RESUMEN
The integrity of epithelia is maintained within dynamic mechanical environments during tissue development and homeostasis. Understanding how epithelial cells mechanosignal and respond collectively or individually is critical to providing insight into developmental and (patho)physiological processes. Yet, inferring or mimicking mechanical forces and downstream mechanical signaling as they occur in epithelia presents unique challenges. A variety of in vitro approaches have been used to dissect the role of mechanics in regulating epithelia organization. Here, we review approaches and results from research into how epithelial cells communicate through mechanical cues to maintain tissue organization and integrity. We summarize the unique advantages and disadvantages of various reduced-order model systems to guide researchers in choosing appropriate experimental systems. These model systems include 3D, 2D, and 1D micromanipulation methods, single cell studies, and noninvasive force inference and measurement techniques. We also highlight a number of in silico biophysical models that are informed by in vitro and in vivo observations. Together, a combination of theoretical and experimental models will aid future experiment designs and provide predictive insight into mechanically driven behaviors of epithelial dynamics.
RESUMEN
Protein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. Measuring force displacement characteristics of holey carbon EM-grids, we found that their effective spring constant is similar to physiological values expected from skin tissues. Despite their apparent smoothness at light-microscopy resolution, spatial boundaries of the protein micropatterns are irregular at nanometer scale. Our protein micropatterning workflow provides the means to steer both positioning and morphology of cell doublets to determine nanometer details of punctate adherens junctions. Our workflow serves as the foundation for studying the fundamental structural changes governing cell-cell signaling.
Asunto(s)
Procesamiento de Imagen Asistido por Computador , Proteínas , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía por Crioelectrón/métodos , Carbono/química , Transducción de SeñalRESUMEN
We designed and fabricated, using low-cost 3D printing technologies, a device that enables direct control of cell density in epithelial monolayers. The device operates by varying the tension of a silicone substrate upon which the cells are adhered. Multiple devices can be manufactured easily and placed in any standard incubator. This allows long-term culturing of cells on pretensioned substrates until the user decreases the tension, thereby inducing compressive forces in plane and subsequent instantaneous cell crowding. Moreover, the low-profile device is completely portable and can be mounted directly onto an inverted optical microscope. This enables visualization of the morphology and dynamics of living cells in stretched or compressed conditions using a wide range of high-resolution microscopy techniques.
Asunto(s)
Fenómenos Biomecánicos/fisiología , Técnicas de Cultivo de Célula/métodos , Microscopía/métodos , Impresión Tridimensional/instrumentación , Animales , Perros , Diseño de Equipo , Células de Riñón Canino Madin DarbyRESUMEN
The use of bacteriophages (phages) for antibacterial therapy is under increasing consideration to treat antimicrobial-resistant infections. Phages have evolved multiple mechanisms to target their bacterial hosts, such as high-affinity, environmentally hardy receptor-binding proteins. However, traditional phage therapy suffers from multiple challenges stemming from the use of an exponentially replicating, evolving entity whose biology is not fully characterized (e.g., potential gene transduction). To address this problem, we conjugate the phages to gold nanorods, creating a reagent that can be destroyed upon use (termed "phanorods"). Chimeric phages were engineered to attach specifically to several Gram-negative organisms, including the human pathogens Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae, and the plant pathogen Xanthomonas campestris The bioconjugated phanorods could selectively target and kill specific bacterial cells using photothermal ablation. Following excitation by near-infrared light, gold nanorods release energy through nonradiative decay pathways, locally generating heat that efficiently kills targeted bacterial cells. Specificity was highlighted in the context of a P. aeruginosa biofilm, in which phanorod irradiation killed bacterial cells while causing minimal damage to epithelial cells. Local temperature and viscosity measurements revealed highly localized and selective ablation of the bacteria. Irradiation of the phanorods also destroyed the phages, preventing replication and reducing potential risks of traditional phage therapy while enabling control over dosing. The phanorod strategy integrates the highly evolved targeting strategies of phages with the photothermal properties of gold nanorods, creating a well-controlled platform for systematic killing of bacterial cells.
Asunto(s)
Antibacterianos/administración & dosificación , Bacteriófagos/fisiología , Oro/química , Hipertermia Inducida , Nanotubos/química , Terapia de Fagos/métodos , Infecciones por Pseudomonas/terapia , Animales , Perros , Farmacorresistencia Bacteriana Múltiple , Humanos , Rayos Infrarrojos , Células de Riñón Canino Madin Darby , Nanopartículas del Metal/química , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiologíaRESUMEN
Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established in vitro cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.