Mutant SWAP-70 Y517F inhibits B-cell polarization in vivo. (A) Wt or Swap-70^−/− B cells were infected with retroviruses expressing GFP, SWAP-70, and GFP or SWAP-70, Y517F, and GFP. After 72 hours, the infected GFP-positive cells were sorted by FACS and then allowed to attach to anti-CD44 coated surfaces. The attached cells were stained with rhodamine-phallodin and images collected by fluorescence microscopy as shown. Representative images from one of 3 independent experiments performed in triplicate. (B) The number of cells with a particular morphology was quantified for each of the experiments. Polarized cells were defined as having one dendrite equal in length to the width of the cell body. Irregular cells were those cells that had several protrusions and or dendrites and whose shape was no longer largely round. Bars show mean ± SEM, and significance was calculated by the use of an unpaired t test. (C) Cells infected with either SWAP-70 or SWAP-70 Y517F expressing retrovirus were labeled with cell tracker red and combined with cells infected with GFP-expressing retrovirus labeled with cell tracker green. Two hours after the cells were injected into the tail vein of Wt mice, lymph nodes and blood were collected and the percentage of B cells determined by FACS analysis of anti-B220 stained samples. The ratio of SWAP-70 infected or SWAP-70Y517F infected to control cells infected with GFP retrovirus was calculated and plotted (data from 5 independent experiments). The value 1 represents the input ratio, and numbers lower than 1 show there are more control cells. The P value represents the comparison between the SWAP-70/GFP ratio to the SWAP-70 Y517F/GFP ratio.