PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line.

Dhurgham Alfahad,Khatab Mawlood,Bandar Alharbi,Philip Dash,Salem Alharethi

doi:10.3906/biy-2004-108

Abstract

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.

Highlights

Focal adhesions (FAs) have been shown to be involved in many aspects of cell physiology, such as signal transduction (Morimatsu et al, 2015), the anchoring of cells to extracellular matrix (ECM), and the cell’s response to mechanical and physical forces (Geiger et al, 2009; Hoffman et al, 2011)
Spatial distribution of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within FAs The distributions of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were monitored in MDA-MB-231 cells with the aid of PLCδ1- Pleckstrin Homology (PH)-GFP/mCherry and Bruton’s tyrosine kinase (Btk)-PH-GFP/mCherry biosensors, respectively
Changes of local PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels during FA assembly and disassembly Since the use of various ECM gave rise to identical spatial distribution across PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in relation to zyxin, further experiments investigating temporal changes of PtdIns(4,5)P2 and PtdIns(3,4,5) P3 levels throughout the lifetime of FA were undertaken with MDA-MB-231 cells cultured on a collagen surface

Summary

Introduction

Focal adhesions (FAs) have been shown to be involved in many aspects of cell physiology, such as signal transduction (Morimatsu et al, 2015), the anchoring of cells to ECM, and the cell’s response to mechanical and physical forces (Geiger et al, 2009; Hoffman et al, 2011). PtdIns(4,5)P2 and PtdIns(3,4,5)P3 are lipids measuring 64.5 ± 28 nm and 125.6 ± 22 nm, respectively; both can be found in distinct and well-restricted regions of pheochromocytorna (PC12) cell membranes (Wang & Richards, 2012). Many signalling pathways have been shown to be regulated by PtdIns(4,5) P2 and PtdIns(3,4,5)P3 through the recruitment of kinases into these regions (Wang & Richards, 2012). Pleckstrin Homology (PH) domains consist of around 120 amino acids and have a seven-strand β-barrel which forms 2 antiparallel β-sheets and a C-terminal α-helix (Figure 1). The β1, β2, β3, and β4 loops form specific

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