RESUMEN
Human germinal center-associated lymphoma (HGAL) is an adaptor protein specifically expressed in germinal center lymphocytes. High expression of HGAL is a predictor of prolonged survival of diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma. Furthermore, HGAL expression is associated with early-stage DLBCL, thus potentially limiting lymphoma dissemination. In our previous studies, we demonstrated that HGAL regulates B-cell receptor signaling and cell motility in vitro and deciphered some molecular mechanisms underlying these effects. By using novel animal models for in vivo DLBCL dispersion, we demonstrate here that HGAL decreases lymphoma dissemination and prolongs survival. Furthermore, by using an unbiased proteomic approach, we demonstrate that HGAL may interact with multiple cytoskeletal proteins thereby implicating a multiplicity of effects in regulating lymphoma motility and spread. Specifically, we show that HGAL interacts with tubulin, and this interaction may also contribute to HGAL effects on cell motility. These findings recapitulate previous observations in humans, establish the role of HGAL in dissemination of lymphoma in vivo, and explain improved survival of patients with HGAL-expressing lymphomas.
Asunto(s)
Proteínas del Citoesqueleto , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfoma de Células B Grandes Difuso , Proteínas de Microfilamentos/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Humanos , Ratones , Ratones Transgénicos , Proteínas de Neoplasias , ProteómicaRESUMEN
The incidence of malignant melanoma has rapidly increased in the last two decades. There are many challenges associated with the current conventional therapies, including tumour size and location, the specificity of treatments, tumour resistance, non-mutually exclusive mutations, drug resistance, and many adverse side effects. Due to conventional therapies having several limitations, we have explored an alternative therapy such as nano-clays; nano-sized natural materials originating from clay fraction of the soil. Recently, clay nanoparticles have increasingly been used as a drug carrier for cancer treatment due to their high absorption, ability to engulf microbes, and low toxicity. In this study, we evaluated the effects of a nano-clays mix on melanoma cell proliferation and cell viability in vitro and melanoma growth in vivo xenograft animal model. The in vitro study revealed that nano-clay treatments significantly reduced melanoma cell proliferation and cell viability in a dosage-dependent manner. The in vivo tumour xenograft model demonstrated that nano-clay mix treatment led to significantly reduced tumour size and weight, decreased tumour cell mitosis, and induced tumour necrosis. These processes owe to the most probable changes in the membrane potential of the cancer cells once nano-clays bind with the former through the high non-specific adhesion characteristic of the cancer cells. As the data suggest an important role of nano-clays as an inhibitor of melanoma cell proliferation and survival, these prove to be a natural and effective medicine for the treatment of melanoma. The proven compatibility of nano-clays with the human cells with little side-effects makes them a highly preferred choice for the treatment of melanoma and probably other types of cancers.
Asunto(s)
Arcilla/química , Terapias Complementarias/métodos , Melanoma/tratamiento farmacológico , Nanopartículas/administración & dosificación , Neoplasias Cutáneas/tratamiento farmacológico , Administración Cutánea , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Nanopartículas/química , Piel/efectos de los fármacos , Piel/patología , Neoplasias Cutáneas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Regulating B-cell receptor (BCR) signaling after antigenic stimulation is essential to properly control immune responses. Currently known mechanisms of inhibiting BCR signaling are via co-receptor stimulation and downstream immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation. Herein we demonstrate that BCR stimulation induces rapid and reversible palmitoylation of the SCF-FBXO10 ubiquitin E3 ligase. This results in FBXO10 relocation to the cell membrane, where it targets the human germinal center-associated lymphoma (HGAL) protein for ubiquitylation and degradation, leading to decreases in both BCR-induced calcium influx and phosphorylation of proximal BCR effectors. Importantly, FBXO10 recognition and degradation of HGAL is phosphorylation independent and instead relies on a single evolutionarily conserved HGAL amino acid residue (H91) and FBXO10 relocalization to the cytoplasmic membrane. Together our findings demonstrate the first evidence of negative BCR signaling regulation from direct BCR stimulation and define the temporospatial functions of the FBXO10-HGAL axis. FBXO10 is infrequently mutated in DLBCL but some of these mutations deregulate BCR signaling. These observations may have important implications on lymphomagenesis and other immune processes.
