Dhurgham Al-Fahad - Department of Pharmaceutical Sciences, College of Pharmacy University of Thi-Qar, Iraq
Firas Alyaseen - Department of Pharmaceutical Sciences, College of Pharmacy University of Thi-Qar, Iraq.
Ahmed Al-Amery - Faculty of Education, Soran University, Erbil, Kurdistan Region, Iraq.
Clementino Ibeas Bin - Ilumix Biotech Ltd, London, United Kingdom.

Background: Focal adhesions (FAs) are highly dynamic complex structures that assembled and disassembled on an ongoing basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion to cell migration. Assembly and disassembly processes of FAs are regulated by a variety of cellular signaling proteins and adaptors. We previously demonstrated that local levels of Phosphatidylinositol ۴,۵‐bisphosphate (PtdIns(۴,۵)P۲) in MDA-MB-۲۳۱ cells increases during FA assembly and declines during disassembly. In this study we aimed to investigate whether PtdIns(۴,۵)P۲ regulates FA turnover. Methods: MDA-MB-۲۳۱ cells were co-transfected with a labeling vinculin (or zyxin) and the PLC𝛅۱-PH biosensor to visualize FA localization and PtdIns(۴,۵)P۲ in the cell membrane. We also used pharmacological inhibitors to determine the mechanism underlying the changes of PtdIns(۴,۵)P۲ level during FA turnover and cell migration. Immunostaining, immunoprecipitation, and Western blotting were used to examine the localization and interaction between phospholipase C (PLC)/phosphatidylinositol ۳-kinase (PI۳K) FA proteins. Results: We showed that inhibition of PLC, PI۳K significantly reduced the decline of PtdIns(۴,۵)P۲ levels within FA disassembly and the slowdown rate of FA turnover and cell migration. We also showed that the inhibition of enzymes implicated in the downstream pathway of PtdIns(۴,۵)P۲, such as diacylglycerol kinase (DAGK) and protein kinase C (PKC) significantly reduced FA turnover time and the speed of cell migration. Additionally, we demonstrated that PLC but not PI۳K interact with FAs. In conclusion, Conclusions: This study suggests that dynamical changes of PtdIns(۴,۵)P۲ might regulate FA turnover and facilitate cell migration.

Cell Migration, Focal Adhesion Turnover, MDA-MB-۲۳۱ Breast Cancer Cell Line, Ptdins(۴, ۵)P۲, PLC, PI۳K.