Schoofs, HansDaubel, NinaSchnabellehner, SarahGrönloh, Max L BPalacios Martínez, SebastiánHalme, AleksiMarks, Amanda MJeansson, MarieBarcos, SaraBrakebusch, CordBenedito, RuiEngelhardt, BrittaVestweber, DietmarGaengel, KonstantinLinsenmeier, FabianSchürmann, SebastianSaharinen, Pipsavan Buul, Jaap DFriedrich, OliverSmith, Richard SMajda, MateuszMäkinen, Taija2025-03-282025-03-282025-03-19Nature. 2025 Mar 19.https://hdl.handle.net/20.500.12105/26586This work was supported by grants from Knut and Alice Wallenberg Foundation (grant nos. 2018.0218 to T.M. and 2020.0057 to T.M. and K.G.), the Swedish Research Council (grant nos. 2020-02692 to T.M. and 2021-04896 to K.G.), Göran Gustafsson foundation (to T.M.), the Swedish Cancer Society (grant nos. 9 0220 Pj, 22 2025 Pj to T.M.), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 814316 (to H.S. and T.M.), the Swiss National Science Foundation (grant no. 310030_189080 to B.E.), and the Research Council of Finland’s Centre of Excellence Program (grant no. 307366 to P.S.). O.F. received funding through an R&D grant by the Bavarian Ministry of Economy & Energy (grant no. 41-6618c/587/2-LSM-2303-0015). R.S.S. was supported by a Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant to the John Innes Centre (grant no. BB/X01102X/1).Lymphatic capillaries continuously take up interstitial fluid and adapt to resulting changes in vessel calibre. The mechanisms by which the permeable monolayer of loosely connected lymphatic endothelial cells (LECs) maintains mechanical stability remain elusive. Here we identify dynamic cytoskeletal regulation of LEC shape, induced by isotropic stretch, as crucial for the integrity and function of dermal lymphatic capillaries. We found that the oak leaf-shaped LECs showed a spectrum of VE-cadherin-based junctional configurations at the lobular intercellular interface and a unique cytoskeletal organization, with microtubules at concave regions and F-actin at convex lobes. Multispectral and longitudinal intravital imaging of capillary LEC shape and actin revealed dynamic remodelling of cellular overlaps in vivo during homeostasis and in response to interstitial fluid volume increase. Akin to puzzle cells of the plant epidermis, LEC shape was controlled by Rho GTPase CDC42-regulated cytoskeletal dynamics, enhancing monolayer stability. Moreover, cyclic isotropic stretch increased cellular overlaps and junction curvature in primary LECs. Our findings indicate that capillary LEC shape results from continuous remodelling of cellular overlaps that maintain vessel integrity while preserving permeable cell-cell contacts compatible with vessel expansion and fluid uptake. We propose a bellows-like fluid propulsion mechanism, in which fluid-induced lumen expansion and shrinkage of LEC overlaps are countered by actin-based lamellipodia-like overlap extension to aid vessel constriction.engVoRhttp://creativecommons.org/licenses/by/4.0/Attribution 4.0 International40108458Natureopen access