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Protein kinase Cs (PKCs) are activated by lipids in the plasma membrane and bind to a scaffold assembled on the epidermal growth factor (EGF) receptor (EGFR). Understanding how this complex is routed is important, because this determines whether EGFR is degraded, terminating signaling. Here, cells were preincubated in EGF-tagged gold nanoparticles, then allowed to internalize them in the presence or absence of a phorbol ester PKC activator. PKC colocalized with EGF-tagged nanoparticles within 5 min and migrated with EGFR-bearing vesicles into the cell. Two conformations of PKC-epsilon were distinguished by different primary antibodies. One, thought to be enzymatically active, was on endosomes and displayed a binding site for antibody RR (R&D). The other, recognized by Genetex green (GG), was soluble, on actin-rich structures, and loosely bound to vesicles. During a 15-min chase, EGF-tagged nanoparticles entered large, perinuclear structures. In phorbol ester-treated cells, vesicles bearing EGF-tagged nanoparticles tended to enter this endocytic recycling compartment (ERC) without the GG form. The correlation coefficient between the GG (inactive) and RR conformations on vesicles was also lower. Thus, active PKC has a Charon-like function, ferrying vesicles to the ERC, and inactivation counteracts this function. The advantage conferred on cells by aggregating vesicles in the ERC is unclear.

A computerized morphometric classification technique based on latent factors reveals major protrusion classes: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.