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, e1000604. size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when cluster size increases beyond the size of a single clathrin-coated pit, 4-Aminobenzoic acid B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton. INTRODUCTION B lymphocytes are critical in adaptive immune responses to pathogens (Trombetta and Mellman, 2005 ). Dysregulation of B cell activity can lead to cancer, 4-Aminobenzoic acid autoimmunity, and allergy (Avalos = 0 min. BCR is shown in green and transmitted light is grayscale. (b) Top left region of cell in a; pink arrow tracks BCRs that cluster and internalize into the cytoplasm. (c) Reconstructed superresolution images of the BCR (magenta) clustering on the bottom Mouse monoclonal to AXL surface of DG-75 cells labeled with anti-human IgM F(ab)-Alexa Fluor 647 and left unstimulated or stimulated with anti-human IgM F(ab)2 at either 2 or 8 g/ml F(ab)2 for 15 min prior to plating, fixation, and imaging. (d) Percentage of total fluorescent area in each cell composed of small (<9600 nm2, orange bar), intermediate (9600C48,000 nm2, pink bar), and large (>48,000 nm2, blue bar) punctae for unstimulated DG-75 cells; cells stimulated with 2 g/ml F(ab)2 for 5 or 15 min; and cells stimulated with 8 g/ml F(ab)2 for 5 or 4-Aminobenzoic acid 15 min (= unstimulated 18 cells47,847 spots; 5 min 2 g/ml 10 cells23,728 spots; 5 min 8 g/ml 12 cells10,497 spots; 15 min 2 g/ml 9 cells14,347 spots; 15 min 8 g/ml 19 cells8237 spots). Plots show the mean (square), median (line), 25/75 percentile range (box), and outliers with a coefficient value of 1 1.5 and data points (circles) from each cell. BCR cluster size is dependent on concentration of F(ab)2 stimulation As a first step in BCR uptake, antigen-bound BCRs aggregate on the plasma membrane (Lee = Unstim. 5 cells, 5 min 2 g/ml 9 cells, 5 min 8 g/ml 6 cells, 15 min 2 g/ml 6 cells, and 15 min 8 g/ml 6 cells). Actin is recruited early to BCR clusters stimulated with a high concentration of F(ab)2 To identify proteins that may be important for generation of the novel clathrin on SRM structures, we used TIRF microscopy to study the colocalization of a panel of candidate endocytic proteins (clathrin, actin, endophilins A1 and A2, synaptojanin 2, and FCHSD2) with BCRs when cells were stimulated with 2 or 8 g/ml F(ab)2 (Supplemental Figure S6). Based on our previous data, at the lower concentration of stimulating antigen, BCRs are localized in single CCS, and at the high concentrations of antigen, BCR clusters are predominantly localized in and around clustered clathrin on SRM structures. Other proteins were imaged but here we are focusing on proteins that exhibited differences in localization between the two concentrations. Our data show that both actin and Endophilin A1 were significantly recruited to BCR clusters stimulated with 8 g/ml F(ab)2 at the 15-min time point. Endophilin A1 is thought to be expressed predominantly in neurons (Ringstad = unstimulated 7 cells15,050 spots, 15 min 8 4-Aminobenzoic acid g/ml 10 cells5676 spots, 15 min 8 g/ml.