Rap1 activation is important for MTOC polarization towards APCs. (A–D) LPS-stimulated primary B cells that were transduced with control siRNA or with Rap1a and Rap1b siRNAs were stained with CellTrace Far Red (pseudocolored blue) and mixed with APCs expressing anti-Igκ (antigen). B-cell–APC conjugates were stained for antigen and α-tubulin. Representative images of B cells that were mixed with APCs for 20 min (A). Arrows indicate the MTOC. Scale bar: 5 µm. PIs for >53 B-cell–APC conjugates from four experiments (B; quantified as in Fig. S1F). ****P<0.0001. C and D show the PI values and percentage of cells with a PI≤0.75 for the full timecourse (mean±s.e.m.; four experiments each with >45 conjugates per point). For APC experiments, 37.5% of cells with a PI≤0.75 would be random MTOC distribution. *P<0.05, **P<0.01, ***P<0.001 compared to control siRNA cells at the same time point. (E–G) Vector control and RapGAPII-expressing A20 cells were mixed with anti-Igκ-expressing APCs and then stained for α-tubulin. For A20 cells mixed with APCs for 30 min, PIs were calculated for >47 B-cell–APC conjugates from three experiments (E). ****P<0.0001. F and G shows the full timecourse (mean±s.e.m.; three experiments, each with >35 conjugates per point). *P<0.05, **P<0.01, ***P<0.001 compared to control cells at the same time point.

Cofilin controls BCR-induced MTOC polarization. (A,B) Primary B cells were treated with DMSO or 2 μM latrunculin A (Lat A) for 5 min, mixed with 4.5-µm anti-IgM-coated beads, and then stained for pericentrin. Representative confocal xy slices overlaid on DIC images (A). For the 30 min time point, PIs and the percentage of cells with a PI≤0.75 (∼20% would be random; see Table S1) were calculated for >156 conjugates from three experiments (B). (C,D) A20 cells expressing either WT or cofilin S3D fused to mCherry were mixed with anti-IgG-coated beads for 30 min. Representative confocal images of α-tubulin and F-actin staining (C). PIs for >79 cells from three experiments (D). (E,F) Primary B cells were treated with the control Q peptide (5 μM) or the M and W cofilin-inhibitory peptides (5 μM each) and then mixed with anti-IgM-coated beads for 30 min. Representative images of pericentrin staining (E). PIs for >43 cells from three experiments (F). (G–I) A20 cells were transduced with control siRNA or cofilin siRNA. Blot shows cofilin knockdown (G). The cells were mixed with anti-IgG-coated beads for 30 min, then stained for pericentrin and for the anti-IgG on the beads. Representative confocal images are shown (H; the dotted line is the outline of the cell) along with PIs for >29 cells. ****P<0.0001. (J,K) RapGAPII-expressing A20 cells transfected with WT cofilin or the constitutively active cofilin S3A fused to mCherry were mixed with anti-IgG-coated beads for 30 min. Representative confocal images of α-tubulin and F-actin staining (J). PIs for >51 cells from three experiments (K). ***P<0.001, ****P<0.0001. Scale bars: 5 μm.

Cofilin-mediated actin reorganization is required for the MTOC to approach the plasma membrane. (A,B) A20 cells were treated with 5 μM of the control Q peptide (A) or with 5 μM each of the M and W cofilin-blocking peptides and then allowed to spread on anti-IgG-coated coverslips for 15 min. Cells were stained for α-tubulin and F-actin and imaged by TIRFM with a 100-nm depth. Fluorescence intensity profiles along the dotted lines are plotted along with the percentage of cells in which the MTOC was in the TIRF plane (B) (mean±s.e.m.; >41 cells per condition in each of three experiments). **P<0.01. (C–E) A20 cells transduced with control siRNA or cofilin siRNA were allowed to spread on anti-IgG-coated coverslips for 15–60 min and imaged by TIRFM with a 100-nm depth. Images of cells at the 15 min time point are shown (C). The percentage of cells with the MTOC within the TIRF plane (D) and the percentage of cells that exhibited a peripheral F-actin ring surrounding a central actin-depleted region (E) are shown for each time point. (F) A20 cells expressing GFP–α-tubulin and F-tractin-tdTomato were treated with control (Q) or cofilin-inhibitory (M and W) peptides, added to anti-Ig-coated coverslips, and imaged in real time for 8 min by confocal microscopy. The kymographs represent a time series of images for the confocal slice closest to the coverslip taken along the white line every 10 s. Scale bars: 10 μm.