Asunto(s)
Proteínas F-Box/metabolismo , Centro Germinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Membrana Celular/metabolismo , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilación/fisiología , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Purpose: To quantify the partition coefficient and the diffusion coefficient of metal-carrier proteins in the human lens capsule as a function of age. Methods: Whole lenses from human donors were incubated overnight in a solution of fluorescently labeled transferrin, albumin, or ceruloplasmin. In the central plane of the capsule thickness, fluorescence recovery after photobleaching (FRAP) experiments were conducted to measure the diffusion of the protein within the lens capsule. The anterior portion of the lens was recorded before the FRAP experiments to locate the boundaries of the anterior lens capsule and to measure the partition coefficient of the labeled proteins. The partition coefficient (P), the time to half maximum recovery of the fluorescent intensity (τ1/2), and the diffusion coefficient (D) for each protein were analyzed as a function of donor age. Results: There was no statistically significant relationship between the half maximum recovery time or the diffusion coefficient and age for transferrin (molecular weight [MW]=79.5 kDa, τ1/2=17.26±4.840 s, D=0.17±0.05 µm2/s), serum albumin (MW=66.5 kDa, τ1/2=18.45±6.110 s, D=0.17±0.06 µm2/s), or ceruloplasmin (MW=120 kDa, τ1/2=36.57±5.660 s, D=0.08±0.01 µm2/s). As expected, the larger protein (ceruloplasmin) took longer to recover fluorescent intensity due to its slower movement within the lens capsule. The partition coefficient statistically significantly increased with age for each protein (Palbumin: 0.09-0.71, Pceruloplasmin: 0.42-0.95, Ptransferrin: 0.19-1.17). Conclusions: The diffusion of heavy-metal protein carriers within the anterior lens capsule is not dependent on age, but it is dependent on the size of the protein. The permeability of the lens capsule to these heavy-metal protein carriers increases with age, suggesting that there will be a higher concentration of heavy metals in the older lens. This behavior may favor the formation of cataract, because heavy metals enhance protein oxidation through the Fenton reaction.
Asunto(s)
Envejecimiento/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo , Cápsula del Cristalino/diagnóstico por imagen , Adulto , Anciano , Albúminas/metabolismo , Ceruloplasmina/metabolismo , Difusión , Humanos , Cápsula del Cristalino/metabolismo , Persona de Mediana Edad , Transferrina/metabolismo , Adulto JovenRESUMEN
Human germinal center (GC)-associated lymphoma (HGAL) is an adaptor protein expressed in GC B cells. HGAL regulates cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. Herein, we demonstrate phosphorylation of HGAL by Syk and Lyn kinases at tyrosines Y80, Y86, Y106Y107, Y128, and Y148. The HGAL YEN motif (amino acids 107-109) is similar to the phosphopeptide motif pYXN used as a binding site to the growth factor receptor-bound protein 2 (Grb2). We demonstrate by biochemical and molecular methodologies that HGAL directly interacts with Grb2. Concordantly, microscopy studies demonstrate HGAL-Grb2 colocalization in the membrane central supramolecular activation clusters (cSMAC) following BCR activation. Mutation of the HGAL putative binding site to Grb2 abrogates the interaction between these proteins. Further, this HGAL mutant localizes exclusively in the peripheral SMAC and decreases the rate and intensity of BCR accumulation in the cSMAC. Furthermore, we demonstrate that Grb2, HGAL, and Syk interact in the same complex, but Grb2 does not modulate the effects of HGAL on Syk kinase activity. Overall, the interplay between the HGAL and Grb2 regulates the magnitude of BCR signaling and synapse formation.
