Live imaging of alterations in cellular morphology and organelles during cornification using an epidermal equivalent model

The stratum corneum plays a crucial role in epidermal barrier function. Various changes occur in granular cells at the uppermost stratum granulosum during cornification. To understand the temporal details of this process, we visualized the cell shape and organelles of cornifying keratinocytes in a living human epidermal equivalent model. Three-dimensional time-lapse imaging with a two-photon microscope revealed that the granular cells did not simply flatten but first temporarily expanded in thickness just before flattening during cornification. Moreover, before expansion, intracellular vesicles abruptly stopped moving, and mitochondria were depolarized. When mitochondrial morphology and quantity were assessed, granular cells with fewer, mostly punctate mitochondria tended to transition to corneocytes. Several minutes after flattening, DNA leakage from the nucleus was visualized. We also observed extension of the cell-flattening time induced by the suppression of filaggrin expression. Overall, we successfully visualized the time-course of cornification, which describes temporal relationships between alterations in the transition from granular cells to corneocytes.

Characteristics of FLG-KD models. a, Quantitative PCR analysis of FLG expression in control and FLG-KD models. The mRNA levels of FLG corresponding to the control were set to 100. All the experimental points were obtained from 8 independent experiments. Wilcoxon rank sum test: p = 0.00015. b, Representative immunostaining images with AKH1 (against the filaggrin monomer, green) and H-300 (against the N-terminal region of filaggrin, red) in addition to Hoechst 33258 staining (blue). c, Representative histological staining. Green arrowheads show nuclear remnants. d, Representative transmission electron microscopy (TEM) images. The red rectangles in the top and the middle panels indicate the regions of the middle and the lower panels, respectively. Red arrowheads show normal corneodesmosomes, and yellow arrowheads show abnormal corneodesmosomes. e, Representative three-dimensional immunostaining against ZO-1 (green) and CDSN (red) in addition to Hoechst 33258 staining (blue) in the uppermost layer. The left and right panels show merged and CDSN images, respectively. f, Quantitative PCR analysis of FLG2, LOR, CDSN, and ABCA12 expression. The mRNA levels corresponding to the control were set to 100. All the experimental points were obtained from at least 7 independent experiments. Wilcoxon rank sum test: p = 0.0093 (FLG2), p = 0.015 (LOR), p = 0.23 (CDSN), p = 0.38 (ABCA12). Scale bar, 50 μm (b), 20 μm (c, e), 4 μm (upper panels in d), 1 μm (middle panels in d), or 400 nm (lower panels in d). p values, * < 0.05, ** < 0.01, *** < 0.001. Images of keratinocytes labelled with TMRE, MTR, and MBG 5 min and 30 min after treatment with a mixture of antimycin A (10 μM) and oligomycin complex (2 μg/mL) to induce mitochondrial depolarization. The TMRE signals almost completely disappeared after the induction of depolarization, while the MTR and MBG signals were slightly visible. Scale bar, 20 μm. Hoechst 33258 (blue) and CTG (green), as shown in Fig. 1a-c. Image size, 160.10 × 160.10 × 63 μm 3 .

Supplementary Movie 2
Representative movie showing the morphological change in the epidermal model labelled with Hoechst 33258 (blue) and CTG (green), as shown in Fig. 1c, d. The video plays at 10 frames per second. Scale bar, 10 μm.

Supplementary Movie 3
Representative movie showing DNA leakage from the nuclear region in the epidermal model labelled with Hoechst 33258 (blue) and CTG (green), as shown in Fig. 1f. The video plays at 10 frames per second. Scale bar, 5 μm.

Supplementary Movie 4
Representative movie showing the degradation of DNA in the epidermal model labelled with Hoechst 33258 (blue) and CTG (green), as shown in Fig. 1g. The video plays at 10 frames per second. Scale bar, 20 μm.

Supplementary Movie 5
Representative movie showing the movement of acidic vesicles and morphological change in the epidermal model labelled with Hoechst 33258 (blue), CTG (green), and LTR (red), as shown in Fig.   2d. The video plays at 10 frames per second. Scale bar, 10 μm.

Supplementary Movie 6
Representative movie showing the disappearance of mitochondrial signals in the epidermal model labelled with Hoechst 33258 (blue), CYTO-ID (green), and TMRE (red), as shown in Fig. 4a. The region designated with a white rectangle on the right is magnified and shown on the left. The video plays at 10 frames per second. Scale bar, 10 μm.

Supplementary Movie 7
Representative movie showing the decrease in mitochondrial signals in the epidermal model labelled with Hoechst 33258 (blue), CYTO-ID (green), and MTR (red), as shown in Fig. 4b. The region designated with a white rectangle on the right is magnified and shown on the left. The video plays at