Figure 7.

PLCγ2 and Vav act in concert in the coordination of B cell spreading in response to membrane-bound antigen. (A–E) B cells were settled onto bilayers containing anti-IgM as antigen (Ag; red) and were analyzed after 3 min. (A and B) PLCγ2-KO DT40 B cells expressing Vav-GFP (green). (A) TIRFM images. A relative fluorescence intensity plot to indicate the distribution of Vav-GFP and antigen is depicted by the diagonal dashed line. Bar, 5 μm. (B) Quantification of Vav-GFP–containing microsignalosomes. Data are representative of two experiments, and the number of antigen microclusters containing Vav-GFP was measured in 15–20 cells of each type, representing a total of 934 microsignalosomes. Mean numbers in WT and PLCγ2-KO are 36 ± 3 and 18 ± 2, respectively. ***, P < 0.001. (C and D) Vav3-KO DT40 B cells expressing PLCγ2-GFP (green). (C) TIRFM images. A relative fluorescence intensity plot to indicate the distribution of PLCγ2-GFP and antigen is depicted by the diagonal dashed line. Bar, 5 μm. (D) Quantification of PLCγ2-GFP–containing microsignalosomes. Data are representative of two experiments, and the number of microsignalosomes containing PLCγ2-GFP was measured in 18–23 cells of each cell type, representing a total of 1,061 microsignalosomes. Mean numbers in WT and Vav3-KO are 32 ± 3 and 22 ± 6, respectively. **, P < 0.005. (E) Quantification of (left) Vav-GFP–containing antigen microclusters in WT and PLCγ2-KO DT40 B cells and (right) PLCγ2-GFP–containing antigen microclusters in WT and Vav3-KO DT40 B cells, expressed as a percentage of total antigen microclusters present. Data are representative of two experiments, and the percentage of antigen microclusters was measured in 15–23 cells of each cell type, representing a total of 1,476–1,811 antigen microclusters. (left) Mean percentages in WT and PLCγ2-KO are 62 ± 3 and 43 ± 2, respectively. ***, P < 0.001. (right) Mean percentages in WT and Vav3-KO are 78 ± 2 and 64 ± 3, respectively. ***, P = 0.0001.