Figure 1.
Cbl-b is required for normal trafficking to late endosomes.
(A–B) Trafficking to Lamp-1+ and Cathepsin L+ endocytic compartments by the ligated BCR in WT and Cblb−/− splenic B cells. (A) Representative confocal images of BCR stimulated cells (30 min) and (B) quantitative analysis of three independent experiments (30 cells/exp) (*p = 0.0003 and **p = 0.0004). Average Mander's coefficient of BCR vs Lamp-1 for WT cells was 0.556±0.12 vs 0.146±0.07 for Cblb−/− cells (p<0.0001). Average Mander's coefficient of Cathepsin-L vs Lamp-1 for WT cells was 0.415±0.05 vs 0.179±0.03 for Cbl-b−/− cells (p<0.002). (C) Ubiquitination of Igβ in Cblb−/− and WT splenocytes. Unstimulated or BCR stimulated splenocytes were lysed, immunoprecipitated with anti-Igβ antibodies, and then sequentially immunoblotted with anti-Ub (upper panel) or anti-Igβ (lower panel) antibodies. Results are representative of three experiments. (D) Cells were stimulated as in “A” for two minutes, fixed, and visualized by confocal microscopy. Shown are the percentages of each cell population that formed a receptor cap on the cell surface containing more than 50% of visualized BCRs (n = 3 experiments, p = 0.005). (E) Internalization of BCR in Cblb−/− (blue, diamonds) and WT (purple, squares) splenocytes in response to anti-IgM F(ab)2 antibodies. Results are representative of three experiments.
Figure 2.
Cbl-b is recruited to the BCR and co-traffics to late endosomes.
WT splenic B cells were stimulated through the BCR for the indicated times. (A) Representative confocal images demonstrating relative locations of BCR (green), Lamp-1 (red), and Cbl-b (blue) over time. (B) Quantitative analysis (n = 3, 30 cells/exp for each condition and time point) of co-localization over time of BCR and Cbl-b (diamond, red), BCR and Lamp-1 (triangle, light blue), and Cbl-b and Lamp-1 (circle, dark blue).
Figure 3.
BCR endocytic trafficking is dependent on the UBA domain of Cbl-b and independent of its ligase activity.
(A) Schematic representation of different Cbl-b mutants that were used to reconstitute Cblb−/− splenocytes (TKB, tyrosine kinase binding domain; RF, ring finger domain; PRO, proline rich regions; UBA, ubiquitin binding domain). Numbers above schematic refer to amino acid positions. (B) Immunoblots of packaging cell lysates with anti-Cbl-b antibodies demonstrating the relative molecular weights of the indicated expressed Cbl-b mutants. (C) Representative confocal images demonstrating the ability of each indicated Cbl-b mutant to reconstitute BCR endocytic trafficking in Cblb−/− splenocytes. For these experiments, cells were stained with anti-BCR (green), anti-Lamp-1 (red), and anti-Cbl-b antibodies (blue). (D) Quantitative analysis (n = 3, 30 cells/exp for each condition) for co-localization of BCR with Lamp-1 (* statistically similar, p<0.001 for Cbl-b WT vs. N1/2, ΔUBA).
Figure 4.
BCR endocytic trafficking is normal in splenocytes expressing an E3 ligase dead mutant of Cbl-b (CblbC373A).
Indicated splenocytes were stimulated through the BCR and imaged after 30(A) Representative images. (B) Quantitative analysis of three independent experiments (p<0.001 for Cblb−/− vs CblbC373A or WT).
Figure 5.
The UBA domain of Cbl-b, in the context of c-Cbl, is sufficient for BCR endocytic trafficking.
(A) Schematic representation of Cbl-b and c-Cbl encoding constructs used to reconstitute Cblb−/− splenocytes. Percentages represent homology between c-Cbl and Cbl-b in indicated domains. (B) Immunoblot of PlatE cell lysates expressing indicated constructs with anti-c-Cbl antibodies. (C) Representative confocal images of Cblb−/− splenocytes reconstituted with virus encoding c-Cbl or c-CblΔUBA-Cbl-b after stimulation through BCR for 30 minutes (n = 3). (D) Quantitation of experiments provided in (C, BCR co-localization with Lamp-1) across three independent experiments (30 cells/exp) (p<0.001).
Figure 6.
Cbl-b is required for the endocytic transit of TLR9.
(A) Representative confocal microscopic images of splenocytes from mice with indicated genotypes. For experiments, cells were stimulated through the BCR (green) for 30 minutes then fixed and stained for TLR9 (blue) and Lamp-1 (red)(n = 3). (B) Quantitation of experiments shown in (A) for fraction of cells demonstrating significant co-localization of TLR9 with Lamp-1 (n = 3, 30 cells/exp) (Cbl-b−/− vs. CblbC373A or WT, p<0.001). (C) Quantitation of fractions of cells in (A) demonstrating significant co-localization between BCR and TLR9 (n = 3, 30 cells/exp). (D) In vitro assay of T-bet induction in response to ODN 1826 or control ODN targeted through the BCR (n = 3, p<0.01).