RESUMEN
Atomic force microscopy is an extremely versatile technique, featuring atomic-scale imaging resolution, and also offering the possibility to probe interaction forces down to few pN. Recently, this technique has been specialized to study the interaction between single living cells, one on the substrate, and a second being adhered on the cantilever. Cell-cell force spectroscopy offers a unique tool to investigate in fine detail intra-cellular interactions, and it holds great promise to elucidate elusive phenomena in physiology and pathology. Here we present a systematic study of the effect of the main measurement parameters on cell-cell curves, showing the importance of controlling the experimental conditions. Moreover, a simple theoretical interpretation is proposed, based on the number of contacts formed between the two interacting cells. The results show that single cell-cell force spectroscopy experiments carry a wealth of information that can be exploited to understand the inner dynamics of the interaction of living cells at the molecular level.
Asunto(s)
Comunicación Celular , Fenómenos Mecánicos , Microscopía de Fuerza Atómica , Análisis EspectralRESUMEN
Lipid membranes play a fundamental role in the pathological development of protein misfolding diseases. Several pieces of evidence suggest that the lipid membrane could act as a catalytic surface for protein aggregation. Furthermore, a leading theory indicates the interaction between the cell membrane and misfolded oligomer species as the responsible for cytotoxicity, hence, for neurodegeneration in disorders such as Alzheimer's and Parkinson's disease. The definition of the mechanisms that drive the interaction between pathological protein aggregates and plasma membrane is fundamental for the development of effective therapies for a large class of diseases. Atomic force microscopy (AFM) has been employed to study how amyloid aggregates affect the cell physiological properties. Considerable efforts were spent to characterize the interaction with model systems, i.e., planar supported lipid bilayers, but some works also addressed the problem directly on living cells. Here, an overview of the main works involving the use of the AFM on both model system and living cells will be provided. Different kind of approaches will be presented, as well as the main results derived from the AFM analysis.
Asunto(s)
Amiloide/metabolismo , Amiloide/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Microscopía de Fuerza Atómica , Animales , Humanos , Agregado de Proteínas , Propiedades de SuperficieRESUMEN
Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation.
Asunto(s)
Amiloidosis/metabolismo , Plasminógeno/metabolismo , Prealbúmina/metabolismo , Amiloide/metabolismo , Bases de Datos de Proteínas , Fibrinolisina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Desnaturalización Proteica , Pliegue de Proteína , Proteolisis , Tripsina/metabolismoRESUMEN
Minimizing the foreign body reaction to polyimide-based implanted devices plays a pivotal role in several biomedical applications. In this work, we propose materials exhibiting nonbiofouling properties and a Young's modulus reflecting that of soft human tissues. We describe the synthesis, characterization, and in vitro validation of poly(carboxybetaine) hydrogel coatings covalently attached to polyimide substrates via a photolabile 4-azidophenyl group, incorporated in poly(carboxybetaine) chains at two concentrations of 1.6 and 3.1 mol %. The presence of coatings was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy. White light interferometry was used to evaluate the coating continuity and thickness (between 3 and 6 µm under dry conditions). Confocal laser scanning microscopy allowed us to quantify the thickness of the swollen hydrogel coatings that ranged between 13 and 32 µm. The different hydrogel formulations resulted in stiffness values ranging from 2 to 19 kPa and led to different fibroblast and macrophage responses in vitro. Both cell types showed a minimum adhesion on the softest hydrogel type. In addition, both the overall macrophage activation and cytotoxicity were observed to be negligible for all of the tested material formulations. These results are a promising starting point toward future advanced implantable systems. In particular, such technology paves the way for novel neural interfaces able to minimize the fibrotic reaction, once implanted in vivo, and to maximize their long-term stability and functionality.
