title: The role of the human Discs large homologue-1 (Dlg1) in the Connexin 43 (Cx43) lifecycle and wound healing in keratinocytes
Creators: name: Scott, Harry
Publisher: name: University of Glasgow
Year: 2024

description: Gap junctions, which are formed by connexin proteins, are responsible for direct intercellular communication between adjacent cells by allowing the exchange of ions, metabolites, secondary messengers and microRNAs. Through this function, gap junctions are essential in maintaining homeostasis. Connexin 43 (Cx43) is a ubiquitous member of the gap junction family of proteins, being expressed in a diverse range of cells such as keratinocytes, cardiomyocytes and neurons. Alterations in Cx43 expression and function are associated with various disease states, including chronic non-healing wounds, which do not progress through the normal stages of wound healing in an orderly fashion. Chronic wounds are especially prevalent in patients suffering from diabetes and impose a massive economic and health burden worldwide, partially due to a lack of effective treatment options. 

Discs large protein was originally discovered in Drosophila and acts as a tumour suppressor. The mammalian homologue of Discs large, human Discs large homologue-1 (Dlg1), is a cell polarity and scaffolding protein which has previously been shown to interact with Cx43 in vitro in various epithelial cell lines and in vivo in human cervical mucosal epithelial tissue. The interaction of Cx43 and Dlg1 was further investigated in this study. 

In order to determine the role of Dlg1 in the Cx43 lifecycle, Dlg1 was depleted in HaCaT cells, a human epithelial cell line. Knockdown of Dlg1 decreased Cx43 on the plasma membrane and reduced overall Cx43 protein levels, but had no effect on Cx43 mRNA levels, indicating that Dlg1 has an important function in the Cx43 lifecycle. Cx43 colocalisation with a marker of the endoplasmic reticulum also decreased, while association with the lysosomes and, particularly, the Golgi increased, revealing Dlg1 depletion causes accumulation of Cx43 in these subcellular compartments. Treatment of cells with a lysosomal inhibitor resulted in a higher fold increase in Cx43 levels in siDlg1-treated cells compared to mock-treated cells, suggesting increased Cx43 degradation through this pathway in cells where Dlg1 is knocked down. Depletion of Dlg1 also caused significantly reduced gap junctional intercellular communication between cells, signifying that Dlg1 controls gap junctional signalling. Based on these data, Dlg1 is suggested to be involved in forward trafficking of Cx43 to the plasma membrane or stabilisation of Cx43 at the plasma membrane, or perhaps a combination of the two.

Due to its role in maintaining Cx43 at the plasma membrane, it was hypothesised that removal of Dlg1 may improve wound healing rates, as Cx43 is frequently upregulated in cells at the wound edge of chronic wounds. Therefore, scratch wound assays were carried out on HaCaT cells with and without siDlg1 treatment. Surprisingly, Dlg1 depletion resulted in reduced wound closure rates due to an inhibition of cell proliferation, demonstrating that Dlg1 has other crucial functions in the cell which outweigh the benefit gained by removal of Cx43 from the plasma membrane. Cx43 and Dlg1 were also observed to colocalise in live cell wound closure experiments using HEK293 cells, with both proteins being detected at the cell membrane and at the tips of cellular protrusions of cells migrating into the wound area. The localisation of the two proteins was also investigated in diabetic and non-diabetic human skin in immunofluorescence experiments. The two proteins were found to colocalise at the plasma membrane of cells in different sublayers of the epidermis and colocalisation was additionally observed in dermal cells and adipocytes, suggesting the interaction is conserved in a range of cell types.

Finally, the interaction between the Cx43 C-terminal tail and Dlg1 SH3/HOOK/pGUK domains was investigated through the use of AlphaFold, which modelled the interaction of the two proteins. Three potential sites of interaction with the Dlg1 SH3/HOOK/pGUK domains were observed within the Cx43 C-terminus. Expression vectors for the Cx43 C-terminus (and deletions of putative interaction sites) and Dlg1 SH3/HOOK/pGUK were designed for experimental testing to confirm the accuracy of the AlphaFold model. Further experiments involving these recombinant proteins will reveal the molecular determinants of Cx43/Dlg1 interaction, as well as whether phosphorylation affects the interaction.

Overall, this thesis builds on previous work on the Cx43/Dlg1 interaction, further defining the role of Dlg1 in the Cx43 lifecycle. It also provides the first demonstration that Dlg1 is an important component of the normal wound healing process in keratinocytes and defines three potential interaction sites with Dlg1 within the Cx43 C-terminus. Future research will determine whether disruption of the Cx43/Dlg1 interaction can improve wound healing rates and therefore whether the interaction may be a viable therapeutic target.