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Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not to other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.