A signaling network is revealed that combines positive and negative feedback to control the activity of the small GTPase Rho in adherent cells. This network generates spontaneous pulses of Rho activity and actomyosin contraction that are modulated by extracellular elasticity.
Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn−/− mice (Lyn−/− BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn−/− BMMCs release more β-hexosaminidase than wild-type BMMCs following FcεRI cross-linking and show that multiple mast cell responses to FcεRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cγ isoforms, the mobilization of Ca2+, the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the α4β1 integrin, VLA-4) are slow to initiate in Lyn−/− BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn−/− BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn−/− BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn−/− BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcεRI signaling in Lyn−/− BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn−/− BMMCs may additionally contribute to their altered signaling properties.
We describe a finite-element model of mast cell calcium dynamics that incorporates the endoplasmic reticulum's complex geometry. The model is built upon a three-dimensional reconstruction of the endoplasmic reticulum (ER) from an electron tomographic tilt series. Tetrahedral meshes provide volumetric representations of the ER lumen, ER membrane, cytoplasm, and plasma membrane. The reaction-diffusion model simultaneously tracks changes in cytoplasmic and ER intraluminal calcium concentrations and includes luminal and cytoplasmic protein buffers. Transport fluxes via PMCA, SERCA, ER leakage, and Type II IP3 receptors are also represented. Unique features of the model include stochastic behavior of IP3 receptor calcium channels and comparisons of channel open times when diffusely distributed or aggregated in clusters on the ER surface. Simulations show that IP3R channels in close proximity modulate activity of their neighbors through local Ca2+ feedback effects. Cytoplasmic calcium levels rise higher, and ER luminal calcium concentrations drop lower, after IP3-mediated release from receptors in the diffuse configuration. Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations.
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