Abstract
Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation 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. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.
Highlights
Akt1 is required for Lamin A/C phosphorylation and Akt1 knockdown prevents Lamin A/C degradation and nuclear removal, suggesting Lamin A/C phosphorylation is important for nuclear lamina breakdown in keratinocyte nuclear removal [10]
We determined that Phosphorylated serine 404 (pSer404) Lamin A/C dispersal to cytoplasmic structures occurred in suprabasal, differentiating rat epidermal keratinocytes (REKs) in monolayer culture, (Fig. 1A, B) and calcium switched human keratinocytes, (Fig. 1C), these structures were less developed
Lamin A/C dispersal was reduced in Akt1 knockdown REK organotypic cultures [10], suprabasal Akt1 knockdown REKs had increased nuclear size in post-confluent cultures compared to controls [10], (Fig. 1E–H), and suprabasal differentiating cells are larger and less granular based on flow cytometry side scatter (SSC) measurements, (Fig. 1G–L)
Summary
The mammalian epidermis is an essential barrier between an organism and its environment [1, 2]. Proteins important in autophagic processes are required for removal of nuclear content; dysregulation of autophagyrelated proteins correlates with decreased nuclear degradation and the autophagic marker LC3 localises close to the nucleus in differentiating cells [12, 13]. We identified Akt1-dependent dispersion of nuclear lamina components throughout the cytoplasm in differentiating keratinocytes that precedes nuclear degradation. We isolated a ‘degradosome’ complex of proteins that associate with Akt, including actin and the actinbinding proteins Myh and Arp that are involved in nuclear removal. These results provide evidence for two nuclear degradation intermediates in keratinocyte differentiation and the characterisation of a protein complex important for nuclear degradation regulation
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