B cell receptor (BCR) cluster formation, B cell spreading, and BCR signalsome are reduced and actin polymerization is enhanced in Rictor knockout (KO) B cells.

Splenic B cells were incubated with Alexa Fluor (AF) 546– monobiotinylated (mB)-Fab′–anti- immunoglobulin (Ig) without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for varying lengths of time. After fixation and permeabilization, cells were stained for Alexa Fluor 488 (AF488)-phallodin. Cells were analyzed using confocal microscopy (CFm) and flow cytometry. Shown are representative images at indicated times (A and B) and the average values (±SD) of mean fluorescence intensity (MFI) of phallodin in the cytoplasm and on the plasma membrane at 10 min (C). About 50 cells from 3 independent experiments using NIS-Elements AR 3.2 software and the MFI of phallodin using flow cytometry (D). Splenic B cells from wild-type (WT) and Rictor KO mice were incubated with AF546-mB-Fab′–anti-Ig tethered to lipid bilayers at 37°C for indicated times. Cells were fixed, permeabilized, and stained. Shown are representative images (E and F) and the average values (± SD) of the MFI of filamentous actin (F-actin) (G), the MFI of the BCR (H), B cell contact area (I), and the MFI of phosphorylated Brutons tyrosine kinase pBtk (J) in the contact zone. Total internal reflection fluorescent microscopy (TIRFm) analysis of the spatial relationship of BCR with phosphotyrosine (pY) and pBtk in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-immunoglobulin (Ig). The colocalization coefficients between BCR and pY and pBtk staining were determined using NIS-Elements AR 3.2 software (K). The data were generated using 20–90 cells from 3 independent experiments. Scale bars, 2.5 μm. Mann–Whitney U test was used to do the statistics, *p < 0.01. The numerical data (for C, D, G, H, I, J, and K) can be found in S1 Data.