Sensing the Spark: Dissecting Electrotaxis Behavior Using Engineered CRISPRi and Synthetic Biosensors in Madin-Darby Canine Kidney Cells

Abstract

Electrotaxis is the directed movement of cells in response to an electric field and is a crucial biological process underlying processes such as tissue morphogenesis, wound healing, and cancer metastasis. Despite its significance, the mechanisms that enable cells to sense the electric field and facilitate directional migration are still not fully understood. Recently, transmembrane protein 154 (TMEM154) was identified as a driver of electrotaxis directionality in the human immune cell line, HL60s, where TMEM154 knockout resulted in the cells no longer exhibiting directed migration under an electric field. However, Madin-Darby Canine Kidney (MDCK) epithelial cells which are also capable of electrotaxis on the collective level, but not as single cells, have very low native expression of the TMEM154 ortholog. Additionally, TMEM154 was not upregulated upon electrical stimulation, signifying that MDCKs do not require TMEM154 for electrotaxis. To understand how TMEM154 abundance shapes electrotaxis behavior, this research will generate a stable MDCK-CRISPR inference (CRISPRi) cell line that can be used to knockdown TMEM154 in high-expressing MDCK-TMEM154 cells to evaluate how knockdowns can alter electrotaxis dynamics. Additionally, TMEM154 will be used as a design scaffold to engineer synthetic biosensors to visualize cell dynamics immediately following electrotaxis to provide another method of understanding cell response dynamics. Through this thesis we were able to create a synthetic TMEM154 inspired biosensor that localizes to the cell membrane and a stable MDCK-CRISPRi cell line. Understanding the mechanisms by which cells sense and react to an electric field has significant implications for fundamental biology, particularly in biological processes where electric fields guide cell migration such as wound healing and tissue patterning. As such, this research will also discuss the relevance of electrotaxis in chronic wound healing and emerging bioelectric therapeutic approaches in the global context.