RESUMEN
Lacrymaria olor cytoskeleton and membrane "origami" enables rapid cell hyperextension.
Asunto(s)
Cilióforos , Citoesqueleto , Membrana Celular/ultraestructura , Citoesqueleto/ultraestructura , Cilióforos/fisiología , Cilióforos/ultraestructuraRESUMEN
The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.
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Arabidopsis/fisiología , Membrana Celular/fisiología , Fluidez de la Membrana/fisiología , Arabidopsis/ultraestructura , Membrana Celular/ultraestructura , Colorantes Fluorescentes/metabolismo , Temperatura , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Vías Secretoras/genética , Sinapsis/metabolismo , Transmisión Sináptica/genética , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Femenino , Colorantes Fluorescentes/química , Expresión Génica , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hipocampo/citología , Hipocampo/metabolismo , Luz , Masculino , Imagen Molecular/métodos , Neuronas/citología , Cultivo Primario de Células , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Receptores AMPA/metabolismo , Sinapsis/ultraestructura , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The elastic properties of cell membranes, particularly the membrane tension and bending modulus, are known to be key regulators of cellular functions. Here, we present a correlative and integrated tool based on optical tweezers and scanning electron microscopy to accurately determine these properties in a variety of cell types. Although there are intrinsic difficulties associated with correlative experiments, we believe that the methods presented can be considered a suitable protocol for determining the elastic properties of cell membranes. For complete details on the use and execution of this protocol, please refer to Soares et al. (2020).
Asunto(s)
Membrana Celular , Microscopía Electrónica de Rastreo , Pinzas Ópticas , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Elasticidad , HumanosRESUMEN
Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.
Asunto(s)
Reacción Acrosómica/genética , Membrana Celular/ultraestructura , Exocitosis/genética , Espermatozoides/ultraestructura , Acrosoma/metabolismo , Membrana Celular/genética , Humanos , Masculino , Espermatozoides/patología , Zona Pelúcida/metabolismoRESUMEN
Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica , Animales , Animales Modificados Genéticamente , Membrana Celular/ultraestructura , Polaridad Celular/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Embrión no Mamífero , Células Epiteliales/enzimología , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Larva/citología , Larva/enzimología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.
Asunto(s)
Bacillus/citología , Bacillus/efectos de los fármacos , Cromo/farmacología , Fenol/farmacología , Bacillus/metabolismo , Biodegradación Ambiental , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Cromo/metabolismo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Ácidos Grasos/química , Fosfolípidos/química , Estrés FisiológicoRESUMEN
The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/ultraestructura , Lectinas/ultraestructura , Perforina/ultraestructura , Conformación Proteica , Secuencia de Aminoácidos/genética , Animales , Membrana Celular/química , Membrana Celular/ultraestructura , Complejo de Ataque a Membrana del Sistema Complemento/química , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Cristalografía por Rayos X , Dimerización , Lectinas/química , Lectinas/inmunología , Modelos Moleculares , Perforina/química , Perforina/inmunología , Subunidades de Proteína/genética , Dispersión del Ángulo Pequeño , Caracoles/ultraestructura , Difracción de Rayos XRESUMEN
Microorganisms offer an alternative green and scalable technology for the synthesis of value added products. Fungi secrete high quantities of bioactive substances, which play dual-functional roles as both reducing and stabilizing agents in the synthesis of colloidal metal nanoparticles such as silver nanoparticles, which display potent antimicrobial properties that can be harnessed for a number of industrial applications. The aim of this work was the production of silver nanoparticles using the extracellular cell free extracts of Phanerochaete chrysosporium, and to evaluate their activity as antimicrobial and antibiofilm agents. The 45-nm diameter silver nanoparticles synthesized using this methodology possessed a high negative surface charge close to -30 mV and showed colloidal stability from pH 3-9 and under conditions of high ionic strength ([NaCl] = 10-500 mM). A combination of environmental SEM, TEM, and confocal Raman microscopy was used to study the nanoparticle-E. coli interactions to gain a first insight into their antimicrobial mechanisms. Raman data demonstrate a significant decrease in the fatty acid content of E. coli cells, which suggests a loss of the cell membrane integrity after exposure to the PchNPs, which is also commensurate with ESEM and TEM images. Additionally, these biogenic PchNPs displayed biofilm disruption activity for the eradication of E. coli and C. albicans biofilms.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Nanopartículas del Metal/química , Phanerochaete/química , Plata/química , Antibacterianos/química , Antibacterianos/farmacología , Antiinfecciosos/química , Candida albicans/fisiología , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Escherichia coli/química , Escherichia coli/fisiología , Escherichia coli/ultraestructura , Ácidos Grasos/análisis , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Concentración de Iones de Hidrógeno , Nanopartículas del Metal/ultraestructura , Microscopía Electrónica de Transmisión de Rastreo , Concentración Osmolar , Tamaño de la Partícula , Espectrometría RamanRESUMEN
In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.
