Keratin Dynamics and the Role of Lipids on Keratinocyte Differentiation and mRNA Stability

PhD ThesisKeratin K2 is a 66 kDa type II intermediate filament protein expressed in differentiating keratinocytes of the epidermis with a very low-level expression in normal oral mucosa. In the epidermis it is expressed in the upper granular and spinous layers and is considered a marker of terminal differentiation. It is known to be upregulated in pre-cancerous lesions of the oral cavity. However, the mechanism of its induction in dysplasia and its role in oral cancer is not known. As it is only expressed in vivo, being down-regulated in cultured keratinocytes, there are no reported studies on the functions of this protein. Point mutations in this protein are associated with the skin condition Ichthyosis Bullosa of Siemens (IBS). Keratin filaments are in a constant state of assembly and disassembly to maintain cell stability and support. Keratins’ reorganisation and dynamics are affected mainly by their state of phosphorylation which explains the difference between health and disease and the molecular interactions between different keratin pairs as well as with other cytoplasmic proteins. Heat shock was shown to re-organises the keratin network which is mediated by phosphorylation. A model has been developed to study keratin filaments dynamics by introducing keratin K2, into a simple epithelial cell line, MCF-7 (breast carcinoma cell line) which lacks K2 expression but expresses other keratins including K8, K18 and K19. Introduction of K2 into MCF-7 cytoplasm allows it to bind and fully integrate into the pre-existing network. To understand the mechanism of K2 integration into the pre-existing filaments, the stability of the network and its phosphorylation state using two phosphatase inhibitors, Calyculin A (CL-A) and Okadaic Acid (OA) was studied. To investigate the response of keratin cytoskeleton to stress, 4 the effect of heat shock on filaments reorganisation was studided using immunocytochemistry and live cell imaging. The expression of K2 mRNA and protein was investigated in keratinocytes cell lines as well as in normal human epidermal keratinocyte (NHEK) along with other terminal differentiation keratins, K1 and K10. The absence of serum lipids (SLP) and phenol red (PR), which are generally used in culture medium, had a significant effect on the expression of these keratins at both mRNA and protein levels in NHEK. The effect was different compared to immortalised cell lines, which could be explained by immortalisation methods altering gene response. Adding back retinoic acid (ATRA) to the culture medium differentially affected the expression of these genes. Adding PR back into PR-free culture medium in NHEK did reduce the expression of K1 and K10 but not K2. To further investigate the effect of SLP, RA and PR, the post-transcriptional stability of K2, K10 and K1 mRNAs using Actinomycin D (AD) in NHEK cells was studied. Interestingly, K2 mRNA was stabilised whereas K1 and K10 mRNAs were destabilised by ATRA. These observations explain the differential effect of ATRA on the expression of these genes previously reported in the literature. Further investigations are required to decipher the mechanism(s) regulating transcriptional changes affected by different culture conditions and by RA. The aim of this project is to study keratin dynamics and the role of lipids on keratinocyte differentiation and mRNA stability

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network

Serum lipids, retinoic acid and phenol red differentially regulate expression of keratins K1, K10 and K2 in cultured keratinocytes

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown. We show for the first time that PR and SLP themselves suppress expression of differentiation-specific keratins K1, K10 and K2 in normal human epidermal keratinocytes (NHEK) and two important cell lines, HaCaT and N/TERT-1. Removal of SLP increased expression of K1, K10 and K2 in 2D and 3D cultures, which was further enhanced in the absence of PR. The effect was reversed for K1 and K10 by adding all-trans retinoic acid (ATRA) but increased for K2 in the absence of PR. Furthermore, retinoid regulation of differentiation-specific keratins involves post-transcriptional mechanisms as we show KRT2 mRNA is stabilised whilst KRT1 and KRT10 mRNAs are destabilised in the presence of ATRA. Taken together, our results indicate that the presence of PR and SLP in cell culture media may significantly impact in vitro studies of keratinocyte differentiation

Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.1432_1440del; p.478_480del was present in all affected individuals with angle-closure disease. We show ubiquitous expression of this transcript in cell lines derived from human tissues and in iris, retina, retinal pigment and ciliary epithelia, cornea and lens. We also identified eight additional mutations in SPATA13 in a cohort of 189 unrelated PACS/PAC/PACG samples. This gene encodes a 1277 residue protein which localises to the nucleus with partial co-localisation with nuclear speckles. In cells undergoing mitosis SPATA13 isoform I becomes part of the kinetochore complex co-localising with two kinetochore markers, polo like kinase 1 (PLK-1) and centrosome-associated protein E (CENP-E). The 9 bp deletion reported in this study increases the RAC1-dependent guanine nucleotide exchange factors (GEF) activity. The increase in GEF activity was also observed in three other variants identified in this study. Taken together, our data suggest that SPATA13 is involved in the regulation of mitosis and the mutations dysregulate GEF activity affecting homeostasis in tissues where it is highly expressed, influencing PACG pathogenesis