Asunto(s)
Linfocitos B/metabolismo , Proteína Adaptadora GRB2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Linfocitos B/inmunología , Línea Celular , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Quinasa Syk/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
PURPOSE: This study aimed to quantify the three-dimensional micromorphology of the surface of the human lens capsule as a function of age. METHODS: Imaging experiments were conducted on whole human lenses received from eight human cadavers (donor age range: 30-88 years). Imaging was performed with an atomic force microscope (AFM) in contact mode in fluid. The porosity and surface roughness were quantified from the height images obtained. A novel approach, based on stereometric and fractal analysis of three-dimensional surfaces developed for use in conjunction with AFM data, was also used to analyze the surface microtexture as a function of age. RESULTS: The AFM images obtained depict a highly ordered fibrous structure at the surface of the lens capsule, although the overall structure visually changes with age. Porosity and roughness were quantified for each image and analyzed as a function of donor age. The interfibrillar spacing revealed an increasing trend with age, although this result was not significant (p = 0.110). The root mean square (RMS) deviation and average deviation significantly decreased with increasing age (p<0.001 for both). The fractal analysis provided quantitative values for 29 amplitude, hybrid, functional, and spatial parameters. All the hybrid parameters decreased with age, although not significantly. Of the functional parameters, the surface bearing index increased significantly with age (p = 0.017) and the summit height exhibited a decreasing trend with age (p = 0.298). Of the spatial parameters, the dominant radial wavelength trend moved toward an increase with age (p = 0.103) and the cross-hatch angle tended toward a decrease with age (p = 0.213). CONCLUSIONS: Significant changes in the three-dimensional surface microtexture of the human lens capsule were found with age, although more experiments on a larger dataset are needed to conclude this with certainty. The analyzed AFM images demonstrate a fractal nature of the surface, which is not considered in classical surface statistical parameters. The surface fractal dimension may be useful in ophthalmology for quantifying human lens architectural changes associated with different disease states to further our understanding of disease evolution.
Asunto(s)
Imagenología Tridimensional/métodos , Cápsula del Cristalino/ultraestructura , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Cadáver , Femenino , Fractales , Humanos , Imagenología Tridimensional/instrumentación , Cápsula del Cristalino/anatomía & histología , Cápsula del Cristalino/diagnóstico por imagen , Masculino , Microscopía de Fuerza Atómica/métodos , Persona de Mediana EdadRESUMEN
GLI1 oncogene has been implicated in the pathobiology of several neoplasms including diffuse large B-cell lymphoma (DLBCL). However, mechanisms underlying GLI1-increased activity in DLBCL are poorly characterized. Herein, we demonstrate that IKKß phosphorylates GLI1 in DLBCL. IKKß activation increased GLI1 protein levels and transcriptional activity, whereas IKKß silencing decreased GLI1 levels and transcriptional activity. Tumor necrosis factor-α (TNFα) mediated IKKß activation-impaired GLI1 binding with the E3 ubiquitin ligase-ITCH, leading to decreased K48-linked ubiquitination/degradation of GLI1. We found 8 IKKß-dependent phosphorylation sites that mediate GLI1 stability. Mutating or deleting these residues facilitated GLI1-ITCH interaction and decreased the protective effect of TNFα on GLI1 stability. IKKß-GLI1 crosstalk is significant because combined inhibition of both molecules resulted in synergistic suppression of DLBCL viability in vivo and in vitro. By linking IKKß-mediated nuclear factor-κB activity with GLI1, we identified a crosstalk between these 2 pathways that can inform the design of novel therapeutic strategies in DLBCL.
