The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.
Bottom Line: Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility.Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR.Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival.
Affiliation: Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.Show MeSH
Mentions: To assess the functional significance of this ezrin- and actin-mediated restriction in BCR diffusion in steady-state B cells, we monitored intracellular calcium flux by flow cytometry upon treatment of cells with pharmacological agents of actin alteration. Latrunculin A (LatA) treatment is sufficient to induce robust calcium signaling comparable to that induced by crosslinking IgM (Figures 5A–5C). Similar results were obtained upon treatment of cells with the fungal toxin Cytochalasin D (CytoD), which binds to the barbed ends of actin filaments and thus disrupts actin polymerization (Figure 5D). Jasplakinolide (JP), an actin-stabilizing drug, in vitro also induced robust calcium signaling (Figure 5E). To determine whether alteration of the actin cytoskeleton was sufficient to induce other signaling pathways, we examined phosphorylation of downstream signaling molecules including ERK and Akt. We observed a rapid induction of phosphorylation of these substrates upon disruption of the actin cytoskeleton (Figures 5F and 5G). Moreover, we detect upregulation of the activation marker CD86 1 day after a brief treatment of primary naive B cells with actin-disrupting agents (Figure 5H). Taken together, these results indicate that the signaling induced by disruption of the actin cytoskeleton is sufficient to induce not only early signaling events but also expression of costimulatory molecules. These observations suggest that the actin cytoskeleton plays an important role in regulating signaling in B cells.
Affiliation: Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.