Asunto(s)
Resinas Acrílicas/farmacología , Adhesión Celular/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Fibroblastos/metabolismo , Hidrogeles/farmacología , Macrófagos/metabolismo , Resinas Acrílicas/síntesis química , Animales , Materiales Biocompatibles Revestidos/síntesis química , Módulo de Elasticidad , Humanos , Hidrogeles/síntesis química , Ratones , Células RAW 264.7RESUMEN
The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/metabolismo , Multimerización de Proteína , Animales , Proteínas Bacterianas/toxicidad , Células CHO , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cricetulus , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
Highly toxic protein misfolded oligomers associated with neurological disorders such as Alzheimer's and Parkinson's diseases are nowadays considered primarily responsible for promoting synaptic failure and neuronal death. Unraveling the relationship between structure and neurotoxicity of protein oligomers appears pivotal in understanding the causes of the pathological process, as well as in designing novel diagnostic and therapeutic strategies tuned toward the earliest and presymptomatic stages of the disease. Here, it is benefited from tip-enhanced Raman spectroscopy (TERS) as a surface-sensitive tool with spatial resolution on the nanoscale, to inspect the spatial organization and surface character of individual protein oligomers from two samples formed by the same polypeptide sequence and different toxicity levels. TERS provides direct assignment of specific amino acid residues that are exposed to a large extent on the surface of toxic species and buried in nontoxic oligomers. These residues, thanks to their outward disposition, might represent structural factors driving the pathogenic behavior exhibited by protein misfolded oligomers, including affecting cell membrane integrity and specific signaling pathways in neurodegenerative conditions.
Asunto(s)
Transferasas de Carboxilo y Carbamoilo/toxicidad , Proteínas de Escherichia coli/toxicidad , Nanopartículas/química , Pliegue de Proteína , Multimerización de Proteína , Espectrometría Raman/métodos , Pliegue de Proteína/efectos de los fármacosRESUMEN
BACKGROUND: Many data highlight the benefits of the Mediterranean diet and its main lipid component, extra-virgin olive oil (EVOO). EVOO contains many phenolic compounds that have been found effective against several aging- and lifestyle-related diseases, including neurodegeneration. Oleuropein, a phenolic secoiroid glycoside, is the main polyphenol in the olive oil. It has been reported that the aglycone form of Oleuropein (OleA) interferes in vitro and in vivo with amyloid aggregation of a number of proteins/peptides involved in amyloid, particularly neurodegenerative, diseases avoiding the growth of toxic oligomers and displaying protection against cognitive deterioration. METHODS: In this study, we carried out a cellular and biophysical study on the relationships between the effects of OleA on the aggregation and cell interactions of the D76N ß2-microglobulin (D76N b2m) variant associated with a familial form of systemic amyloidosis with progressive bowel dysfunction and extensive visceral amyloid deposits. RESULTS: Our results indicate that OleA protection against D76N b2m cytotoxicity results from i) a modification of the conformational and biophysical properties of its amyloid fibrils; ii) a modification of the cell bilayer surface properties of exposed cells. CONCLUSIONS: This study reveals that OleA remodels not only D76N b2m aggregates but also the cell membrane interfering with the misfolded proteins-cell membrane association, in most cases an early event triggering amyloid-mediated cytotoxicity. GENERAL SIGNIFICANCE: The data provided in the present article focus on OleA protection, featuring this polyphenol as a promising plant molecule useful against amyloid diseases.
Asunto(s)
Acetatos/farmacología , Amiloide/efectos adversos , Amiloidosis/prevención & control , Apoptosis/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Piranos/farmacología , Amiloidosis/metabolismo , Amiloidosis/patología , Monoterpenos Ciclopentánicos , Humanos , Membranas Artificiales , Neuroblastoma/metabolismo , Neuroblastoma/patología , Células Tumorales Cultivadas , Microglobulina beta-2/metabolismoRESUMEN
BACKGROUND: Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aß peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS: Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aß fibrillogenesis. RESULTS: We found that Hsp60 powerfully inhibits Aß amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS: We observe that Hsp60 inhibits Aß aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE: Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aß peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration.
Asunto(s)
Amiloide/química , Chaperonina 60/química , Amiloide/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Chaperonina 60/metabolismo , Humanos , Unión ProteicaRESUMEN
Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (â¼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.
Asunto(s)
Elementos de Facilitación Genéticos , Lamina Tipo B/genética , Enfermedad de Pelizaeus-Merzbacher/genética , Eliminación de Secuencia , Animales , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Femenino , Expresión Génica , Regulación de la Expresión Génica , Estudios de Asociación Genética , Humanos , Lamina Tipo B/metabolismo , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Datos de Secuencia Molecular , LinajeRESUMEN
The localized formation of gold nanostructures with controlled size and shape on chitosan films doped with gold precursor upon electromagnetic irradiation of various types is demonstrated here. Such controlled formation is achieved by tuning the wavelength, the energy and the interaction time of the radiation with the composite films. In particular, the use of a single UV nanosecond laser pulse results in the formation of gold sub-micron platelets with specific crystal structure, while increasing the number of pulses, further precursor reduction and photofragmentation induce the formation of gold nanoparticles. Using x-ray radiation as an alternative energy source, the reduction of the gold precursor and the subsequent formation of particles follow a different pathway. Specifically, x-ray-induced photo-reduction triggers the selective formation of gold sub-micron platelets with a very well defined {111} crystal phase. In this case, the density of crystal platelets increases by increasing the irradiation time of the films, while no photofragmentation process is observed. The gold structures pre-formed by x-ray radiation can be fragmented by subsequent pulsed UV laser irradiation forming nanoparticles with much narrower size distribution compared to that obtained via exclusive UV irradiation. Thanks to the perfect coupling between the natural polymeric matrix and gold nanostructures, the bionanocomposite systems developed could find various applications in biomaterial science and in biosensors field.