Asunto(s)
Quinasa I-kappa B/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Humanos , Linfoma de Células B Grandes Difuso/genética , FN-kappa B/metabolismo , Fosforilación , Estabilidad Proteica , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteína con Dedos de Zinc GLI1RESUMEN
PURPOSE: To image the ultrastructure of the anterior lens capsule at the nanoscale level using atomic force microscopy (AFM). METHODS: Experiments were performed on anterior lens capsules maintained in their in situ location surrounding the lens from six human cadavers (donor age range: 44-88 years), four cynomolgus monkeys (Macaca fascicularis age range: 4.83-8.92 years), and seven pigs (<6 months). Hydration of all samples was maintained using Dulbecco's Modified Eagle Medium (DMEM). Whole lenses were removed from the eye and placed anterior side up in agarose gel before gel hardening where only the posterior half of the lens was contained within the gel. After the gel hardened, the Petri dish was filled with DMEM until the point where the intact lens was fully submerged. AFM was used to image the anterior lens surface in contact mode. An integrated analysis program was used to calculate the interfibrillar spacing, fiber diameter, and surface roughness of the samples. RESULTS: The AFM images depict a highly ordered fibrous structure at the surface of the lens capsule in all three species. The interfibrillar spacing for the porcine, cynomolgus monkey, and human lens capsules was 0.68±0.25, 1.80±0.39, and 1.08±0.25 µm, respectively. In the primate, interfibrillar spacing significantly decreased linearly as a function of age. The fiber diameters ranged from 50 to 950 nm. Comparison of the root mean square (RMS) and average deviation demonstrate that the surface of the porcine lens capsule is the smoothest, and that the human and cynomolgus monkey capsules are significantly rougher. CONCLUSIONS: AFM was successful in providing high-resolution images of the nanostructure of the lens capsule samples. Species-dependent differences were observed in the overall structure and surface roughness.
Asunto(s)
Cápsula del Cristalino/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Animales , Humanos , Macaca fascicularis , Microscopía de Fuerza Atómica , Persona de Mediana Edad , Especificidad de la Especie , Propiedades de Superficie , PorcinosRESUMEN
The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand-binding domain, the I-domain, of integrin αMß2. Single-molecule force measurements of αM I-domain-iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I-domain via its C-terminus resulted in a 1.3-fold to 1.5-fold increase in unbinding force compared with I-domains immobilized via the N-terminus. The force spectra (unbinding force versus loading rate) of the I-domain-iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4-fold increase in the lifetime of the I-domain-iC3b bond. Given the structural and functional similarity of all integrin I-domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C-terminus of the I-domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration.
Asunto(s)
Complemento C3b/química , Proteínas Inmovilizadas/química , Antígeno de Macrófago-1/química , Humanos , Ligandos , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Human germinal center-associated lymphoma (HGAL) is specifically expressed only in germinal center (GC) B lymphocytes and GC-derived lymphomas. HGAL protein decreases lymphocyte motility by inhibiting the ability of myosin to translocate actin via direct interaction with F-actin and myosin II and by activating RhoA signaling via direct interactions with RhoA-specific guanine nucleotide exchange factors. HGAL protein also regulates B-cell receptor (BCR) signaling by directly binding to and enhancing Syk kinase activity and activation of its downstream effectors. Herein we demonstrate that HGAL protein can be myristoylated and palmitoylated and that these modifications localize HGAL to cellular membrane raft microdomains with distinct consequences for BCR signaling and chemoattractant-induced cell mobility. In BCR signaling, raft localization of HGAL facilitates interaction with Syk and modulation of the BCR activation and signaling, which induces HGAL phosphorylation and redistribution from lipid raft to bulk membrane and cytoplasm, followed by degradation. In contrast, HGAL myristoylation and palmitoylation avert its inhibitory effects on chemoattractant-induced cell motility. These findings further elucidate the growing and complex role of HGAL in B-cell biology and suggest that membrane-bound and cytoplasmic HGAL protein differently regulates distinct biological processes.
