RESUMEN
INTRODUCTION: Glioblastoma (GBM) is an aggressive primary brain cancer. Lack of effective therapy is related to its highly invasive nature. GBM invasion has been studied with reductionist systems that do not fully recapitulate the cytoarchitecture of the brain. We describe a human-derived brain organotypic model to study the migratory properties of GBM IDH-wild type ex vivo. METHODS: Non-tumor brain samples were obtained from patients undergoing surgery (n = 7). Organotypic brain slices were prepared, and green fluorescent protein (GFP)-labeled primary human GBM IDH-wild type cells (GBM276, GBM612, GBM965) were placed on the organotypic slice. Migration was evaluated via microscopy and immunohistochemistry. RESULTS: After placement, cells migrated towards blood vessels; initially migrating with limited directionality, sending processes in different directions, and increasing their speed upon contact with the vessel. Once merged, migration speed decreased and continued to decrease with time (p < 0.001). After perivascular localization, migration is limited along the blood vessels in both directions. The percentage of cells that contact blood vessels and then continue to migrate along the vessel was 92.5% (- 3.9/ + 2.9)% while the percentage of cells that migrate along the blood vessel and leave was 7.5% (- 2.9/ + 3.9) (95% CI, Clopper-Pearson (exact); n = 256 cells from six organotypic cultures); these percentages are significantly different from the random (50%) null hypothesis (z = 13.6; p < 10-7). Further, cells increase their speed in response to a decrease in oxygen tension from atmospheric normoxia (20% O2) to anoxia (1% O2) (p = 0.033). CONCLUSION: Human organotypic models can accurately study cell migration ex vivo. GBM IDH-wild type cells migrate toward the perivascular space in blood vessels and their migratory parameters change once they contact vascular structures and under hypoxic conditions. This model allows the evaluation of GBM invasion, considering the human brain microenvironment when cells are removed from their native niche after surgery.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patología , Encéfalo/patología , Células Tumorales Cultivadas , Movimiento Celular/fisiología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Microambiente TumoralRESUMEN
In adipose tissue, resistance to insulin's ability to increase glucose uptake can be induced by multiple factors, including obesity. Impaired insulin action may take place at different spatial loci at the cellular or subcellular level. To begin to understand the spatial response to insulin in human subcutaneous adipose tissue (hSAT), we developed a quantitative imaging method for activation of a major signaling node in the glucoregulatory insulin signaling pathway. After treatment with insulin or control media, biopsied tissues were immunostained for Akt phosphorylation at Thr-308/9 (pAkt) and then imaged by confocal fluorescence microscopy automated to collect a large grid of high resolution fields. In hSAT from 40 men and women with obesity, substantial heterogeneity of pAkt densities in adipocyte membranes were quantified in each image mosaic using a spatial unit of at least twice the size of the point spread function. Statistical analysis of the distribution of pAkt spatial units was best fit as the weighted sum of two separate distributions, corresponding to either a low or high pAkt density. A "high pAkt fraction" metric was calculated from the fraction of high pAkt distributed units over the total units. Importantly, upon insulin stimulation, tissues from the same biopsy showed either a minimal or a substantial change in the high pAkt fraction. Further supporting a two-state response to insulin stimulation, subjects with similar insulin sensitivity indices are also segregated into either of two clusters identified by the amount of membrane-localized pAkt.
Asunto(s)
Adipocitos/metabolismo , Insulina/metabolismo , Obesidad/metabolismo , Grasa Subcutánea/metabolismo , Adipocitos/enzimología , Adulto , Anciano , Membrana Celular/metabolismo , Estudios de Cohortes , Activación Enzimática , Femenino , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Resistencia a la Insulina , Masculino , Microscopía Confocal , Microscopía Fluorescente , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Grasa Subcutánea/enzimología , Adulto JovenRESUMEN
Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 degrees C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 degrees C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.
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Lípidos de la Membrana/química , Microdominios de Membrana/química , Orthomyxoviridae/química , Fosfolípidos/química , Temperatura , Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Membranas Artificiales , Microscopía Fluorescente , Tamaño de la Partícula , Estándares de Referencia , Propiedades de Superficie , Factores de Tiempo , Internalización del VirusRESUMEN
A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.