Asunto(s)
Campos Electromagnéticos , Oro/química , Membranas Artificiales , Nanopartículas del Metal/químicaRESUMEN
The first genetic variant of ß2 -microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild-type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1-enriched domains only, thus establishing a link between aggregate-membrane contact and cell damage.
Asunto(s)
Amiloide/toxicidad , Proteínas Mutantes/toxicidad , Microglobulina beta-2/toxicidad , Fenómenos Biofísicos/efectos de los fármacos , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Difusión , Gangliósido G(M1) , Humanos , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza Atómica , Agregado de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We report the nontemplated colloidal synthesis of single crystal CsPbBr3 perovskite nanosheets with lateral sizes up to a few micrometers and with thickness of just a few unit cells (i.e., below 5 nm), hence in the strong quantum confinement regime, by introducing short ligands (octanoic acid and octylamine) in the synthesis together with longer ones (oleic acid and oleylamine). The lateral size is tunable by varying the ratio of shorter ligands over longer ligands, while the thickness is mainly unaffected by this parameter and stays practically constant at 3 nm in all the syntheses conducted at short-to-long ligands volumetric ratio below 0.67. Beyond this ratio, control over the thickness is lost and a multimodal thickness distribution is observed.
RESUMEN
The control of neuron-substrate adhesion has been always a challenge for fabricating neuron-based cell chips and in particular for multielectrode array (MEA) devices, which warrants the investigation of the electrophysiological activity of neuronal networks. The recent introduction of high-density chips based on the complementary metal oxide semiconductor (CMOS) technology, integrating thousands of electrodes, improved the possibility to sense large networks and raised the challenge to develop newly adapted functionalization techniques to further increase neuron electrode localization to avoid the positioning of cells out of the recording area. Here, we present a simple and straightforward chemical functionalization method that leads to the precise and exclusive positioning of the neural cell bodies onto modified electrodes and inhibits, at the same time, cellular adhesion in the surrounding insulator areas. Different from other approaches, this technique does not require any adhesion molecule as well as complex patterning technique such as µ-contact printing. The functionalization was first optimized on gold (Au) and silicon nitride (Si3N4)-patterned surfaces. The procedure consisted of the introduction of a passivating layer of hydrophobic silane molecules (propyltriethoxysilane [PTES]) followed by a treatment of the Au surface using 11-amino-1-undecanethiol hydrochloride (AT). On model substrates, well-ordered neural networks and an optimal coupling between a single neuron and single micrometric functionalized Au surface were achieved. In addition, we presented the preliminary results of this functionalization method directly applied on a CMOS-MEA: the electrical spontaneous spiking and bursting activities of the network recorded for up to 4 weeks demonstrate an excellent and stable neural adhesion and functional behavior comparable with what expected using a standard adhesion factor, such as polylysine or laminin, thus demonstrating that this procedure can be considered a good starting point to develop alternatives to the traditional chip coatings to provide selective and specific neuron-substrate adhesion.
RESUMEN
Plasma membrane is a complex structure, mainly composed by lipids and proteins, which plays a pivotal role in cell metabolism by regulating its selective permeability to ions and molecules. According to the "raft hypothesis", lipids in the bilayer are not forming a structurally passive solvent, but are rather organized in specific domains, which present different structural and functional characteristics. The mechanical properties of the lipid part of plasma membrane have been recently characterized through Atomic Force Microscopy, by analyzing the features of force vs distance curves collected on supported lipid bilayers (SLBs). In case of lipid domains sizing from tens to hundreds of nanometers, which mimic in a good way the lateral organization of real membranes, a high lateral resolution and a large number of curves are often required for properly expressing the complexity of the system, with a consequent exponential growth of acquisition and processing time. In this paper we propose a method, based on a recently developed high speed Force Volume technique and on home-built data processing software, for the mechanical characterization of nanostructured SLBs. With our software we have been able to process data set composed by tens of thousands of curves, collected with a spatial resolution ranging from 8 to 40 nm/pixel. Multiparametric maps and distribution histograms produced by our analysis allowed identifying a specific behavior for each lipid phase in the investigated model membranes, even in presence of nanosized features.
