Investigating the role of End-Binding Protein-2 in epithelial tissue architecture

Taylor, Maddison Paige (2022) Investigating the role of End-Binding Protein-2 in epithelial tissue architecture. Masters thesis, University of East Anglia.

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Microtubules (MTs) and their dynamic instability govern several cellular processes such as a cell’s intrinsic polarity, differentiation, division, migration, organisation, and protein/molecule trafficking. The End-Binding protein family (EB1-3) are one group of MT plus-end tracking proteins, also known as +TIPs, that play a role in MT dynamics. EB1 and EB3 demonstrate functional redundancies and associate strongly with the plus-ends of MTs. EB2, on the other hand, appears to be more divergent in function and associates more strongly along the MT lattice. EB2 demonstrates a role in epithelial differentiation, cell migration and MT dynamics and has been linked to cancer progression and more recently with Fibroblast Growth Factors (FGFs) and their receptors (FGFRs). This investigation has demonstrated that EB2 localises along the MT lattice while FGFR3 accumulates at the cell peripheries in TC7 cells. TC7 cells were used as a model for epithelial colorectal cancer cells and are derived from the Caco-2 cell line. Inhibition of all FGFRs resulted in a significant increase in the mean EB2 fluorescence intensity compared to the DMSO control; therefore, suggesting a role for FGF signalling in the regulation of EB2 expression.

Preliminary data (Mogensen lab) and published data suggest that an upregulation in EB2 is associated with loss of normal tissue architecture, increased cell migration and MT dynamics. In this study the possible mechanisms behind the effect of high EB2 expression on cell shape and unjamming is explored in partially polarised MDCKII cells. Unjamming describes a mechanism in which cells of an epithelial tissue transition from a jammed ‘solid-like’ state, to an unjammed ‘fluid-like’ state. When jammed, cells are closely packed together and have reduced motility. During the transition to an unjammed state, cells become more motile, and their apical surface area increases. Here shape index analysis indicated that EB2 overexpression promotes unjamming. Some other cellular changes included, the polarity marker ZO-1, displaying more undulating labelling. Therefore, suggesting that high EB2 expression compromises junction integrity and function. A reduction in junctional mDia-1 was also seen, suggesting that actin nucleation at adherens junctions is affected by overexpression of EB2. In addition, junctional GEF-H1 was decreased when EB2 was overexpressed, and activation of Rho resulted in the partial rescue of cell shape index. However, no change in junctional pMLC was seen when EB2 was overexpressed, suggesting that actomyosin contraction is possibly affected by abnormal actin and non-muscle myosin organisation.

Interestingly, MDCKII cells overexpressing EB2 revealed more dynamin-2 at the junctions compared to control. Previous data by Ben Rix (Mogensen lab) showed a greater spread in E-cadherin across junctions, suggesting increased E-cadherin internalisation. Dynamin inhibition led to a partial rescue of cell shape, and a decrease in junctional E-cadherin. Therefore, suggesting that EB2 overexpression results in the disruption of normal dynamin-mediated E-cadherin endocytosis. Perhaps, indicating that this disruption allows the permanent changes in junction length which allows the increase in shape index and thus contribute to further unjamming. However, previous research also implicates dynamin-2 with actomyosin contraction at junctions (Chua et al., 2009). Thus, proposing several mechanisms at work.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 19 Apr 2023 10:00
Last Modified: 19 Apr 2023 10:00

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