Asunto(s)
Linfocitos B/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/fisiología , Actinas/genética , Actinas/inmunología , Actinas/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Factores Quimiotácticos/genética , Factores Quimiotácticos/inmunología , Factores Quimiotácticos/metabolismo , Citoplasma/genética , Citoplasma/inmunología , Citoplasma/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoilación/genética , Lipoilación/inmunología , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , Proteínas de Microfilamentos , Miosina Tipo II/genética , Miosina Tipo II/inmunología , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Transporte de Proteínas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Quinasa Syk , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/inmunología , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Inflammatory conditions induce redistribution of junctional adhesion receptors toward the apical regions of endothelial cells promoting lymphocyte TEM. Much of the molecular structures of TEM have been revealed; however, the biophysical mechanisms underlying this process remain to be fully elucidated. Here, we used immunofluorescence microscopy and AFM to study endothelial distribution of adhesion molecules upon lymphocyte activation and transmigration. Our immunofluorescence results revealed redistribution of JAM-A and PECAM-1 but not ICAM-1 or VCAM-1 toward the apical junctional regions of HUVECs following a 6-h stimulation with TNF-α and IFN-γ. Consistently, our SCFS studies revealed that Jurkat cell adhesion to stimulated HUVEC monolayers was significantly greater in junctional regions. Enhanced adhesion was mediated mostly by JAM-A receptors. Further AFM adhesion mapping of the homophilic JAM-A/JAM-A interaction on the surfaces of HUVECs revealed a greater number of JAM-A receptors available for binding along junctional regions after TNF-α and IFN-γ stimulation. Our data reveal for the first time that adhesion "hot spots" of JAM-A receptors are involved in initiating lymphocyte TEM under inflammatory conditions.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/citología , Uniones Intercelulares/metabolismo , Interferón gamma/farmacología , Linfocitos/citología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Uniones Intercelulares/efectos de los fármacos , Células Jurkat , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Microscopía de Fuerza Atómica , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores de Superficie Celular/metabolismo , Análisis EspectralRESUMEN
Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn(2+) additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1(-/-) neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn(2+)-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn(2+) induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.
Asunto(s)
Benzoatos/farmacología , Membrana Celular/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno de Macrófago-1/metabolismo , Tiohidantoínas/farmacología , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos , Animales , Benzoatos/química , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/química , Leucocitos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/química , Manganeso/metabolismo , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Neutrófilos/metabolismo , Unión Proteica , Tiohidantoínas/químicaRESUMEN
The α4ß1 integrin facilitates both monocyte rolling and adhesion to the vascular endothelium and is physiologically activated by monocyte chemoattractant protein (MCP-1). The current study investigated the initial events in the adhesion of THP-1 cells to immobilized Vascular Cell Adhesion Molecule 1 (VCAM-1). Using AFM force measurements, cell adhesion was shown to be mediated by two populations of α4ß1/VCAM-1 complexes. A low affinity form of α4ß1 was anchored to the elastic elements of the cytoskeleton, while a higher affinity conformer was coupled to the viscous elements of the cell membrane. Within 100 ms of contact, THP-1 cells, stimulated by co-immobilized MCP-1, exhibited a tremendous increase in adhesion to VCAM-1. Enhanced cell adhesion was accompanied by a local decoupling of the cell membrane from the cytoskeleton and the formation of long membrane tethers. The tethers were individually anchored by multiple α4ß1/VCAM-1 complexes that prolonged the extension of the viscous tethers. In vivo, the formation of these membrane tethers may provide the quantal structural units for the arrest of rolling monocytes within the blood vessels.
Asunto(s)
Adhesión Celular/fisiología , Membrana Celular/metabolismo , Endotelio Vascular/fisiología , Monocitos/fisiología , Complejos Multiproteicos/metabolismo , Línea Celular Tumoral , Humanos , Integrina alfa4beta1/metabolismo , Microscopía de Fuerza Atómica , Estadísticas no Paramétricas , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Cells are constantly under the influence of various external forces in their physiological environment. These forces are countered by the viscoelastic properties of the cytoskeleton. To understand the response of the cytoskeleton to biochemical and mechanical stimuli, GFP-tubulin expressing CHO cells were investigated using scanning laser confocal microscopy. Cells treated with nocodazole revealed disruption in the microtubule network within minutes of treatment while keeping the cell shape intact. By contrast, trypsin, a proteolytic agent, altered the shape of CHO cells by breaking the peptide bonds at adhesion sites. CHO cells were also stimulated mechanically by applying an indentation force with an atomic force microscope (AFM) and by shear stress in a parallel plate flow chamber. Mechanical stimulation applied using AFM showed two distinct cytoskeletal responses to the applied force: an immediate response that resulted in the depolymerization and displacement of the microtubules out of the contact zone, and a slower response characterized by tubulin polymerization at the periphery of the indented area. Flow chamber experiments revealed that shear force did not induce formation of new microtubules in CHO cells and that detachment of adherent cells from the substrate occurred independent from the flow direction. Overall, the experimental system described here allows real-time characterization of dynamic changes in cell cytoskeleton in response to the mechano-chemical stimuli and, therefore, provides better understanding of the biophysical and functional properties of cells.