IQGAP1 and CLIP-170 are required for BCR-induced MTOC reorientation. (A,B) A20 cells were treated with DMSO, 10 µM EHNA or 5 µM nocodazole for 30 min and then mixed with anti-Ig-coated beads for 30 min. Representative confocal images of pericentrin and DAPI staining (A) are shown along with MTOC PIs for >30 cells (B). (C,D) A20 cells were treated with DMSO, 10 µM EHNA or 20 µM ciliobrevin D for 40 min and then mixed for 30 min with beads that had been coated with Alexa Fluor 647-conjugated anti-IgG. Representative xy confocal slices of pericentrin-stained cells (C, upper panels; dotted circles indicate the periphery of the cell) are shown along with 3D reconstructions of cells that had been stained for F-actin (C, lower panels). MTOC PIs for >33 cells (D). (E–I) A20 cells were transduced with lentiviruses containing the empty pGipZ vector, IQGAP1 shRNAs or CLIP-170 shRNA. Blots show IQGAP1 (E) and CLIP-170 (F) expression. The cells were mixed with anti-IgG-coated beads for 30 min and stained for pericentrin (G). Graphs show MTOC PIs for control versus IQGAP1 shRNA-expressing cells (H; >127 cells from three experiments) or control versus CLIP-170 shRNA-expressing cells (I; >126 cells from three experiments). The percentage of cells with a PI≤0.75 is indicated (∼20% would be random distribution; see Table S1). (J,K) Vector control, IQGAP1 shRNA and CLIP-170 shRNA cells were stained with CMFDA and then mixed with anti-Igκ-expressing APCs for 30 min. xy confocal slices of cells stained for α-tubulin and antigen (H). White arrows indicate the MTOC. For each cell population, MTOC PIs were quantified for >81 cells from three experiments (I). The percentage of cells with a PI≤0.75 is indicated (37.5% would be a random distribution). ****P<0.0001. Scale bars: 5 μm.

Colocalization of IQGAP1, CLIP-170, and F-actin. B cells that had been added to anti-IgG-coated coverslips for 15 min were imaged by STED microscopy. (A) A20 cells that had been transfected with CLIP-170–GFP were stained with Rhodamine–phalloidin and with anti-α-tubulin antibody plus an Alexa Fluor 532-conjugated secondary antibody. CLIP-170–GFP fluorescence was imaged directly. An enlarged merged image is shown along with the individual channels. Scale bars: 10 µm. (B) A20 cells were stained with Rhodamine–phalloidin, anti-IQGAP1 antibody plus an Alexa 532-conjugated secondary antibody, and anti-α-tubulin plus an Alexa 488-conjugated secondary antibody. Scale bar: 5 μm. (C) A20 cells expressing CLIP-170–GFP were stained with Rhodamine–phalloidin and with anti-IQGAP1 plus an Alexa Fluor 532-conjugated secondary antibody. CLIP-170–GFP fluorescence was imaged directly. A 7× enlargement of the area in the white box is shown. All images are representative of multiple cells.

Rap1 promotes IQGAP1 accumulation at the IS by controlling actin organization. (A) A20 cells were treated with DMSO or 2 μM latrunculin A for 5 min, then mixed with anti-IgG-coated beads for 5 min. Cells were stained for IQGAP1, α-tubulin and F-actin. Confocal xy slices of bead–cell conjugates are shown. Dotted circles indicate the bead. (B–D) Vector control and RapGAPII-expressing A20 cells were mixed with anti-IgG-coated beads for 5 min, then stained for IQGAP1 and F-actin. For each conjugate, the corrected fluorescence intensity of F-actin (C) and IQGAP1 (D) within the white circle was quantified (in arbitrary units, AU) for >65 cells from two experiments. (E) LPS-activated primary B cells transfected with control siRNA or with Rap1a and Rap1b siRNAs were mixed with anti-Igκ-expressing APCs for 5 min and then stained for IQGAP1, F-actin and antigen. Arrows show the contact site between the B cell and the APC. (F–I) Vector control and RapGAPII-expressing A20 cells were allowed to spread on anti-IgG-coated coverslips for 15 min, then stained for IQGAP1 and F-actin and imaged by TIRFM or confocal microscopy. TIRFM images (F) and fluorescence profiles along the dotted lines are shown for representative cells (G). Confocal images were used to calculate Pearson's (H) and Manders' (I) coefficients for colocalization of IQGAP1 and F-actin. ****P<0.0001. Scale bars: 5 μm.