Abstract
Focal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.
Highlights
Vinculin are among the proteins with the most potential binding partners within FAs6
The study presented here was motivated by observations we made in pilot experiments using dual colour superresolution (SIM) to visualise the distribution of paxillin-mCherry and zyxin-GFP within focal adhesions
We hypothesised that the observed shift along the long focal adhesions (FAs) axis could be due to the fact that paxillin and zyxin are found in different FA z-layers, which may move with respect to each other during FA function
Summary
Vinculin are among the proteins with the most potential binding partners within FAs6 In line with their linking, structural, role they are amongst the first proteins to be recruited to assembling FA complexes, especially the directly integrin-binding paxillin[12,13,14,15,16]. Zyxin and VASP are recruited to assembling FAs at much later stages than paxillin or vinculin and are more closely linked to a ctin[15]. In response to mechanical cues and during TGF-β induced EMT zyxin, VASP and vinculin stimulate actin polymerisation in a co-dependent m anner[25,26,27,28,29,30,31]. Stimulation with HGF strongly increases the motility of cells in both 2D and 3D environments[35,38,39,40]
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