Asunto(s)
Membrana Celular/ultraestructura , Extensiones de la Superficie Celular/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Algoritmos , Animales , Animales Modificados Genéticamente , Vesículas Citoplasmáticas/ultraestructura , Embrión no Mamífero , Humanos , Microvellosidades/ultraestructura , Seudópodos/ultraestructura , Pez Cebra/embriologíaRESUMEN
Mast cells are connective tissue resident cells with morphological and functional characteristics that contribute to their role in allergic and inflammatory processes, host defense and maintenance of tissue homeostasis. Mast cell activation results in the release of pro-inflammatory mediators which are largely responsible for the physiological functions of mast cells. The lectin ArtinM, extracted from Artocarpus heterophyllus (jackfruit), binds to D-manose, thus inducing degranulation of mast cells. ArtinM has several immunomodulatory properties including acceleration of wound healing, and induction of cytokine release. The aim of the present study was to investigate the role of ArtinM in the activation and proliferation of mast cells. The rat mast cell line RBL-2H3 was used throughout this study. At a low concentration (0.25µg/mL), ArtinM induced mast cell activation and the release of IL-6 without stimulating the release of pre-formed or newly formed mediators. Additionally, when the cells were activated by ArtinM protein tyrosine phosphorylation was stimulated. The low concentration of ArtinM also activated the transcription factor NFkB, but not NFAT. ArtinM also affected the cell cycle and stimulated cell proliferation. Therefore, ArtinM may have therapeutic applications by modulating immune responses due to its ability to activate mast cells and promote the release of newly synthesized mediators. Additionally, ArtinM could have beneficial effects at low concentrations without degranulating mast cells and inducing allergic reactions.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Lectinas/farmacología , Proteínas de Plantas/farmacología , Animales , Artocarpus/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Proliferación Celular/efectos de los fármacos , Interleucina-6/metabolismo , Mastocitos/citología , Mastocitos/metabolismo , Mitosis/efectos de los fármacos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , RatasRESUMEN
Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 µm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.
Asunto(s)
Acrosoma/ultraestructura , Cabeza del Espermatozoide/ultraestructura , Cola del Espermatozoide/ultraestructura , Espermatozoides/ultraestructura , Acrosoma/fisiología , Animales , Axonema/ultraestructura , Membrana Celular/ultraestructura , Núcleo Celular/ultraestructura , Humanos , Masculino , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Mitocondrias/ultraestructura , Semen/citología , Cabeza del Espermatozoide/fisiología , Motilidad Espermática , Cola del Espermatozoide/fisiología , Espermatozoides/fisiologíaRESUMEN
We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable by two-photon laser sources, making it easy to combine CAPRYDAA with either blue or red emission probes. In GUVs or cells, CAPRYDAA can discriminate the lipid phases and liquid-liquid phase heterogeneity. This new membrane probe shows the bathochromic properties of the PRODAN-based probes designed by Weber, overcoming the need for UV or two-photon excitation and facilitating the studies on the membrane properties using regular confocal microscopes.
Asunto(s)
2-Naftilamina/análogos & derivados , Antracenos/química , Membrana Celular/química , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Espectrometría de Fluorescencia/métodos , Animales , Membrana Celular/ultraestructura , Lípidos de la Membrana/análisis , Ratones , Células 3T3 NIHRESUMEN
The ultrastructural characteristics of the mature spermatozoon of the aspidogastrean Rohdella amazonica (Aspidogastridae, Rohdellinae) were studied by means of transmission electron microscopy. The sperm cell shows two axonemes of the 9 + '1' trepaxonematan pattern of Platyhelminthes, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, one mitochondrion, an electron-dense ring, a nucleus and granules of glycogen. The present results were compared with those observed in the aspidogastreans studied to date and in other Platyhelminthes. The lateral expansion and the electron-dense ring are typical characters for aspidogastreans. Although a lateral expansion has been described in other Platyhelminthes such as monogeneans and digeneans, the Aspidogastrea shows a much larger lateral expansion with both peripheral and internal microtubules. The dense ring is observed as a cylinder in a longitudinal view and shows a more granular appearance in sperm cells from the seminal vesicle in comparison to a more electron-dense appearance in sperm cells from the seminal uterine receptacle.
Asunto(s)
Axonema/ultraestructura , Espermatozoides/ultraestructura , Tetraodontiformes/parasitología , Trematodos/ultraestructura , Animales , Membrana Celular/ultraestructura , Núcleo Celular/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Microtúbulos/ultraestructura , Mitocondrias/ultraestructuraRESUMEN
Arterial hypertension (HTN) can lead to serious organ damage. Several mechanisms have been implicated in the pathogenesis of HTN including constitutive activation of platelets, which increases the risk of aggregation and clot formation. We recently demonstrated the plasma membranes of platelets from patients with HTN exhibit modified structural and physicochemical properties; Raman and Fourier transform infrared by attenuated total reflectance (FTIR-ATR) spectroscopy also indicated lipid content and protein structure alterations. This study aimed to precisely quantify the constituents of the main structural phospholipids and cholesterol in the plasma membranes of platelets from patients with HTN and normotensive individuals. We also assessed the consequence of these alterations on platelet structure and function. Liquid chromatography coupled to triple quadrupole mass spectrometry revealed the plasma membranes of HTN platelets contained less cholesterol and phosphatidylcholine, more phosphatidylserine and phosphatidylethanolamine and had similar sphingosine contents. Atomic force microscopy revealed HTN platelets exhibited increased surface roughness and more pleats. Transmission electron microscopy revealed diminution of the internal membranous structures in HTN platelets. Our findings strongly suggest plasma membrane lipid content alterations-including cholesterol depletion-occur in HTN, and these alterations may induce morphological and physiological abnormalities that participate in the functional changes associated with hypertension.
