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

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.

Keywords: Focal adhesions turnover; PtdIns(3,4,5)P3; PtdIns(4,5)P2.

Figure 1

Structure of two ph domains in interaction with ptdins. The PH domain consists of an α-helix (pink colour) and 7 β-strands (yellow colour), forming a β-barrel. PI binds to the PH domain through the loops of the β-barrel. (a) Btk-PH-Ins(1,3,4,5)P4 interaction; (b) PLC𝛅-PH PtdIns(4,5)P2 interaction (Bunney and Katan, 2011; Wang et al., 2015).

Figure 2

Flow diagram depicting the experimental procedures. This diagram shows the design of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 biosensors, which were in turn used together with FAs (zyxin/ paxillin)-GFP or -RFP fusion genes to transfect MDA-MB-231 cells cultured upon different cell surfaces. Fixed and live cells were visualised by confocal microscopy to assess the colocalisation between FA proteins and PtdIns(4,5)P2/PtdIns(3,4,5)P3, as well as to quantify the local levels of both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 during FA assembly and disassembly.

Figure 3

(a) Spatial colocalisation between PtdIns(4,5)P2\ PtdIns(3,4,5)P3 and a single FA in MDA-MB-231 cells cultured on different surfaces. MDA-MB-231 cells plated on 20-μg/mL fibronectin, 0.2% w/v gelatine and 2-mg/mL collagen were cotransfected with PLC𝛅1-PHGFP\ Btk-PH-GFP (green) and zyxin-mCherry(red). The highlighted areas show a magnification of the spatial colocalisation region between PtdIns(4,5)P2 and a single FA. Three independent experiments were performed. Representative pictures are shown. The scale bar is indicative of a 6-μm length. Quantification of spatial colocalisation between (b) PtdIns(4,5)P2, (c) PtdIns(3,4,5)P3, and zyxin. Spearman’s (rho) correlation coefficient analysis was used to determine the strength colocalisation between PtdIns(4,5)P2 and zyxin. The pooled data shows a colocalisation between PtdIns(4,5)P2 and zyxin, as well as between PtdIns(3,4,5)P3 and zyxin on different surfaces.

Figure 4

Quantification of the local levels of PtdIns(4,5)P2 within a single FA. Confocal live imaging showing the local levels of PtdIns(4,5)P2 throughout the turnover of zyxin turnover. MDA-MB-231 cells were plated on collagen (2mg/mL) and cotransfected with PLC𝛅1-PH-GFP and zyxin-RFP. Z-Stacks were taken at a 0.15-μm distance with time-lapse series acquired over a 10-min period with an interval of 15 s. The first column refers to local levels of PtdIns(4,5)P2, and the second column refers to zyxin assembly and disassembly. The highlighted areas show the ROI that was drawn closely around the zyxin and the PtdIns(4,5)P2 to measure the specific localisation of local levels of PtdIns(4,5)P2 within a single zyxin. The zyxin selected for measuring should be absent both at the beginning and end of the time-lapse series.

Figure 5

Dynamic change of local levels of PtdIns(4,5)P2 within and around FA turnover. The green curve refers to the level of PtdIns(4,5)P2 within FAs, The brown curves refer to the level of PtdIns(4,5)P2 around zyxin and paxillin turnover, and the red curve refers to zyxin turnover over time (105 s). (a/b) Dynamic change of PtdIns(4,5)P2 within and around zyxin and paxilin during assembly and disassembly. The local levels of PtdIns(4,5)P2 were increased within zyxin and paxillin during assembly and declined during disassembly, while around zyxin the local levels of PtdIns(4,5)P2 were slightly changed. (c) Percentage of change of local levels of PtdIns(4,5)P2 within zyxin was significantly higher than around zyxin. (c) Quantification of the dynamic change of PtdIns(4,5)P2 at different focal planes within zyxin during assembly and disassembly. Data are representative of N = 30 FA in 10 migrating MDA-MB-231 cells. Statistical analysis was performed with the aid of one-way ANOVA with Tukey’s multiple comparison test. P-values showing statistical difference were labelled as (**) for P < 0.01 and (***) for P < 0.0005. Data presented as mean ± SEM of 3 independent experiments.

Figure 6

Dynamic change of local levels of PtdIns(3,4,5)P3 within and around FA turnover. The green curve refers to the level of PtdIns(3,4,5)P3 within FA, the brown curves refer to the level of PtdIns(3,4,5)P3 around zyxin and paxillin turnover, and the red curve refers zyxin and paxillin turnover over time (105 s). (a/b) Quantification of the dynamic change of PtdIns(3,4,5)P3 within and around zyxin and paxillin assembly and disassembly. PtdIns(3,4,5)P3 was at a constant level during zyxin and paxillin turnover. (c) Percentage change of local levels of PtdIns(3,4,5)P3 within and around zyxin was not significantly different. (d) Dynamic change of PtdIns(3,4,5)P3 within zyxin and paxillin at different focal plane. Data are representative of N = 30 FA intensity profiles in 10 migrating MDA-MB-231 cells. Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparison test. Statistical significance was accepted at P < 0.05. Data presented as mean ± SEM of 3 independent experiments.

Figure 7

Diagram summarising the main findings of this study. Both PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were found to be colocalised with zyxin and paxillin; however, only the level of PtdIns(4,5)P2 was found to change with the assembly and disassembly of FA proteins.