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Microtúbulos/metabolismo , Animales , Fenómenos Biomecánicos , Células CHO , Cricetinae , Cricetulus , Módulo de Elasticidad , Humanos , Mecanotransducción Celular , Microscopía de Fuerza Atómica , Microscopía Confocal , Microtúbulos/ultraestructura , Proteolisis , Tripsina/químicaRESUMEN
PURPOSE: To quantify the cut quality of lamellar dissections made with the femtosecond laser using atomic force microscopy (AFM). SETTING: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA. DESIGN: Experimental study. METHODS: Experiments were performed on 3 pairs of human cadaver eyes. The cornea was thinned to physiologic levels by placing the globe, cornea side down, in 25% dextran for 24 hours. The eyes were reinflated to normal pressures by injecting a balanced salt solution into the vitreous cavity. The eyes were placed in a holder, the epithelium was removed, and the eyes were cut with a Visumax femtosecond laser. The energy level was 180 nJ for the right eye and 340 nJ for the left eye of each pair. The cut depths were 200 µm, 300 µm, and 400 µm, with the cut depth maintained for both eyes of each pair. A 12.0 mm trephination was then performed. The anterior portion of the lamellar surface was placed in a balanced salt solution and imaged with AFM. As a control, the posterior surface was placed in 2% formalin and imaged with environmental scanning electron microscopy (SEM). Four quantitative parameters (root-mean-square deviation, average deviation, skewness, kurtosis) were calculated from the AFM images. RESULTS: From AFM, the 300 µm low-energy cuts were the smoothest. Similar results were seen qualitatively in the environmental SEM images. CONCLUSION: Atomic force microscopy provided quantitative information on the quality of lamellar dissections made using a femtosecond laser, which is useful in optimizing patient outcomes in refractive and lamellar keratoplasty surgeries.
Asunto(s)
Sustancia Propia/cirugía , Sustancia Propia/ultraestructura , Disección/métodos , Terapia por Láser/métodos , Microscopía de Fuerza Atómica , Anciano , Anciano de 80 o más Años , Cadáver , Trasplante de Córnea , Humanos , Microscopía Electrónica de Rastreo , Proyectos Piloto , Donantes de TejidosRESUMEN
Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.
Asunto(s)
Péptidos/química , Streptococcus pyogenes/metabolismo , Adhesinas Bacterianas/metabolismo , Amidas/química , Biofisica/métodos , Membrana Celular/metabolismo , Fibronectinas/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica/métodos , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray/métodos , TemperaturaRESUMEN
Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.
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Microscopía de Fuerza Atómica/métodos , Polietilenglicoles/química , Proteínas/química , Sefarosa/química , Albúmina Sérica Bovina/química , Adsorción , Vidrio/química , Microesferas , Propiedades de SuperficieRESUMEN
Protein-protein interactions are the basis of both biochemical and biophysical signaling of living cells. In many cases, the receptor is present on the cell surface while the ligand is in solution or linked to another support (extracellular matrix or another cell). In the case of cellular adhesion, forces are continuously applied to receptor-ligand complexes and, as a consequence, the dissociation kinetics of the bonds may change. It is, thus, relevant to study the kinetics of protein-protein interactions in response to applied forces, as this is the most physiologically relevant situation. The atomic force microscope (AFM) was one of the first nanotools to be applied to this end. However, new approaches need to be developed to better understand the complex energy landscape of molecular interactions under applied stress. In this chapter, we described the use of the AFM to carry out force-clamp measurements on receptor-ligand bonds. Force-clamp measurements on bonds consist of applying a constant and controlled force to a receptor-ligand bond and measure the resulting dissociation lifetime. The described methods include the required materials, functionalization of tips and substrates, force-clamping measurements, and processing and interpretation of the results. An illustrative example is given with the well-studied streptavidin-biotin complex.
