CCR2 deficiency boosts F-actin accumulation and BCR
internalization
Actin remodeling has been shown to play a crucial role during the
process of BCR clustering and formation of B cell morphological
features.6 To examine
the involvement of CCR2 in actin remodeling and polymerization, we
assessed F-actin accumulation and the activation of actin nucleation
promoting factor-WASP in CCR2 KO B cells after sAg stimulation using
confocal microscopy (CFm). The F-actin levels observed in activated CCR2
deficient B cells were consistently higher than those of the control B
cells (Fig. 4A-B ). The colocalization between F-actin and BCR
was significantly increased after 5 min of stimulation (Fig.
4C ). Similarly, the pWASP levels in activated CCR2 deficient B cells
remained higher than those in WT B cells (Fig. 4D ).
Furthermore, after 5 min of stimulation, the colocalization of pWASP and
BCR in CCR2 KO B cells was enhanced (Fig. 4E ). Additionally,
phosflow was performed to measure the pWASP and F-actin levels which
coincided with the CFm results. Examining the dynamics of actin
polymerization, we found that the mean fluorescence intensity (MFI) of
F-actin in CCR2 KO B cells increased slower during the first 5 min of
stimulation, after which it accelerated rapidly and peaked at 10
min
(Fig. 4F ). The MFI of pWASP raised rapidly during the first 5
min of stimulation and declined thereafter (Fig. 4G ). Further,
the Mst1-Dock8-WASP axis protein expression levels were examined using
western blotting. In the absence of CCR2, the levels of Dock8, pMst1,
and pWASP were increased (Fig. 4H ). To get clearer image of the
B cell membrane morphology, we utilized scanning electron microscopy
(SEM) to observe B cell morphology and quantify their filopodia
(Fig. 4I ). Compared to the WT B cells, the filopodia number and
length were remarkably increased in CCR2 KO B cells (Fig.
4J-K ). Furthermore, in order to illustrate the latent mechanism through
which CCR2 regulates the BCR signaling via the Mst1/Dock8/WASP axis, we
pretreated CCR2 KO B cells with the Mst1 inhibitor, XMU-MP-1, and
reexamined the BCR signaling molecules upon sAg stimulation. Remarkably,
the levels of Dock8, pMst1, pSHIP, pBtk, pPI3K, pAKT, pFoxo-1, pmTOR,
and pWASP in CCR2 KO B cells decreased following XMU-MP-1 treatment
(Fig. 4L ). Subsequently, to verify whether the inhibition of
mTORC1 in CCR2 KO B cells could also adjust the Dock8/WASP axis, we
pretreated CCR2 KO B cells with rapamycin. After the inhibition, the
levels of Dock8, pMst1, and pWASP in CCR2 KO B cells were rescued to a
comparable degree to WT B cells (Fig. 4M ). Taken together, CCR2
deletion augments actin accumulation, which is achieved through the
modulation of the Dock8-WASP activity, and the latter, at least in part,
implemented via the Mst1-mTORC1 pathway.