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Membrana Celular/metabolismo , Colesterol/análisis , Fibroblastos/metabolismo , Adsorción , Animales , Adhesión Celular , Técnicas de Cultivo de Célula , Línea Celular , Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , Células Clonales , Materiales Biocompatibles Revestidos/metabolismo , Medio de Cultivo Libre de Suero , Vidrio/química , Ratones , Células 3T3 NIH , Orgánulos/metabolismo , Fosfolípidos/análisis , Fosfolípidos/metabolismo , Polilisina/metabolismo , Propiedades de Superficie , Temperatura , Factores de Tiempo , Transfección , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologíaRESUMEN
In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca(2+). Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.
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Astrocitos/metabolismo , Señalización del Calcio , Calcio/metabolismo , Sistema Nervioso Central/metabolismo , Animales , Traumatismos por Explosión , Células Cultivadas , Sistema Nervioso Central/citología , Explosiones , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratas Sprague-Dawley , Receptores Purinérgicos/metabolismo , Transducción de Señal , Estrés MecánicoRESUMEN
Blast-Induced Traumatic Brain Injury (bTBI) describes a spectrum of injuries caused by an explosive force that results in changes in brain function. The mechanism responsible for primary bTBI following a blast shockwave remains unknown. We have developed a pneumatic device that delivers shockwaves, similar to those known to induce bTBI, within a chamber optimal for fluorescence microscopy. Abrupt changes in pressure can be created with and without the presence of shear forces at the surface of cells. In primary cultures of human central nervous system cells, the cellular calcium response to shockwaves alone was negligible. Even when the applied pressure reached 15 atm, there was no damage or excitation, unless concomitant shear forces, peaking between 0.3 to 0.7 Pa, were present at the cell surface. The probability of cellular injury in response to a shockwave was low and cell survival was unaffected 20 hours after shockwave exposure.
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Encéfalo/metabolismo , Encéfalo/patología , Calcio/metabolismo , Explosiones , Presión , Resistencia al Corte , Células Cultivadas , Humanos , Microscopía , ReologíaRESUMEN
Synaptic vesicles contain a variety of proteins and lipids that mediate fusion with the pre-synaptic membrane. Although the structures of many synaptic vesicle proteins are known, an overall picture of how they are organized at the vesicle surface is lacking. In this paper, we describe a better method for the isolation of squid synaptic vesicles and characterize the results. For highly pure and intact synaptic vesicles from squid optic lobe, glycerol density gradient centrifugation was the key step. Different electron microscopic methods show that vesicle membrane surfaces are largely covered with structures corresponding to surface proteins. Each vesicle contains several stalked globular structures that extend from the vesicle surface and are consistent with the V-ATPase. BLAST search of a library of squid expressed sequence tags identifies 10 V-ATPase subunits, which are expressed in the squid stellate ganglia. Negative-stain tomography demonstrates directly that vesicles flatten during the drying step of negative staining, and furthermore shows details of individual vesicles and other proteins at the vesicle surface.
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Biología/métodos , Decapodiformes/ultraestructura , Animales , Centrifugación por Gradiente de Densidad/métodos , Microscopía Electrónica/métodos , Lóbulo Óptico de Animales no Mamíferos/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(-/-) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.
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Apoptosis , Proteína Ligando Fas/metabolismo , Células Madre/metabolismo , Caspasa 8/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Humanos , Inmunosupresores/farmacología , Células Jurkat , Linfocitos/metabolismo , Modelos Biológicos , ARN/metabolismo , ARN Interferente Pequeño/metabolismo , Células Madre/citología , Factores de TiempoRESUMEN
Borrelia burgdorferi sensu stricto is an etiological agent of Lyme disease. The lack of an adequate ex vivo system for human tissue infection is an obstacle to fully understanding the molecular mechanisms of invasion of tissue by B. burgdorferi and its adaptation within the human host. Here, we report on the development of such a system. We inoculated blocks of human tonsillar tissue with B. burgdorferi spirochetes, cultured them in a low-shear rotating wall vessel (RWV) bioreactor, and analyzed them using light and electron microscopy, nested polymerase chain reaction (PCR), and quantitative real-time PCR. Also, we evaluated the expression of the outer surface proteins (Osps) OspA and OspC by use of quantitative Western blotting. Light and electron microscopic analysis revealed multiple spirochetes localized extracellularly within the tissue, and their identity was confirmed by PCR. Quantification of spirochetes inside the RWV-cultured tonsillar tissue demonstrated that the number of B. burgdorferi exceeded the initial inoculum by an order of magnitude, indicating that spirochetes replicated in the tissue. Electron microscopic analysis showed that some spirochetes were arranged in cystic structures and that invading spirochetes differentially expressed surface proteins; both of these features have been described for infected tissues in vivo. The system we have developed can be used to study B. burgdorferi pathogenesis under controlled conditions ex vivo, in particular to explore the gene activation responsible for the adaptation of B. burgdorferi to human tissue that leads to Lyme disease.
