RESUMO
Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells ('transcellular' route) or through the inter-endothelial junctions ('paracellular' route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium, a process we term 'tenertaxis'.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Linfócitos/metabolismo , Animais , Fenômenos Biomecânicos , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Leucócitos/metabolismo , Linfócitos/citologia , RatosRESUMO
There is wide anecdotal recognition that biological cell viability and behavior can vary significantly as a function of the source of commercial tissue culture polystyrene (TCPS) culture vessels to which those cells adhere. However, this marked material dependency is typically resolved by selecting and then consistently using the same manufacturer's product - following protocol - rather than by investigating the material properties that may be responsible for such experimental variation. Here, we quantified several physical properties of TCPS surfaces obtained from a wide range of commercial sources and processing steps, through the use of atomic force microscopy (AFM)-based imaging and analysis, goniometry and protein adsorption quantification. We identify qualitative differences in surface features, as well as quantitative differences in surface roughness and wettability that cannot be attributed solely to differences in surface chemistry. We also find significant differences in cell morphology and proliferation among cells cultured on different TCPS surfaces, and resolve a correlation between nanoscale surface roughness and cell proliferation rate for both cell types considered. Interestingly, AFM images of living adherent cells on these nanotextured surfaces demonstrate direct interactions between cellular protrusions and topographically distinct features. These results illustrate and quantify the significant differences in material surface properties among these ubiquitous materials, allowing us to better understand why the dish can make a difference in biological experiments.
Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Materiais Biocompatíveis/química , Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Proliferação de Células , Sobrevivência Celular/fisiologia , Poliestirenos/química , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , Camundongos , Células NIH 3T3 , Propriedades de SuperfícieRESUMO
Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.
Assuntos
Matriz Extracelular/fisiologia , Substâncias Macromoleculares/metabolismo , Células-Tronco Mesenquimais/fisiologia , Interações Microbianas/fisiologia , Engenharia Tecidual/métodos , Western Blotting , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Ficoll , Humanos , Imuno-Histoquímica , Microscopia de Força Atômica , Modelos BiológicosRESUMO
The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5ß1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.
Assuntos
Fibrinogênio/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Hidrogéis/química , Polietilenoglicóis/química , Albuminas/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Masculino , Microscopia de Fluorescência , Ratos , Ratos Endogâmicos F344 , Fator de Crescimento Transformador alfa/metabolismo , Ureia/metabolismoRESUMO
Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Here, we demonstrate a complex consisting of two recombinant proteins, phospholipids and a carbon nanotube that mimics this process. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble upon its removal over an indefinite number of cycles. The assembly is thermodynamically metastable and can only transition reversibly if the rate of surfactant removal exceeds a threshold value. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in an increased photoconversion efficiency of more than 300% over 168 hours and an indefinite extension of the system lifetime.
Assuntos
Eletroquímica , Fotoquímica , Energia Solar , Bicamadas Lipídicas , Nanotubos de Carbono , Fosfolipídeos/química , Proteínas Recombinantes/químicaRESUMO
Pericytes surround capillary endothelial cells and exert contractile forces modulating microvascular tone and endothelial growth. We previously described pericyte contractile phenotype to be Rho GTPase- and α-smooth muscle actin (αSMA)-dependent. However, mechanisms mediating adhesion-dependent shape changes and contractile force transduction remain largely equivocal. We now report that the neutral cysteine protease, calpain, modulates pericyte contractility and cellular stiffness via talin, an integrin-binding and F-actin associating protein. Digital imaging and quantitative analyses of living cells reveal significant perturbations in contractile force transduction detected via deformation of silicone substrata, as well as perturbations of mechanical stiffness in cellular contractile subdomains quantified via atomic force microscope (AFM)-enabled nanoindentation. Pericytes overexpressing GFP-tagged talin show significantly enhanced contractility (~two-fold), which is mitigated when either the calpain-cleavage resistant mutant talin L432G or vinculin are expressed. Moreover, the cell-penetrating, calpain-specific inhibitor termed CALPASTAT reverses talin-enhanced, but not Rho GTP-dependent, contractility. Interestingly, our analysis revealed that CALPASTAT, but not its inactive mutant, alters contractile cell-driven substrata deformations while increasing mechanical stiffness of subcellular contractile regions of these pericytes. Altogether, our results reveal that calpain-dependent cleavage of talin modulates cell contractile dynamics, which in pericytes may prove instrumental in controlling normal capillary function or microvascular pathophysiology.