Asunto(s)
Plaquetas/metabolismo , Membrana Celular/ultraestructura , Hipertensión/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Anciano , Plaquetas/ultraestructura , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Humanos , Masculino , Fluidez de la Membrana , Persona de Mediana EdadRESUMEN
Movement and phagocytosis are clue events in colonisation and invasion of tissues by Entamoeba histolytica, the protozoan causative of human amoebiasis. During phagocytosis, EhRab proteins interact with other functional molecules, conducting them to the precise cellular site. The gene encoding EhrabB is located in the complementary chain of the DNA fragment containing Ehcp112 and Ehadh genes, which encode for the proteins of the EhCPADH complex, involved in phagocytosis. This particular genetic organisation suggests that the three corresponding proteins may be functionally related. Here, we studied the relationship of EhRabB with EhCPADH and actin during phagocytosis. First, we obtained the EhRabB 3D structure to carry out docking analysis to predict the interaction sites involved in the EhRabB protein and the EhCPADH complex contact. By confocal microscopy, transmission electron microscopy, and immunoprecipitation assays, we revealed the interaction among these proteins when they move through different vesicles formed during phagocytosis. The role of the actin cytoskeleton in this event was also confirmed using Latrunculin A to interfere with actin polymerisation. This affected the movement of EhRabB and EhCPADH, as well as the rate of phagocytosis. Mutant trophozoites, silenced in EhrabB gene, evidenced the interaction of this molecule with EhCPADH and strengthened the role of actin during erythrophagocytosis.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Entamoeba histolytica/metabolismo , Fagocitosis/genética , Trofozoítos/ultraestructura , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actinas/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidad , Entamoeba histolytica/ultraestructura , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Humanos , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Mutación , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trofozoítos/efectos de los fármacos , Trofozoítos/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide-lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling-at the biophysical level-the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Dobles de Lípidos/química , Lipopéptidos/farmacología , Péptidos Cíclicos/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/química , Bacterias/química , Bacterias/efectos de los fármacos , Bacterias/ultraestructura , Membrana Celular/química , Membrana Celular/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Lipopéptidos/biosíntesis , Lipopéptidos/química , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cilios/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucosa/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cerebelo/citología , Cerebelo/metabolismo , Cilios/ultraestructura , Forma BB de la Creatina-Quinasa/genética , Forma BB de la Creatina-Quinasa/metabolismo , Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa-6-Fosfatasa/genética , Glucólisis , Masculino , Microsomas/metabolismo , Microsomas/ultraestructura , Neuronas Receptoras Olfatorias/citología , Fosforilación Oxidativa , Ratas , Ratas Sprague-Dawley , Técnicas de Cultivo de TejidosRESUMEN
The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.
Asunto(s)
Proteínas Algáceas/química , Membrana Celular/química , Chlorophyceae/química , ATPasas de Translocación de Protón Mitocondriales/química , Subunidades de Proteína/química , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Chlorophyceae/enzimología , Chlorophyceae/genética , Chlorophyceae/ultraestructura , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Near infrared (NIR) laser light can have important reactions on live cells. For example, in a macroscopic scale, it is used therapeutically to reduce inflammation and in a single-cell scale, NIR lasers have been experimentally used to guide neuronal growth. However, little is known about how NIR lasers produce such behaviours on cells. In this paper we report effects of focussing a continuous wave 810-nm wavelength laser on in vivo 3T3 cells plasma membrane. Cell membranes were labelled with FM 4-64, a dye that fluoresces when associated to membrane lipids. Confocal microscopy was used to image cell membranes and perform fluorescence recovery after photobleaching (FRAP) experiments. We found that the NIR laser produces an increase of the fluorescence intensity at the location of laser spot. This intensity boost vanishes once the laser is turned off. The mean fluorescence increase, calculated over 75 independent measurements, equals 19%. The experiments reveal that the fluorescence rise is a growing function of the laser power. This dependence is well fitted with a square root function. The FRAP, when the NIR laser is acting on the cell, is twice as large as when the NIR laser is off, and the recovery time is 5 times longer. Based on the experimental evidence and a linear fluorescence model, it is shown that the NIR laser provokes a rise in the number of molecular associations dye-lipid. The results reported here may be a consequence of a combination of induced increments in membrane fluidity and exocytosis.