Actin Dynamics and Signaling Activation of B Lymphocytes Respond to Substrate Topography

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B cells are activated by the binding of membrane-bound antigen to the B cell receptor (BCR), which induces actin dynamics, reorganization of receptors into signaling microclusters, and cell spreading. In vivo, B cells gather antigen from a variety of sources which may have different physical characteristics such as mobility, stiffness or topography. However, the effect of these physical parameters on BCR clustering and signaling activation is not understood. Here we have studied the role of topography of the stimulating surface on cell spreading, actin polymerization and signaling activation. BCR ligand coated substrates presenting ridges of variable spacing were used to probe the interaction of B cells with non-planar surfaces. Using high-resolution TIRF and confocal microscopy of live cells, we followed the movement of BCR clusters and actin dynamics. We found that small ridge separations induced actin waves that travel parallel to the ridges, resulting in protrusions and retractions of the cell edge. Large ridge separations result in global oscillations of actin intensity in the vicinity of ridges. We further investigated the temporal dynamics of calcium enrichment after antigen engagement in B cells. On flat substrates we measured periodic oscillations of calcium influx with a period of about 30 s, consistent with previously observed values. Interestingly, we found the period of calcium enrichment was dependent on ridge spacing, with increasing time intervals on smaller spacings. Drugs that inhibited actin dynamics slowed down the observed oscillations of calcium. Our results indicate that B cells are sensitive to topographical features, resulting in modulated actin dynamics and that calcium signaling is coupled to substrate-proximal actin dynamics.