VE-cadherin tailless sensor was were used as a zero-force control. FRET index was measured at > 20 cell-cell junctions per condition. (F) BAECs expressing VEcadTS were subjected to 15 dynes/cm 2 shear stress for the indicated times. Similar results were obtained in 3 independent experiments. > 20 junctions were measured per condition, values are means ± standard error from one experiment. The FRET index was also measured from junctions of BAEC expressing the VE-cadherin tailless control and cells expressing soluble TS module. (E) Quantification of the FRET index from cell junctions of BAEC expressing VEcadTS untreated or treated with 10 µM Y27632 or 10 µM of MLCK inhibitor ML7 for 30 minutes. (D) Corrected FRET images in pseudocolor (right) or background subtracted acceptor images (left) of BAEC expressing VEcadTS with or without 10 µM ROCK inhibitor Y27632, or the tailless control. The graph is averaged from four separate photobleaching experiments per condition. A region in the junction was photobleached and the fluorescence recovery measured. (C) BAECs were transfected with either VE-cadherin tension sensor or VE-cadherin C-terminal tagged venus. (B) Localization of VEcadTS in VE-cadherin −/− endothelial cells (left) and endogenous VE-cadherin in HUVECs (right). The sensor module was inserted into the VE-cadherin cytoplasmic tail between the p120-catenin and beta-catenin binding domains. (A) Schematic of the VE-cadherin tension sensor (VEcadTS) and the tailless control. The VE-cadherin tension sensor is therefore functional in flow sensing.ĭesign and validation of a VE-cadherin tension sensor effects of flow on junctional forces VECadTS restored alignment similarly to wild-type VE-cadherin, whereas the tailless control was inactive ( supplemental figure 1A, quantified in supplemental figure 1B). To test its function in flow sensing, VE-cadherin −/− cells were reconstituted with VECadTS or wild-type VE-cadherin with a C-terminal Venus fluorescent protein and exposed to 15 dynes/cm 2 shear stress for 24 hours. The VE-cadherin tension sensor (VECadTS), expressed in VE-cadherin (−/−) endothelial cells, localized to cell junctions and distributed similarly to endogenous VE-cadherin in human umbilical vein endothelial cells (HUVECs) ( FIGURE 1B). We also constructed a zero-force (high FRET) control in which the C-terminal β-catenin-binding domain was deleted. The optimal construct had the tension sensor between the p120 binding domain and the β-catenin binding domain in the cytoplasmic tail ( FIGURE 1A). We initially screened expression and localization of constructs in which the tension sensor module was inserted into multiple sites within VE-cadherin (not shown). However, they also argue against the current model of passive transfer of force through the cytoskeleton to the junctions, showing instead that flow triggers cytoskeletal remodeling, which alters forces across the junctional receptors.ĭevelopment of a VE-cadherin tension sensor These data confirm the prediction that shear increases force on PECAM-1.
Tension on PECAM-1 was mediated by flow-stimulated association with vimentin. Flow triggered a simultaneous increase in tension across junctional PECAM-1, while non-junctional PECAM-1 was unaffected. Onset of shear stress triggered a rapid (<30 sec) decrease in tension across VE-cadherin, which paralleled a decrease in total cell-cell junctional tension. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no detectable tension on VE-cadherin outside of junctions. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo. Previous work suggested that flow increases force on PECAM-1, which initiates signaling. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2. Elucidating how ECs sense flow important for understanding both normal vascular function and disease. FSS applied to endothelial cells (EC) triggers signaling events including opening of ion channels, activation of signaling pathways and changes in gene expression. Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure and atherosclerosis.
0 kommentar(er)
