Mote, R. D. | Search bioRxiv

bioRxiv2021

E-cadherin regulates the stability and transcriptional activity of β-catenin in embryonic stem cells

E-CADHERIN is abundantly expressed in embryonic stem cells (ESCs) and plays an important role in the maintenance of cell-cell adhesions. However, the exact function of this molecule beyond cell adhesion, in the context of cell fate decisions is largely unknown. Using mouse ESCs (mESCs), we demonstrate that E-CADHERIN and {beta}- CATENIN interact at the membrane and continue to do so upon internalization within the cell. Knockout of the gene encoding E-CADHERIN, Cdh1, in mESCs resulted in a failure to form tight colonies, accompanied by altered expression of differentiation markers, and retention of pluripotency factor expression during differentiation. Interestingly, Cdh1-/- mESCs showed a dramatic reduction in {beta}-CATENIN levels. Transcriptional profiling of Cdh1-/- mESCs displayed a significant alteration in the expression of a subset of {beta}-CATENIN targets, in a cell-state dependent manner. While treatment with a pharmacological inhibitor against GSK3{beta} could rescue levels of {beta}-CATENIN in Cdh1-/- mESCs, expression of downstream targets were altered in a context-dependent manner, indicating an additional layer of regulation within this subset. Together, our results reveal the existence of a cell-state-dependent regulation of {beta}-CATENIN and its transcriptional targets in an E-CADHERIN dependent manner. Our findings hint at hitherto unknown roles played by E- CADHERIN in regulating the activity of {beta}-CATENIN in ESCs. Significance StatementAre cell adhesions only responsible for maintaining tissue architecture, or do they also regulate cell fate decisions during early embryonic stages by modulating the output of specific signalling pathways? In this study, we study the role of E- CADHERIN, a crucial component of cell-cell adhesions in the context of mouse embryonic stem cells (mESCs). We find that E-CADHERIN regulates the stability and activity of {beta}-CATENIN in mESCs through physical interactions. However, the loss of E-CADHERIN affected the expression of only a subset of downstream targets of {beta}-CATENIN in a cell-state dependent manner. This study highlights a critical cross-talk between molecules involved in cell-cell adhesion and the underlying signalling network critical for establishing cell fate during early mammalian development.

cell biology↗

bioRxiv2021

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

molecular biology↗

bioRxiv2020

Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Youngs modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Youngs modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.

cell biology↗