FIGURE 3.

Immuno-EM demonstrates localization of NaV1.6 to the electron dense podosome structure. The podosome rosette structure was analyzed in epon-embedded samples (A and B), and NaV1.6 and β-actin subcellular localization were analyzed by cryo-immuno-EM (C-E) in differentiated THP-1 that had been treated with m-CSF (50 ng/ml for 5 min) to enhance podosome and podosome rosette formation. Under these conditions, podosome rosettes appear as circular electron dense structures. A, low power view of a cell demonstrates a podosome rosette as an electron dense structure. Note the mitochondria adjacent to the structure. Scale bar, 1 μm. B, a higher power view shows preserved membrane structure within the rosette. Scale bar, 500 nm. C, cryo-immuno-EM revealed intense immuno-gold staining (black dots) for NaV1.6 within the electron dense podosome rosette structure with less intense staining elsewhere in the cell. Scale bar, 1 μm. D, the electron dense podosome structure also demonstrated intense staining for β-actin. Scale bar, 500 nm. E, double labeling for both NaV1.6 (larger 12-nm gold particles) and actin (smaller 6-nm gold particles) revealed co-localization within the electron dense podosome structure (lower left) and in small membrane vesicles (30-80 nm in diameter) outside of the rosette (arrows). Scale bar, 500 nm.