30 µm h(-1). At pH 7, hyphal growth predominated and growth was invasive, whereas at pH 4 only yeast and pseudohyphal cells were present and growth was noninvasive. Both qualitative mass spectrometric analysis of the wall proteome by tandem mass spectrometry and relative quantification of individual wall proteins (pH 7/pH 4), using Fourier transform mass spectrometry (FT-MS) and a reference mixture of (15)N-labelled yeast and hyphal walls, identified similar sets of >20 covalently linked wall proteins. The adhesion proteins Als1 and Als3, Hyr1, the transglucosidase Phr1, the detoxification enzyme Sod5 and the mammalian transglutaminase substrate Hwp1 (immunological detection) were only present at pH 7, whereas at pH 4 the level of the transglucosidase Phr2 was >35-fold higher than at pH 7. Sixteen out of the 22 proteins identified by FT-MS showed a greater than twofold change. These results demonstrate that ambient pH strongly affects the wall proteome of C. albicans, show that our quantitative approach can give detailed insights into the dynamics of the wall proteome, and point to potential vaccine targets.
Asunto(s)
Candida albicans/química , Candida albicans/fisiología , Pared Celular/química , Proteínas Fúngicas/análisis , Regulación Fúngica de la Expresión Génica , Proteoma/análisis , Estrés Fisiológico , Concentración de Iones de Hidrógeno , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría de Masas en TándemRESUMEN
Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth.
Asunto(s)
Proteínas Fúngicas/fisiología , Fusarium/genética , Fusarium/patogenicidad , Interacciones Huésped-Parásitos/genética , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Clonación Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/crecimiento & desarrollo , Fusarium/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/fisiología , Solanum lycopersicum/parasitología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Organismos Modificados Genéticamente , Filogenia , Raíces de Plantas/parasitología , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
The 'Omics' revolution has brought a wealth of new mechanistic insights in many fields of biology. It offers options to base predictions of microbial behaviour on mechanistic insight. As the cellular mechanisms involved often turn out to be highly intertwined it is crucial that model development aims at identifying the level of complexity that is relevant to work at. For the prediction of microbiologically stable foods insight in the behaviour of bacterial spore formers is crucial. Their chances of germination and likelihood of outgrowth are major food stability indicators, as well as the transition from outgrowth to first cell division and vegetative growth. Current available technology to assess these parameters in a time-resolved manner at the single spore level will be discussed. Tools to study molecular processes operative in heat induced damage will be highlighted.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Microbiología de Alimentos/métodos , Modelos Biológicos , Bacillus subtilis/ultraestructura , Calor , Microscopía de Contraste de Fase , Esporas Bacterianas/crecimiento & desarrollo , Esporas Bacterianas/ultraestructuraRESUMEN
The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores' germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that >80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores' 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.
Asunto(s)
Bacillus subtilis/fisiología , Membrana Celular/metabolismo , Esporas Bacterianas/fisiología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Fluorescencia , Imagenología TridimensionalRESUMEN
Fusarium oxysporum is an asexual, soil inhabiting fungus that comprises many different formae speciales, each pathogenic towards a different host plant. In absence of a suitable host all F. oxysporum isolates appear to have a very similar lifestyle, feeding on plant debris and colonizing the rhizosphere of living plants. Upon infection F. oxysporum switches from a saprophytic to an infectious lifestyle, which probably includes the reprogramming of gene expression. In this work we show that the expression of the known effector gene SIX1 of F. oxysporum f. sp. lycopersici is strongly upregulated during colonization of the host plant. Using GFP (green fluorescent protein) as reporter, we show that induction of SIX1 expression starts immediately upon penetration of the root cortex. Induction requires living plant cells, but is not host specific and does not depend on morphological features of roots, since plant cells in culture can also induce SIX1 expression. Taken together, F. oxysporum seems to be able to distinguish between living and dead plant material, preventing unnecessary switches from a saprophytic to an infectious lifestyle.