When lymphocytes encounter APCs bearing cognate Ag, they spread across the surface of the APC to scan for additional Ags. This is followed by membrane contraction and the formation of Ag receptor microclusters that initiate the signaling reactions that lead to lymphocyte activation. Breakdown of the submembrane cytoskeleton is likely to be required for the cytoskeleton reorganization that drives cell spreading and for removing physical barriers that limit Ag receptor mobility. In this report, we show that Ag receptor signaling via the Rap GTPases promotes the dephosphorylation and activation of the actin-severing protein cofilin and that this results in increased severing of cellular actin filaments. Moreover, we show that this cofilin-mediated actin severing is critical for the changes in actin dynamics that drive B and T cell spreading, for the formation of BCR microclusters, and for the increased mobility of BCR microclusters within the plasma membrane after BCR engagement. Finally, using a model APC, we show that activation of this Rap–cofilin signaling module controls the amount of Ag that is gathered into BCR microclusters and that this is directly related to the magnitude of the resulting BCR signaling that is initiated during B cell–APC interactions. Thus, Rap-dependent activation of cofilin is critical for the early cytoskeletal changes and BCR reorganization that are involved in APC-dependent lymphocyte activation.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

B lymphocytes spread and extend membrane processes when searching for antigens and form immune synapses upon contacting cells that display antigens on their surface. Although these dynamic morphological changes facilitate B cell activation, the signaling pathways underlying these processes are not fully understood. We found that activation of the Rap GTPases was essential for these changes in B cell morphology. Rap activation was important for B cell receptor (BCR)- and lymphocyte-function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune-synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane-process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens but not soluble antigens. Thus by controlling B cell morphology and cytoskeletal organization, Rap might play a key role in the activation of B cells by particulate and cell-associated antigens.

SummaryThe gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells and forms intercellular channels for the transfer of small molecules between adjacent cells, as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 regulates cell adhesion and migration in neurons and glioma cells, and we now show that Cx43 influences BCR-, LFA-1-and CXCL12-mediated activation of the Rap1 GTPase. Using shRNA knockdown of Cx43 in WEHI 231 cells, we show that Cx43 is required for sustained Rap1 activation and BCR-mediated spreading. To determine the domains of Cx43 that are important for this effect, Cx43-null J558 m3 B cells (which express a wild-type IgM BCR) were transfected with wildtype Cx43-GFP or a C-terminal-truncated Cx43 (Cx43T-GFP). Expression of wild-type Cx43-GFP, but not Cx43T-GFP, was sufficient to restore sustained, BCR-mediated Rap1 activation and cell spreading. Cx43, and specifically the C-terminal domain, was also important for LFA-1-and CXCL12-mediated Rap1 activation, spreading and adhesion to an endothelial cell monolayer. These data show that Cx43 has an important and previously unreported role in B-cell processes that are essential to normal B-cell development and immune responses.

B cells that bind antigens displayed on antigen-presenting cells (APCs) form an immune synapse, a polarized cellular structure that optimizes the dual functions of the B cell receptor (BCR), signal transduction and antigen internalization. Immune synapse formation involves polarization of the microtubule-organizing center (MTOC) towards the APC. We now show that BCR-induced MTOC polarization requires the Rap1 GTPase (which has two isoforms, Rap1a and Rap1b), an evolutionarily conserved regulator of cell polarity, as well as cofilin-1, an actin-severing protein that is regulated by Rap1. MTOC reorientation towards the antigen contact site correlated strongly with cofilin-1-dependent actin reorganization and cell spreading. We also show that BCR-induced MTOC polarization requires the dynein motor protein as well as IQGAP1, a scaffolding protein that can link the actin and microtubule cytoskeletons. At the periphery of the immune synapse, IQGAP1 associates closely with Factin structures and with the microtubule plus-end-binding protein CLIP-170 (also known as CLIP1). Moreover, the accumulation of IQGAP1 at the antigen contact site depends on F-actin reorganization that is controlled by Rap1 and cofilin-1. Thus the Rap1-cofilin-1 pathway coordinates actin and microtubule organization at the immune synapse.