Regulation and Function of Syk Tyrosine Kinase in Mast Cell Signaling and Beyond
Figure 3
Reconstitution of Syk-mediated actin redistribution in Syk−/− mast cells: C4A2 Syk−/− mast cells were transfected with empty plasmids or plasmids that express wild-type Syk [25]. These cells were cultured with antigen-specific IgE, washed, and then either incubated with medium alone ((a) and (c)) or medium containing antigen. After 10 min, the cells were immunostained as described previously [76] using mAb anti-Syk (26B61A6) and F(ab')2 donkey antimouse IgG conjugated with Alexa 488; actin staining was with phalloidin conjugated with Alexa-633. In nonactivated Syk−/− cells transfected with empty vector (a) or wild-type Syk (c), actin was uniformly distributed on the cell periphery (arrow). After receptor activation, in these cells expressing wild-type Syk ((d), arrow), there was redistribution of actin which follows the ruffled cell membrane, characteristic of activated mast cells; while minimal or no redistribution of actin was observed in cells that did not express Syk ((d), asterisk) or in cells transfected with empty vector (b). In nonstimulated cells expressing wild-type Syk, the Syk was found throughout the cytoplasm ((c), arrow head); after receptor activation, part of the cytoplasmic Syk was recruited to the plasma membrane and colocalized with actin ((d), arrow head). Bar: 10 μm.
