Biophysical Regulation of Nuclear Architecture in Human Keratinocytes.
Abstract
Epidermal homeostasis is an essential process in the maintenance of healthy skin, and it is regulated by the fine tuning of the proliferation and terminal differentiation of keratinocytes. Various biochemical and biophysical cues regulate the balance between growth and differentiation within the epidermis, and limited adhesion to the extracellular matrix (ECM) is a key trigger for terminal differentiation. Previous studies in our laboratory have shown that keratinocyte adhesion and spreading also influence the size and shape of the nucleus, potentially impacting chromatin remodelling and epigenetic gene regulation. Here, we investigated the direct impact of simple mechanical stimuli on the nuclear architecture of human keratinocytes (HK) using micropatterned substrates to control the adherent surface and cell morphology. When cultured on small micropatterns (20 μm diameter), HKs adopted a rounded morphology and induced terminal differentiation within 24h, while on larger patterns (50 μm diameter) HKs were able to spread and remained undifferentiated. Nuclear morphology is also altered as the nuclear cross-sectional area and volume are both reduced on small patterns.
Immunofluorescence imaging of the nucleoskeletal proteins Lamin A/C showed a clear redisposition towards the nuclear periphery after 24h in HKs on the small patterns but not large patterns. Analysis of immunofluorescence intensity levels of closed and open chromatin markers showed a significant reduction of chromatin markers H3K27Ac, H3K27me3 and H3K9me3 on small patterns after 24h. Furthermore, closed chromatin marker H3K27me3 and H3K9me3 showed differential association into foci; clusters of the marker seem to condense together into fewer and larger foci after 24h on small patterns. Interestingly, we also observed nucleoli fusing on the small patterns, resulting in less numerous and larger nucleoli and reduced translational activity of the HKs. RNAi knockdown of Nesprin-2 and plectin-KO mice cell lines further revealed that these responses depend specifically on the size and shape of the nucleus as influenced by its linkage to the F-actin network. Finally, transcriptional profiling identified corresponding differences in downstream gene expression patterns, notably in ribosome biogenesis, DNA damage repair and retinoic acid signalling pathways accompanied by measurable changes in cellular phenotype.
Together, these findings indicate that biophysical cues directly regulate nuclear architecture in HKs and are translated in differential expression of genes.
Authors
Pundel., Oscar Jose.Collections
- Theses [4201]