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Técnicas Bacteriológicas/métodos , Borrelia burgdorferi/fisiología , Antígenos Bacterianos/metabolismo , Antígenos de Superficie/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas , Borrelia burgdorferi/ultraestructura , Expresión Génica , Humanos , Técnicas In Vitro , Lipoproteínas/metabolismo , Tonsila Palatina/microbiologíaRESUMEN
The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca(2+)-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-gamma-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca(2+)-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Fusión de Membrana , Óvulo/citología , Óvulo/metabolismo , Erizos de Mar/citología , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/deficiencia , Adenosina Trifosfato/farmacología , Adenilil Imidodifosfato/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Membrana Celular/metabolismo , Clonación Molecular , Citosol/metabolismo , Dextranos/farmacología , Etilmaleimida/farmacología , Exocitosis/efectos de los fármacos , Femenino , Fusión de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Óvulo/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Alineación de Secuencia , Análisis de Secuencia de Proteína , Estilbenos/farmacología , Ácidos Sulfónicos/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/inmunología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Although immunoblotting (Western blotting) is widely used for the detection of specific proteins, it is often thought to be an inadequate technique for accurate and precise measurements of protein concentration. However, an accurate and precise technique is essential for quantitative testing of hypotheses, and thus for the analysis and understanding of proposed molecular mechanisms. The analysis of Ca(2+)-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents, requires such an unambiguous identification and a quantitative assessment of the membrane surface density of specific molecules. Newly refined immunoblotting and analysis approaches permit a quantitative analysis of the SNARE protein complement (VAMP, SNAP-25, and syntaxin) of functional secretory vesicles. The method illustrates the feasibility of the routine quantification of femtomole to attomole amounts of known proteins by immunoblotting. The results indicate that sea urchin egg secretory vesicles and synaptic vesicles have markedly similar SNARE densities.
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Exocitosis/fisiología , Proteínas de la Membrana/análisis , Proteínas del Tejido Nervioso/análisis , Vesículas Secretoras/química , Proteínas de Transporte Vesicular , Animales , Western Blotting , Calcio/farmacología , Electroforesis en Gel de Poliacrilamida , Exocitosis/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteínas SNARE , Erizos de Mar , Vesículas Sinápticas/química , Proteína 25 Asociada a SinaptosomasRESUMEN
A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.
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Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/aislamiento & purificación , Focalización Isoeléctrica , Peso Molecular , Proteínas/química , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , UltrafiltraciónRESUMEN
SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using previously established fusion assays and a high sensitivity immunoblotting protocol, we analyzed the relationship between these SNARE proteins and Ca2+-triggered membrane fusion. Neither the extent of fusion nor the number of intermembrane fusion complexes per vesicle were correlated with the measured density of identified egg cortical vesicle (CV) SNAREs. Without syntaxin, CVs remained fusion competent. Surprisingly, for one (but not another) protease the Ca2+ dependence of fusion was correlated with CV SNARE density, suggesting a native protein complex that associates with SNAREs, the architecture of which ensures high Ca2+ sensitivity. As SNAREs may function during CV docking in vivo, and as further proteolysis after SNARE removal eventually ablates fusion, we hypothesize that the triggered steps of regulated fusion (Ca2+ sensitivity and the catalysis and execution of fusion) require additional proteins that function downstream of SNAREs.