212 Regulated mitochondrial fission via NIX and DRP1 in keratinocytes drives epidermal differentiation

Abstract

Epidermal keratinocytes initiate wholesale organelle degradation during cornification, but the mechanisms driving this process are poorly understood. Using live confocal microscopy, we imaged single organelle dynamics in organotypic human epidermis focusing on mitochondria, which are known to regulate keratinocyte differentiation. Mitochondria in the lower epidermis underwent cyclic fission and fusion, but in the upper layers, they became stably fragmented, depolarized, and acidified, leading us to investigate if they were targeted for autophagic degradation. We found differentiating keratinocytes up-regulate the autophagy receptor NIX, which is concentrated on granular layer mitochondria in both organotypic epidermis and human skin biopsies. Thus, we hypothesized NIX could serve as an initiation signal for mitochondrial degradation during cornification. Accordingly, ectopic expression of NIX in undifferentiated keratinocytes was sufficient to induce mitochondrial fragmentation and led to premature differentiation of epidermal cultures. To test the requirement for NIX in epidermal morphogenesis, we used CRISPR/Cas9 gene editing to generate NIX-deficient human organotypic epidermis. NIX-targeted cultures exhibited impaired expression of epidermal differentiation markers and aberrant mitochondrial degradation seen by electron microscopy. Delineating the underlying mechanism, we showed that NIX enhanced mitochondrial localization of FIS1, a receptor for the membrane fission GTPase DRP1. Further, over-expression of FIS1 induced fragmentation of keratinocyte mitochondria similar to NIX. Finally, we showed that compromising DRP1 function directly using a dominant-negative mutant blocked mitochondrial fragmentation in the upper epidermal layers, resulting in abnormal morphology and differentiation of suprabasal keratinocytes. In sum, we report a novel pathway in differentiating keratinocytes driving break-down of mitochondria via the autophagy receptor NIX and DRP1 in order to support proper epidermal morphogenesis.