451 Human epidermal differentiation requires autophagy and lysosomal trafficking

Abstract

The regulation of the pH is an important element in various biological functions. The pH distribution of the stratum corneum (SC) is considered as the gradient decreasing along surface in general while precise SC pH distribution and its dynamic changes under different environmental conditions remain to be clarified. The purpose of this study is to produce a transgenic mouse as a tool to visualize the intact SC pH in vivo. We generated a ratiometric pH-biosensor with pH-sensitive fluorescent protein, Venus H148G fused with pH-insensitive protein, mCherry (Venus H148G-mCherry), expressed specifically in the uppermost layer of stratum granulosum (SG1) using knock-in construct of SASPase gene by modified CRISPR/Cas9 system. Noninvasive confocal microscopic analysis of SC pH in living Venus H148G-mCherry mouse ear skin observed with dry objective demonstrated that upper SC is neutral compared to middle SC. Immersion of water onto SC surface enabled highresolution deeper range SC pH imaging and revealed that the SC and SG1 layer are divided into three zones with distinct pH properties rather than gradual changes across the layers. Namely, the SG1 was neutral, the middle SC was acidic, and neutral pH was again observed in the upper SC. Analysis of the 3D pH distribution of whole SC showed that the upper neutral SC zone distributed unevenly by differentiated units, and the acidic middle SC was wellmaintained. When ear skin was exposed to phosphate buffers with various pH (pH5.4, 6.6 and 7.4) by topical application, the pH of upper SC dynamically changed according to the pH applied, while the middle SC maintained its acidic pH. These findings indicated that the SC is a highly organized tissue with tightly-regulated pH layers having the distinct functions, and that the 3D in vivo pH imaging mice will provide a valuable tool to dissect the homeostatic mechanisms of SC by pH regulation.