Asunto(s)
Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , Modelos Moleculares , Nanoestructuras/química , Programas Informáticos , Membrana CelularRESUMEN
The architecture and structural mechanics of the cell nucleus are defined by the nuclear lamina, which is formed by A- and B-type lamins. Recently, gene duplication and protein overexpression of lamin B1 (LB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LB1 affects nuclear mechanics and function and how it may result in pathology remain unexplored. Here, we report that in primary human skin fibroblasts derived from ADLD patients, LB1, but not other lamins, is overexpressed at the nuclear lamina and specifically enhances nuclear stiffness. Transient transfection of LB1 in HEK293 and neuronal N2a cells mimics the mechanical phenotype of ADLD nuclei. Notably, in ADLD fibroblasts, reducing LB1 protein levels by shRNA knockdown restores elasticity values to those indistinguishable from control fibroblasts. Moreover, isolated nuclei from ADLD fibroblasts display a reduced nuclear ion channel open probability on voltage-step application, suggesting that biophysical changes induced by LB1 overexpression may alter nuclear signaling cascades in somatic cells. Overall, the overexpression of LB1 in ADLD cells alters nuclear mechanics and is linked to changes in nuclear signaling, which could help explain the pathogenesis of this disease.
Asunto(s)
Núcleo Celular/patología , Embrión de Mamíferos/citología , Fibroblastos/patología , Lamina Tipo B/metabolismo , Enfermedad de Pelizaeus-Merzbacher/patología , Piel/citología , Adulto , Animales , Western Blotting , Estudios de Casos y Controles , Permeabilidad de la Membrana Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Lamina Tipo B/antagonistas & inhibidores , Lamina Tipo B/genética , Masculino , Ratones , Persona de Mediana Edad , Técnicas de Placa-Clamp , Enfermedad de Pelizaeus-Merzbacher/genética , Enfermedad de Pelizaeus-Merzbacher/metabolismo , Fenotipo , ARN Interferente Pequeño/genética , Piel/metabolismoRESUMEN
Grasslike compliant micro/nano crystals made of diarylethene (DAE) photochromic molecules are spontaneously formed on elastomer films after dipping them in a solution containing the photochromic molecules. The frictional forces of such micro- and nanofibrillar surfaces are reversibly tuned upon ultraviolet (UV) irradiation and dark storage cycles. This behavior is attributed to the Young's modulus variation of the single fibrils due to the photoisomerization process of the DAE molecules, as measured by advanced atomic force microscopy (AFM) techniques. In fact, a significant yet reversible decrease of the stiffness of the outer part of the fibrils in response to the UV light irradiation is demonstrated. The modification of the molecular structure of the fibrils influences their mechanical properties and affects the frictional behavior of the overall fibrillar surfaces. These findings provide the possibility to develop a system that controllably and accurately generates both low and high friction forces.
Asunto(s)
Etilenos/química , Fricción , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Procesos Fotoquímicos , Propiedades de Superficie , Rayos UltravioletaRESUMEN
The molecular mechanism at the basis of the neurodegenerative process related to Alzheimer's disease (AD) is triggered by the local composition of the neural plasma membrane. The role of cholesterol is controversial. In this investigation the interaction of the AD peptide amyloid-beta (1-42) with model membranes containing lipid rafts has been investigated by atomic force microscopy techniques. Supported lipid membranes made of phospholipids/sphingomyelin/cholesterol have been investigated as a function of the molar content of cholesterol, in a range spanning the phase diagram of the lipid system. The administration of amyloid-beta induced a phase reorganization of the lipid domains, when the cholesterol molar fraction was below 5%. At the same time, a mechanical destabilization and an appreciable thinning of the membrane induced by the peptide were detected. The major interaction was observed in the presence of the gel phase Lß, and was enhanced by a low cholesterol amount. With the appearance of the liquid ordered phase Lo, the effect was hindered. At high cholesterol content (20% mol), no detectable effects in the bilayer morphology or in its mechanical stability were recorded. These findings give new insights on the molecular mechanism of the amyloid/membrane interaction, highlighting the peculiar role of cholesterol.
