Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

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 minutes precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1associated proteins prevented nuclear volume reduction. Single cell RNA sequencing of differentiating keratinocytes identified gene changes correlated with lamin dispersal, which we propose are due to changes in lamina-associated domains upon Lamin A/C dispersal. 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.

Perspective: Controlling Epidermal Terminal Differentiation with Transcriptional Bursting and RNA Bodies

“…Ablation of these bodies results in reduced epidermal barrier function, suggesting they have a key role in proper epidermal development. Our proposal is that these bodies are a form of biomolecular condensate and could be related to other bodies we have observed in granular keratinocytes group ([ 5 ], Figure 1 ).…”

“…As nuclear export of mRNA is slow compared to transcription, this rate-limiting step effectively acts as a buffer for transcript abundance in polysomes. However, this then presents an interesting idea in keratinocytes as their nuclear lamins are phosphorylated and degraded during nuclear destruction [ 4 , 5 ]. If transcription continues beyond this point, there will be no buffering provided by the nuclear lamina, and protein abundance could be affected also.…”

“…AKT1-phosphorlyated HSP27 stops stabilising cortical actin leading to collapse of the network concomitant with nuclear destruction [ 127 ]. The ARP2/3 complex has been shown to be involved in normal epidermal differentiation of keratinocytes [ 5 , 128 ]. Additionally, experiments have shown that binding partners of the ARP2/3 complex undergo phase transition [ 103 ], suggesting that actin could be a key contributor to condensate formation in terminally differentiating keratinocytes.…”

“…Epidermal differentiation involves nuclear degradation (ND) and the loss of intracellular organelles (adapted from Rogerson et al, 2018). ( B ) pSer404 Lamin A expression (yellow) in the differentiating keratinocytes expressing keratin 10 (magenta) is observed in spherical cytoplasmic bodies [ 4 , 5 ]. ( C ) Expression of these bodies increases over time in confluent culture, peaking at 72 h post-confluency.…”

Phosphoproteomic analysis of the AKT signalling axis in cutaneous squamous carcinoma progression reveals novel therapeutic targets

“…Notably, these were the only changes shared by all three kinases. AKT phosphorylation is altered in cSCC (O’Shaughnessy et al, 2007; Sully et al, 2013), with a switch from AKT1, which is important in epidermal barrier function (O’Shaughnessy et al, JBC, 2007a; Naeem et al,2015; Naeem et al, 2017; Rogerson et al, 2021), to AKT2, which is associated with development and wound healing (Pankow et al ., 2006). AKT phosphorylation was significantly increased in both the Met1 and Met2 lines (Figure 1E and F).…”