Ezrin enhances line tension along transcellular tunnel edges via NMIIa driven actomyosin cable formation

Abstract

Transendothelial cell macroaperture (TEM) tunnels control endothelium barrier function and are triggered by several toxins from pathogenic bacteria that provoke vascular leakage. Cellular dewetting theory predicted that a line tension of uncharacterized origin works at TEM boundaries to limit their widening. Here, by conducting high-resolution microscopy approaches we unveil the presence of an actomyosin cable encircling TEMs. We develop a theoretical cellular dewetting framework to interpret TEM physical parameters that are quantitatively determined by laser ablation experiments. This establishes the critical role of ezrin and non-muscle myosin II (NMII) in the progressive implementation of line tension. Mechanistically, fluorescence-recovery-after-photobleaching experiments point for the upstream role of ezrin in stabilizing actin filaments at the edges of TEMs, thereby favouring their crosslinking by NMIIa. Collectively, our findings ascribe to ezrin and NMIIa a critical function of enhancing line tension at the cell boundary surrounding the TEMs by promoting the formation of an actomyosin ring.

Force-induced transcellular tunnel formation in endothelial cells

“…We observed a gradual accumulation of actin at the site of tip indentation, with some tunnel formation events showing stronger actin enrichment than others ( Figure 4B ). While actin cable formation around transcellular tunnels has recently been observed ( Stefani et al , 2017 ), the degree of actin accumulation in control cells compared with toxin expressing cells is not known. We quantified the level of actin enrichment by measuring the fold change in mean intensity around the site of tip indentation with respect to the mean intensity on initial tip contact with the cell.…”

“…A lower surface tension would also in principle lead to more narrow tunnels ( Gonzalez-Rodriguez et al , 2012 ). However, recent results ( Stefani et al , 2017 ) suggest that line tension, which limits tunnel opening, varies with time and might reach its plateau value over a longer period when the cytoskeleton is affected by EDIN, thus leading to larger tunnels.…”

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function

“…This offers the possibility to observe de novo accumulation of curvature‐sensing and deforming protein domains, such as the inverse‐BAR domain of missing in metastasis (MIM) and actin‐bundling protein with BAIAP2 homology (ABBA) (Maddugoda et al, ). Accumulation of MIM and ezrin along TEM edges controls two types of actin cytoskeleton superstructures that can be observed by scanning electron microscopy (Stefani et al, ). MIM controls the extension of membrane waves supported by the ARP 2/3 ‐driven polymerisation of branched actin filaments, whereas ezrin supports the assembly of an actomyosin cable that encircles TEMs, probably by promoting the stabilisation of actin filaments along TEM edges.…”

“…Physical laws describing the dewetting of liquids provide a theoretical framework to define the relationship between centripetal forces generated by the spreading of intoxicated cells, underlying the nucleation and growth of tunnels, and forces acting against the widening of TEMs, referred to as line tension (Gonzalez‐Rodriguez, Maddugoda, et al, ). The balance between these antagonist forces accounts for the stabilisation of TEMs and can be quantitatively studied by a laser ablation‐based approaches pointing for the importance of a time‐dependent build‐up of actomyosin cable encircling TEMs to stop their enlargement (Stefani et al, ). Noteworthy, TEM tunnels are also induced by a group of bacterial toxins catalysing a rise of cyclic‐AMP concentration, including the oedema toxin from Bacillus anthracis (Maddugoda et al, ; Figure ).…”

Cell surface mechanics gate stem cell differentiation

“…2A,B). Ezrin links the plasma membrane to the underlying cortex and CAEzrin is the current gold standard in the field to experimentally increase MCA (13,14). Upon CAEzrin expression, mESC cells maintained a high MCA 48h after 2i/LIF removal ( Supplementary Fig.…”