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Biotina/metabolismo , Microscopía de Fuerza Atómica/métodos , Estreptavidina/metabolismo , Biotina/química , Cinética , Ligandos , Unión Proteica , Pliegue de Proteína , Estreptavidina/química , Propiedades de SuperficieRESUMEN
The purpose of this project is to measure the elasticity of the human and non-human primate lens capsule at the microscopic scale using Atomic Force Microscopy (AFM). Elasticity measurements were performed using AFM on the excised anterior lens capsule from 9 cynomolgus monkey (5.9-8.0 years), 8 hamadryas baboon (2.8-10.1 years), and 18 human lenses (33-79 years). Anterior capsule specimens were obtained by performing a 5 mm continuous curvilinear capsulorhexis and collecting the resulting disk of capsular tissue. To remove the lens epithelial cells the specimen was soaked in 0.1% trypsin and 0.02% EDTA for 5 min, washed, and placed on a Petri dish and immersed in DMEM. Elasticity measurements of the capsule were performed with a laboratory-built AFM system custom designed for force measurements of ophthalmic tissues. The capsular specimens were probed with an AFM cantilever tip to produce force-indentation curves for each specimen. Young's modulus was calculated from the force-indentation curves using the model of Sneddon for a conical indenter. Young's modulus of elasticity was 20.1-131 kPa for the human lens capsule, 9.19-117 kPa for the cynomolgus lens capsule, and 13.1-62.4 kPa for the baboon lens capsule. Young's modulus increased significantly with age in humans (p = 0.03). The age range of the monkey and baboon samples was not sufficient to justify an analysis of age dependence. The capsule elasticity of young humans (<45 years) was not statistically different from that of the monkey and baboon. In humans, there is an increase in lens capsule stiffness at the microscale that could be responsible for an increase in lens capsule bulk stiffness.
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Envejecimiento/fisiología , Elasticidad/fisiología , Cápsula del Cristalino/fisiología , Microscopía de Fuerza Atómica , Adulto , Anciano , Animales , Capsulorrexis , Módulo de Elasticidad , Tejido Elástico , Humanos , Macaca fascicularis , Persona de Mediana Edad , PapioRESUMEN
In response to external stimuli, cells modulate their adhesive state by regulating the number and intrinsic affinity of receptor/ligand bonds. A number of studies have shown that cell adhesion is dramatically reduced at room or lower temperatures as compared with physiological temperature. However, the underlying mechanism that modulates adhesion is still unclear. Here, we investigated the adhesion of the monocytic cell line THP-1 to a surface coated with intercellular adhesion molecule-1 (ICAM-1) as a function of temperature. THP-1 cells express the integrin lymphocyte function-associated antigen-1 (LFA-1), a receptor for ICAM-1. Direct force measurements of cell adhesion and cell elasticity were carried out by atomic force microscopy. Force measurements revealed an increase of the work of de-adhesion with temperature that was coupled to a gradual decrease in cellular stiffness. Of interest, single-molecule measurements revealed that the rupture force of the LFA-1/ICAM-1 complex decreased with temperature. A detailed analysis of the force curves indicated that temperature-modulated cell adhesion was mainly due to the enhanced ability of cells to deform and to form a greater number of longer membrane tethers at physiological temperatures. Together, these results emphasize the importance of cell mechanics and membrane-cytoskeleton interaction on the modulation of cell adhesion.