RESUMEN
The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here, we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young's modulus of â¼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced myosin-II independent nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading-edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimental observations. Together, these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which, in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness-sensitive processes.
Asunto(s)
Miosina Tipo II/química , Miosina Tipo II/metabolismo , Seudópodos/química , Seudópodos/metabolismo , Animales , Fenómenos Biomecánicos , Adhesión Celular , Movimiento Celular , Matriz Extracelular/metabolismo , Ratones , Células 3T3 NIHRESUMEN
Activating B-Raf mutations that deregulate the MAPK pathway commonly occur in cancer. Whether additional proteins modulate the enzymatic activity of oncogenic B-Raf is unknown. Here we show that the proto-oncogene C-Raf paradoxically inhibits B-Raf(V600E) kinase activity through the formation of B-Raf(V600E)-C-Raf complexes. Although all Raf family members associate with oncogenic B-Raf, this inhibitory effect is specific to C-Raf. Indeed, a B-Raf(V600E) isoform with impaired ability to interact with C-Raf exhibits elevated oncogenic potential. Human melanoma cells expressing B-Raf(V600E) display a reduced C-Raf:B-Raf ratio, and further suppression of C-Raf increases MAPK activation and proliferation. Conversely, ectopic C-Raf expression lowers ERK phosphorylation and proliferation. Moreover, both oncogenic Ras and Sorafenib stabilize B-Raf(V600E)-C-Raf complexes, thereby impairing MAPK activation. This inhibitory function of C-Raf on B-Raf(V600E)-mediated MAPK activation may explain the lack of co-occurrence of B-Raf(V600E) and oncogenic Ras mutations, and influence the successful clinical development of small molecule inhibitors for B-Raf(V600E)-driven cancers.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Bencenosulfonatos/farmacología , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Activación Enzimática , Humanos , Inmunoprecipitación , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Mutación , Células 3T3 NIH , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Fosforilación , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-raf/genética , Piridinas/farmacología , Sorafenib , Transfección , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties. To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces. Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.
Asunto(s)
Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/fisiología , Resinas Acrílicas/química , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas de la Matriz Extracelular/química , Adhesiones Focales/fisiología , Humanos , Microscopía Confocal/métodos , Osteosarcoma/patologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.