Figure 3.

Btk shuttling/scaffold activity regulates vinculin recruitment and F-actin polymerization at the B cell IS. (A) DIC and fluorescence images of vinculin (green), F-actin (red) and su-Ag (white) for representative IS-forming WT, Xid, and Ibru-B cells, fixed at 10 min; antigen, 20 molec/μm². Values of (B) total F-actin FL and (C) total vinculin FL (in AU) at the IS. (D) DIC and fluorescence images of PIP₂ (green), CellVue (red) and su-Ag (white) for representative WT, Xid and Ibru-B cells, fixed at 2 min; antigen, 20 molec/μm². (E) Quantification of total PIP₂ FL at the B cell-artificial membrane contact plane (left) and of PIP₂ FL/area unit (right). (F) DIC and fluorescence images for Btk (green) at the contact and mid-cell planes for representative WT, Xid and Ibru-B cells. Bottom, ratio of total Btk fluorescence at the contact plane (CP) to that at the mid-cell plane (MP) for each B cell type. (G) DIC and fluorescence images for phosphor-Y223-Btk (p-Btk; green) at the contact and mid-cell planes for representative WT, Xid, and Ibru-B cells. Bottom, quantification of total p-Btk fluorescence at the CP and MP; dotted line, basal p-Btk signal in absence of su-Ag. Each dot represents a single cell in (B,C,E–G). Data from a representative experiment are shown in (B–G) (n = 2). Bar 2 μm. ^**p < 0.01; ^****p < 0.0001 by Student's t-test with WT in each case.