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We hereby present a novel "grafting-to"-like approach for the covalent attachment of plasmonic nanoparticles (PNPs) onto whispering gallery mode (WGM) silica microresonators. Mechanically stable optoplasmonic microresonators were employed for sensing single-particle and single-molecule interactions in real time, allowing for the differentiation between binding and non-binding events. An approximated value of the activation energy for the silanization reaction occurring during the "grafting-to" approach was obtained using the Arrhenius equation; the results agree with available values from both bulk experiments and ab initio calculations. The "grafting-to" method combined with the functionalization of the plasmonic nanoparticle with appropriate receptors, such as single-stranded DNA, provides a robust platform for probing specific single-molecule interactions under biologically relevant conditions.
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Cells exert traction forces on the extracellular matrix to which they are adhered through the formation of focal adhesions. Spatial-temporal regulation of traction forces is crucial in cell adhesion, migration, cellular division, and remodeling of the extracellular matrix. By cultivating cells on polyacrylamide hydrogels of different stiffness we were able to investigate the effects of substrate stiffness on the generation of cellular traction forces by Traction Force Microscopy (TFM), and characterize the molecular dynamics of the focal adhesion protein zyxin by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). As the rigidity of the substrate increases, we observed an increment of both, cellular traction generation and zyxin residence time at the focal adhesions, while its diffusion would not be altered. Moreover, we found a positive correlation between the traction forces exerted by cells and the residence time of zyxin at the substrate elasticities studied. We found that this correlation persists at the subcellular level, even if there is no variation in substrate stiffness, revealing that focal adhesions that exert greater traction present longer residence time for zyxin, i.e., zyxin protein has less probability to dissociate from the focal adhesion.
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Estresse Mecânico , Zixina/química , Citoesqueleto de Actina/efeitos dos fármacos , Amidas/farmacologia , Animais , Bovinos , Adesão Celular , Citocalasina D/farmacologia , Células Endoteliais , Recuperação de Fluorescência Após Fotodegradação , Adesões Focais , Proteínas de Fluorescência Verde , Microscopia Intravital , Cinética , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Piridinas/farmacologia , Proteínas Recombinantes de Fusão/química , Vinculina/química , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
This work reports on a novel and versatile approach to control the structure of metal-organic framework (MOFs) films by using polymeric brushes as 3D primers, suitable for triggering heterogeneous MOF nucleation. As a proof-of-concept, this work explores the use of poly(1-vinylimidazole) brushes primer obtained via surface-initiated atom transfer radical polymerization (SI-ATRP) for the synthesis of Zn-based ZIF-8 MOF films. By modifying the grafting density of the brushes, smooth porous films were obtained featuring inherently hydrophobic microporosity arising from ZIF-8 structure, and an additional constructional interparticle mesoporosity, which can be employed for differential adsorption of targeted adsorbates. It was found that the grafting density modulates the constructional porosity of the films obtained; higher grafting densities result in more compact structures, while lower grafting density generates increasingly inhomogeneous films with a higher proportion of interparticle constructional porosity.
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Layer by layer assembly of polyelectrolytes with proteins is a convenient tool for the development of functional biomaterials. Most of the studies presented in the literature are based on the electrostatic interaction between components of opposite charges, limiting the assembly possibilities. However, this process can be tuned by modifying the environment where the main constituents are dissolved. In this work, the electron transfer behavior between an electroactive polyelectrolyte (polyallylamine derivatized with an osmium complex) and a redox enzyme (glucose oxidase) is studied by assembling them in the presence of phosphate ions at different ionic strengths. Our results show that the environment from which the assembly is constructed has a significant effect on the electrochemical response. Notably, the polyelectrolyte dissolved in the presence of phosphate at high ionic strength presents a globular structure which is preserved after adsorption with substantial effects on the buildup of the multilayer system, improving the electron transfer process through the film.
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Supramolecular self-assembly of molecular building blocks represents a powerful "nanoarchitectonic" tool to create new functional materials with molecular-level feature control. Here, we propose a simple method to create tunable phosphate/polyamine-based films on surfaces by successive assembly of poly(allylamine hydrochloride) (PAH)/phosphate anions (Pi) supramolecular networks. The growth of the films showed a great linearity and regularity with the number of steps. The coating thickness can be easily modulated by the bulk concentration of PAH and the deposition cycles. The PAH/Pi networks showed chemical stability between pH 4 and 10. The transport properties of the surface assemblies formed from different deposition cycles were evaluated electrochemically by using different redox probes in aqueous solution. The results revealed that either highly permeable films or efficient anion transport selectivity can be created by simply varying the concentration of PAH. This experimental evidence indicates that this new strategy of supramolecular self-assembly can be useful for the rational construction of single polyelectrolyte nanoarchitectures with multiple functionalities.
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The modulation of cell adhesion via biologically inspired materials plays a key role in the development of realistic platforms to envisage not only mechanistic descriptions of many physiological and pathological processes but also new biointerfacial designs compatible with the requirements of biomedical devices. In this work, we show that the cell adhesion and proliferation of three different cell lines can be easily manipulated by using a novel biologically inspired supramolecular coating generated via dip coating of the working substrates in an aqueous solution of polyallylamine in the presence of phosphate anions-a simple one-step modification procedure. Our results reveal that selective cell adhesion can be controlled by varying the deposition time of the coating. Cell proliferation experiments showed a cell type-dependent quasi-exponential growth demonstrating the nontoxic properties of the supramolecular platform. After reaching a certain surface coverage, the supramolecular films based on phosphate-polyamine networks displayed antiadhesive activity towards cells, irrespective of the cell type. However and most interestingly, these antiadherent substrates developed strong adhesive properties after thermal annealing at 37 °C for 3 days. These results were interpreted based on the changes in the coating hydrophilicity, topography and stiffness, with the latter being assessed by atomic force microscopy imaging and indentation experiments. The reported approach is simple, robust and flexible, and would offer opportunities for the development of tunable, biocompatible interfacial architectures to control cell attachment for various biomedical applications.
