Transfection of cells with aplasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations.C ytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence.A ni ncrease in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as af usion protein with mCherry.

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCβ. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCβ from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that -deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.