Asunto(s)
Péptidos beta-Amiloides/química , Colesterol/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Fragmentos de Péptidos/química , Humanos , Microdominios de Membrana/ultraestructura , Microscopía de Fuerza Atómica , Fosfolípidos/química , Esfingomielinas/químicaRESUMEN
Ataxin 1 (ATXN1) is the protein involved in spinocerebellar ataxia type 1, one of nine dominantly inherited neurodegenerative diseases triggered by polyglutamine expansion. One of the isolated polyglutamine tracts properties is to interact with lipid bilayers. Here we used a multidisciplinary approach to test whether one of the mechanisms responsible for neuronal degeneration involves the destabilization of the nuclear membrane. We thus analyzed the interaction between ATXN1 and lipid membranes, both on cellular models and on artificial lipid bilayers, comparing pathological expanded polyglutamine and histidine interrupted non-harmful polyglutamine tracts of the same length. The toxicity of the different constructs was tested in transiently transfected COS1 cells. Cells expressing pathological ATXN1 presented a significantly higher frequency of anomalous nuclei with respect to those expressing non-harmful ATXN1. Immunofluorescence and electron microscopy showed severe damage in the nuclear membrane of cells expressing the pathological protein. Atomic force microscopy on artificial membranes containing interrupted and non-interrupted partial ATXN1 peptides revealed a different arrangement of the peptides within the lipid bilayer. Force-distance measurements indicated that membrane fragility increases with the lengthening of the uninterrupted glutamine. Transmembrane electrical measurements were performed on artificial bilayers and on the inner nuclear membrane of ATXN1 full length transfected cells. Both artificial lipid bilayers and cellular models demonstrated the dynamic appearance of ionic pathways. Uninterrupted polyglutamines showed not only a larger ionic flow, but also an increase in the single event conductance. Collectively, our results suggest that expanded ATXN1 may induce unregulated ionic pathways in the nuclear membrane, causing severe damage to the cell.
Asunto(s)
Núcleo Celular/ultraestructura , Membrana Dobles de Lípidos/análisis , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Ataxinas , Células COS , Chlorocebus aethiops , Histidina/metabolismo , Microscopía de Fuerza Atómica , Péptidos/química , Ataxias Espinocerebelosas/patologíaRESUMEN
We review the advances obtained by using Atomic Force Microscopy (AFM)-based approaches in the field of cell/tissue mechanics and adhesion, comparing the solutions proposed and critically discussing them. AFM offers a wide range of detectable forces with a high force sensitivity, thus allowing a broad class of biological issues to be addressed. Furthermore, it allows for the accurate control of the probe position during the experiments, providing spatially resolved mechanical maps of the biological samples with subcellular resolution. Nowadays, mechanobiology is recognized as a subject of great relevance in biotechnological and biomedical fields. Focusing on the past decade, we discuss the intriguing issues of cellular mechanosensing, i.e., how cells sense and adapt to their mechanical environment. Next, we examine the relationship between cell mechanical properties and pathological states, focusing on cancer and neurodegenerative diseases. We show how AFM has contributed to the characterization of pathological mechanisms and discuss its role in the development of a new class of diagnostic tools that consider cell mechanics as new tumor biomarkers. Finally, we describe the unique ability of AFM to study cell adhesion, working quantitatively and at the single-cell level. Again, we relate cell adhesion experiments to the study of mechanisms directly or secondarily involved in pathologies.
RESUMEN
The surface properties of drug containers should reduce the adsorption of the drug and avoid packaging surface/drug interactions, especially in the case of biologically-derived products. Here, we developed a multi-technique approach that combined Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM), Contact Angle (CA), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoemission Spectroscopy (XPS) to investigate the interactions of rhNGF on different pharma grade polymeric materials. Polypropylene (PP)/polyethylene (PE) copolymers and PP homopolymers, both as spin-coated films and injected molded samples, were evaluated for their degree of crystallinity and adsorption of protein. Our analyses showed that copolymers are characterized by a lower degree of crystallinity and lower roughness compared to PP homopolymers. In line with this, PP/PE copolymers also show higher contact angle values, indicating a lower surface wettability for the rhNGF solution on copolymers than PP homopolymers. Thus, we demonstrated that the chemical composition of the polymeric material and, in turn, its surface roughness determine the interaction with the protein and identified that copolymers may offer an advantage in terms of protein interaction/adsorption. The combined QCM-D and XPS data indicated that protein adsorption is a self-limiting process that passivates the surface after the deposition of roughly one molecular layer, preventing any further protein adsorption in the long term.