PKCα diffusion and translocation are independent of an intact cytoskeleton

Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKC alpha while manipulating the cytoskeleton. Photolytic Ca2+ uncaging allowed us to quantify the kinetics of PKCa redistribution to the plasma membrane when fused to monomeric, dimeric and tetrameric fluorescence proteins. Results indicated that translocation kinetics were modulated by the state of oligomerization as expected for varying Stokes' radii of the participating proteins. Following depolymerization of the microtubules and the actin filaments we found that Ca2+ induced membrane accumulation of PKCa was independent of the filamentous state of the cytoskeleton. Fusion of PKCa to the photo-convertible fluorescent protein Dendra2 enabled the investigation of PKC alpha-cytoskeleton interactions under resting conditions. Redistribution following spatially restricted photoconversion showed that the mobility of the fusion protein was independent of the state of the cytoskeleton. Our data demonstrated that in living cells neither actin filaments nor microtubules contribute to PKC alpha's cytosolic mobility or Ca2+-induced translocation to the plasma membrane. Instead translocation is a solely diffusion-driven process.