A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization.

Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca2+-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins.DOI: http://dx.doi.org/10.7554/eLife.00926.001

In a manner dependent on CD4 T cell help and Toll-like receptor signals, B cells lacking WASp induce autoantibody production and autoimmune disease in mice.

The activation of the B-cell receptor (BCR), which initiates B-cell activation, is triggered by antigen-induced self-aggregation and clustering of receptors at the cell surface. While antigen-induced actin reorganization is known to be involved in BCR clustering in response to membrane-associated antigen, the underlying mechanism that links actin reorganization to BCR activation remains unknown. Here we show that both the stimulatory Bruton’s tyrosine kinase (Btk) and the inhibitory SH2-containing inositol-5 phosphatase-1 (SHIP-1) are required for efficient BCR self-aggregation. In Btk-deficient B cells, the magnitude of BCR aggregation into clusters and B-cell spreading in response to antigen-tethered lipid bilayer is drastically reduced, compared to that observed in wild type B-cells. In SHIP-1−/− B-cells, while surface BCRs aggregate into microclusters, the centripetal movement and growth of BCR clusters are inhibited and B-cell spreading is increased. The persistent BCR microclusters in SHIP−/− B-cells exhibit higher levels of signaling than merged BCR clusters. Contrast to the inhibition of actin remodeling in Btk-deficient B-cells, actin polymerization, F-actin accumulation, and WASP phosphorylation are enhanced in SHIP-1−/− B-cells in a Btk-dependent manner. Thus, a balance between positive and negative signaling regulates the spatiotemporal organization of the BCR at the cell surface by controlling actin remodeling, which potentially regulates the signal transduction of the BCR. This study suggests a novel feedback loop between BCR signaling and the actin cytoskeleton.

A cell biology study using conditional gene knockout mouse models reveals a novel mechanism by which the actin cytoskeleton negatively regulates the signal transduction of the B cell antigen receptor.

B-cells encounter both soluble (sAg) and membrane-associated antigens (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B-cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg- and sAg-stimulated B-cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAg-stimulated B-cells are greater than those in sAg-stimulated B-cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg- and sAg-stimulated B-cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but in much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg.

A clathrin homolog encoded on human chromosome 22 (CHC22) displays distinct biochemistry, distribution and function compared with conventional clathrin heavy chain (CHC17), encoded on chromosome 17. CHC22 protein is upregulated during myoblast differentiation into myotubes and is expressed at high levels in muscle and at low levels in non-muscle cells, relative to CHC17. The trimeric CHC22 protein does not interact with clathrin heavy chain subunits nor bind significantly to clathrin light chains. CHC22 associates with the AP1 and AP3 adaptor complexes but not with AP2. In non-muscle cells, CHC22 localizes to perinuclear vesicular structures, the majority of which are not clathrin coated. Treatments that disrupt the actin-myosin cytoskeleton or affect sorting in the trans-Golgi network (TGN) cause CHC22 redistribution. Overexpression of a subdomain of CHC22 induces altered distribution of TGN markers. Together these results implicate CHC22 in TGN membrane traffic involving the cytoskeleton.

The 17β-estradiol-treated mouse model is the only small animal model of gonococcal genital tract infection. Here we show gonococci localized within vaginal and cervical tissue, including the lamina propria, and high numbers of neutrophils and macrophages in genital tissue from infected mice. Infection did not induce a substantial or sustained increase in total or gonococcal-specific antibodies. Mice could be reinfected with the same strain and repeat infection did not boost the antibody response. However, intravaginal immunization of estradiol-treated mice induced gonococcal-specific primary and secondary serum antibody responses. We conclude that similar to human infection, experimental murine infection induces local inflammation but not an acquired immune response or immunological memory.