Figure 1. Keratinocyte mitochondria undergo fragmentation, depolarization, and acidification in the upper layers of organotypic epidermis.

(A) NHEKs with labeled mitochondria (green cells) progressively differentiate as they stratify (arrow) to form organotypic epidermis, imaged by SDC microscopy.

(B) SDC images of Mito-dsRed in lower versus upper layers of epidermal cultures.

(C and D) Mitochondrial perimeter and (D) area quantification in cells relative to their z-distance from the top of the tissue (mean ± SD, n = 60 fields, 4 experiments (expts.), ***p < 0.0001).

(E) SDC images of TOM20-mCh-labeled mitochondria in the upper versus lower layers of organotypic epidermis.

(F) SDC images of GFP-labeled mitochondria and TMRE dye in transitional layers containing cells with or without fragmented mitochondria.

(G) Quantification of relative TMRE intensity in pairs of cells having fragmented versus non-fragmented mitochondria (mean ± SD, n = 24 cell pairs, 4 expts., ***p < 0.0001).

(H) The tandem fluorophores GFP-mCh are imported into mitochondria by the Cox8 targeting sequence. Under neutral pH, both fluorophores are active, whereas acidic pH quenches GFP upon routing to lysosomes (LYSO).

(I) SDC images of organotypic epidermis expressing Cox8-GFP-mCh in upper versus lower layers.

(J) TEM image of the upper layers of organotypic epidermis with spherical mitochondrial fragments within a second membrane (arrowheads), indicative of mitophagy.

White scale bars, 10 μm; black scale bar, 500 nm.