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.

Analysis of three different Rab-RabGEF pairs reveals that RabGEFs contain the minimal targeting machinery for recruiting Rabs to specific membranes.

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.

No abstract

Diffusion parameters of the extracellular space (ECS) are changed in many brain pathologies, disturbing synaptic as well as extrasynaptic “volume” transmission, which is based on the diffusion of neuroactive substances in the ECS. Amyloid deposition, neuronal loss, and disturbed synaptic transmission are considered to be the main causes of Alzheimer's disease dementia. We studied diffusion parameters in the cerebral cortex of transgenic APP23 mice, which develop a pathology similar to Alzheimer's disease. The real-time tetramethylammonium (TMA) method and diffusion-weighted MRI were used to measure the ECS volume fraction (α = ECS volume/total tissue volume) and the apparent diffusion coefficients (ADCs) of TMA (ADC TMA ), diffusing exclusively in the ECS and of water (ADC W ). Measurements were performed in vivo in 6-, 8-, and 17- to 25-month-old hemizygous APP23 male and female mice and age-matched controls. In all 6- to 8-month-old APP23 mice, the mean ECS volume fraction, ADC TMA , and ADC W were not significantly different from age-matched controls (α = 0.20 ± 0.01; ADC TMA , 580 ± 16 μm 2 ·s -1 ; ADC W , 618 ± 19 μm 2 ·s -1 ). Aging in 17- to 25-month-old controls was accompanied by a decrease in ECS volume fraction and ADC W , significantly greater in females than in males, but no changes in ADC TMA . ECS volume fraction increased (0.22 ± 0.01) and ADC TMA decreased (560 ± 7 μm 2 ·s -1 ) in aged APP23 mice. The impaired navigation observed in these animals in the Morris water maze correlated with their plaque load, which was twice as high in females (20%) as in males (10%) and may, together with changed ECS diffusion properties, account for the impaired extrasynaptic transmission and spatial cognition observed in old transgenic females.

We provide evidence for anisotropic diffusion in rat corpus callosum and hippocampus. The preferential diffusion pathway in corpus callosum is along the myelinated axon fibres; in the hippocampus diffusion is easier along the transversal axis (x) than along the sagittal (y) or vertical (z) axes. In all areas studied, i.e. in the cortex, corpus callosum and hippocampus, the mean ECS volume fraction alpha (alpha = ECS volume/total tissue volume) ranged between 0.20 and 0.22 and mean non-specific uptake k' was between 4.0 and 5.9 x 10(-3) s-1. Diffusional anisotropy in the hippocampus may be of importance for extrasynaptic transmission and in the 'cross-talk' between synapses.

The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (l) and the volume fraction (a). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of l and a were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA + ). In normal medium (305 mosmol kg _1 ), the average value of l was 1.69 and of a was 0.24. Reducing osmolality to 150 mosmol kg _1 , increased l to 1.86 and decreased a to 0.12. Increasing osmolality to 350 mosmol kg _1 , reduced l to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg _1 . In contrast, a increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M r dextran to measure l, showed the same behaviour as for TMA + , including the same constant l in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that l and a behave differently as the ECS geometry varies. When a decreases, l increases but when a increases, l rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of l.