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Materiais Biocompatíveis/química , Substâncias Macromoleculares/química , Fosfatos/química , Poliaminas/química , Células 3T3 , Absorção Fisiológica , Animais , Materiais Biocompatíveis/síntese química , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Células HeLa , Humanos , Cinética , Substâncias Macromoleculares/síntese química , Camundongos , Microscopia de Força Atômica , Tamanho da Partícula , MolhabilidadeRESUMO
Mechanical stimuli play a key role in many cell functions such as proliferation, differentiation and migration. In the mammary gland, mechanical signals such as the distension of mammary epithelial cells due to udder filling are proposed to be directly involved during lactation and involution. However, the evolution of focal adhesions -specialized multiprotein complexes that mechanically connect cells with the extracellular matrix- during the mammary gland development, as well as the influence of the mechanical stimuli involved, remains unclear. Here we present the use of an equibiaxial stretching device for exerting a sustained normal strain to mammary epithelial cells while quantitatively assessing cell responses by fluorescence imaging techniques. Using this approach, we explored changes in focal adhesion dynamics in HC11 mammary cells in response to a mechanical sustained stress, which resembles the physiological stimuli. We studied the relationship between a global stress and focal adhesion assembly/disassembly, observing an enhanced persistency of focal adhesions under strain as well as an increase in their size. At a molecular level, we evaluated the mechanoresponses of vinculin and zyxin, two focal adhesion proteins postulated as mechanosensors, observing an increment in vinculin molecular tension and a slower zyxin dynamics while increasing the applied normal strain.
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Células Epiteliais/metabolismo , Adesões Focais/metabolismo , Imageamento Tridimensional , Glândulas Mamárias Animais/citologia , Mecanotransdução Celular , Estresse Mecânico , Animais , Sobrevivência Celular , Feminino , Fluorescência , Recuperação de Fluorescência Após Fotodegradação , Cinética , Camundongos Endogâmicos BALB C , Fibras de Estresse/metabolismo , Vinculina/metabolismo , Zixina/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.
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Actinas/metabolismo , Axônios/patologia , Membrana Celular/metabolismo , Cones de Crescimento/patologia , Plasticidade Neuronal , Degeneração Retrógrada , Espectrina/metabolismo , Citoesqueleto de Actina , Animais , Axônios/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Ratos , Ratos WistarRESUMO
Responsive interfacial architectures of practical interest commonly require the combination of conflicting properties in terms of their demand upon material structure. Switchable stiffness, wettability, and permeability, key features for tissue engineering applications, are in fact known to be exclusively interdependent. Here, we present a nanoarchitectonic approach that decouples these divergent properties by the use of thermoresponsive microgels as building blocks for the construction of three-dimensional arrays of interconnected pores. Layer-by-layer assembled poly( N-isopropylacrylamide- co-methacrylic acid) microgel films were found to exhibit an increase in hydrophobicity, stiffness, and adhesion properties upon switching the temperature from below to above the lower critical solution temperature, whereas the permeability of redox probes through the film remained unchanged. Our findings indicate that the switch in hydrophilicity and nanomechanical properties undergone by the microgels does not compromise the porosity of the film, thus allowing the free diffusion of redox probes through the polymer-free volume of the submicrometer pores. This novel approach for decoupling conflicting properties provides a strategic route for creating tailorable scaffolds with unforeseen functionalities.
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The possibility of combining more than one stimulus-responsive property into a single material holds interesting potential for the creation of adaptive devices to be used in diverse fields such as drug delivery, nanomedicine and tissue engineering. This paper describes a novel material based on thermo-responsive PNIPAm nanopillars with amplified surface properties through the incorporation of Fe3O4 nanoparticles. The incorporation of magnetic nanoparticles into the nanopillars, prepared via surface-initiated atom-transfer radical polymerization in anodized aluminum oxide templates, sharply increased their stiffness and hydrophobicity when increasing the temperature above the volume phase transition temperature. Furthermore, their magnetic response turned out to be proportional to the amount of the incorporated nanoparticles. The possibility of sharply increasing the stiffness with a temperature variation close to the human body temperature paves the way to the application of these substrates as "smart" scaffolds for cell culture. Additionally, the presence of superparamagnetic nanoparticles in the nanopillars offers the possibility of using these nanostructured systems for magnetic hyperthermia.
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The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction.
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Adesões Focais , Mecanotransdução Celular , Proteínas/química , Fenômenos Fisiológicos CelularesRESUMO
Optical printing has been proved a versatile and simple method to fabricate arbitrary arrays of colloidal nanoparticles (NPs) on substrates. Here, we show that is also a powerful tool for studying chemical reactions at the single NP level. We demonstrate that 60 nm gold NPs immobilized by optical printing can be used as seeds to obtain larger NPs by plasmon-assisted reduction of aqueous HAuCl4. The final size of each NP is simply controlled by the irradiation time. Moreover, we show conditions for which the growth occurs preferentially in the direction of light polarization, enabling the in situ anisotropic reshaping of the NPs in predetermined orientations.
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Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.
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Processamento Alternativo , Histona-Lisina N-Metiltransferase/genética , Animais , Azepinas/farmacologia , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Éxons , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Células HeLa , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinazolinas/farmacologia , Interferência de RNA , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tretinoína/farmacologiaRESUMO
During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.