111 Single cell RNA seq revealed altered cytoskeletal dynamics and the formation of cytoplasmic RNA-Lamin A bodies during keratinocyte terminal differentiation

Abstract

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, requiring degradation of organelles, including the nucleus. However, the underlying molecular mechanisms are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. Using this model, we have shown that the actomyosin network and the non-muscle Myosin IIa - Myh9 are required for key processes involved in nuclear degradation, comprising Akt1-mediated phosphorylation of Lamin A and the dispersal of phosphorylated Lamin A into the cytoplasmic bodies followed by rapid nuclear shrinkage. Based on these finding, we hypothesised that specific changes in actomyosin dynamics and the actin cytoskeleton are required for Lamin dispersal and nuclear shrinkage during epidermal terminal differentiation. We have designed a single-cell transcriptomics pipeline to better analyse these ’sub-optimal’, dying/cornifying cells, and high-throughput confocal imaging to analyse the actin cytoskeleton and related markers during nuclear degradation. We identified changes in expression of actin remodelling genes that coincided with nuclear degradation processes and altered distribution of the actin cytoskeleton in terminally differentiating keratinocytes. We also identified widespread up-regulation of ribosomal protein mRNAs, that correlated with the formation cytoplasmic RNA bodies associating with phosphorylated lamin A. Therefore, actin dynamics, increased translation, and increased cytoplasmic RNA are all potentially involved, and may serve as signposts for key stages in nuclear degradation and end-stage terminal differentiation in keratinocytes.