Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.

CEITEC Central European Institute of Technology Masaryk University Brno 62500 Czechia

Department of Experimental Biology Faculty of Science Masaryk University Brno 62500 Czechia

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 62500 Czechia

Zobrazit více v PubMed

Murrell M, Oakes PW, Lenz M, Gardel ML. 2015. Forcing cells into shape: the mechanics of actomyosin contractility. Nat. Rev. Mol. Cell Biol. 16, 486–498. (10.1038/nrm4012) PubMed DOI PMC

Heissler SM, Sellers JR. 2014. Myosin light chains: teaching old dogs new tricks. Bioarchitecture 4, 169–188. (10.1080/19490992.2015.1054092) PubMed DOI PMC

Kinoshita N, Sasai N, Misaki K, Yonemura S. 2008. Apical accumulation of rho in the neural plate is important for neural plate cell shape change and neural tube formation. Mol. Biol. Cell 19, 2289–2299. (10.1091/mbc.e07-12-1286) PubMed DOI PMC

Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR. 2009. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 10, 778–790. (10.1038/nrm2786) PubMed DOI PMC

Shutova MS, Svitkina TM. 2018. Common and specific functions of nonmuscle myosin II paralogs in cells. Biochem. Mosc. 83, 1459–1468. (10.1134/S0006297918120040) PubMed DOI PMC

Greene NDE, Copp AJ. 2014. Neural tube defects. Annu. Rev. Neurosci. 37, 221–242. (10.1146/annurev-neuro-062012-170354) PubMed DOI PMC

Nikolopoulou E, Galea GL, Rolo A, Greene NDE, Copp AJ. 2017. Neural tube closure: cellular, molecular and biomechanical mechanisms. Development 144, 552–566. (10.1242/dev.145904) PubMed DOI PMC

Sutherland A, Lesko A. 2020. Pulsed actomyosin contractions in morphogenesis. F1000Res. 9, F1000 Faculty Rev-142. (10.12688/f1000research.20874.1) PubMed DOI PMC

Vijayraghavan DS, Davidson LA. 2017. Mechanics of neurulation: from classical to current perspectives on the physical mechanics that shape, fold, and form the neural tube. Birth Defects Res. 109, 153–168. (10.1002/bdra.23557) PubMed DOI PMC

Hildebrand JD. 2005. Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network. J. Cell. Sci. 118, 5191–5203. (10.1242/jcs.02626) PubMed DOI

Hildebrand JD, Soriano P. 1999. Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. Cell 99, 485–497. (10.1016/S0092-8674(00)81537-8) PubMed DOI

Matsuda M, Sokol SY. 2021. Xenopus neural tube closure: a vertebrate model linking planar cell polarity to actomyosin contractions. Planar Cell Polarity during Dev. 145, 41–60. (10.1016/bs.ctdb.2021.04.001) PubMed DOI

Butler MT, Wallingford JB. 2017. Planar cell polarity in development and disease. Nat. Rev. Mol. Cell Biol. 18, 375–388. (10.1038/nrm.2017.11) PubMed DOI PMC

Cai CQ, Shi OY. 2014. Genetic evidence in planar cell polarity signaling pathway in human neural tube defects. Front. Med. 8, 68–78. (10.1007/s11684-014-0308-4) PubMed DOI

Humphries AC, Narang S, Mlodzik M. 2020. Mutations associated with human neural tube defects display disrupted planar cell polarity in Drosophila. eLife 9, e53532. (10.7554/eLife.53532) PubMed DOI PMC

Simons M, Mlodzik M. 2008. Planar cell polarity signaling: from fly development to human disease. Annu. Rev. Genet. 42, 517–540. (10.1146/annurev.genet.42.110807.091432) PubMed DOI PMC

Adler PN. 2012. The frizzled/stan pathway and planar cell polarity in the Drosophila wing. Curr. Top. Dev. Biol. 101, 1–31. (10.1016/B978-0-12-394592-1.00001-6) PubMed DOI PMC

Devenport D. 2014. The cell biology of planar cell polarity. J. Cell Biol. 207, 171–179. (10.1083/jcb.201408039) PubMed DOI PMC

Hale R, Strutt D. 2015. Conservation of planar polarity pathway function across the animal kingdom. Annu. Rev. Genet. 49, 529–551. (10.1146/annurev-genet-112414-055224) PubMed DOI

Humphries AC, Mlodzik M. 2018. From instruction to output: Wnt/PCP signaling in development and cancer. Curr. Opin. Cell Biol. 51, 110–116. (10.1016/j.ceb.2017.12.005) PubMed DOI PMC

Koca Y, Collu GM, Mlodzik M. 2022. Wnt-frizzled planar cell polarity signaling in the regulation of cell motility. Curr. Top. Dev. Biol. 150, 255–297. (10.1016/bs.ctdb.2022.03.006) PubMed DOI PMC

Peng Y, Axelrod JD. 2012. Asymmetric protein localization in planar cell polarity: mechanisms, puzzles, and challenges. Curr. Top. Dev. Biol. 101, 33–53. (10.1016/B978-0-12-394592-1.00002-8) PubMed DOI PMC

Strutt H, Strutt D. 2021. How do the Fat-Dachsous and core planar polarity pathways act together and independently to coordinate polarized cell behaviours? Open Biol. 11, 200356. (10.1098/rsob.200356) PubMed DOI PMC

Yang Y, Mlodzik M. 2015. Wnt-frizzled/planar cell polarity signaling: cellular orientation by facing the wind (Wnt). Annu. Rev. Cell Dev. Biol. 31, 623–646. (10.1146/annurev-cellbio-100814-125315) PubMed DOI PMC

Yang YZ. 2012. Preface. Plan. Cell Polarity during Dev. 101, xi–xiii. (10.1016/B978-0-12-394592-1.09991-9) PubMed DOI

Habas R, Kato Y, He X. 2001. Wnt/frizzled activation of Rho regulates vertebrate gastrulation and requires a novel formin homology protein Daam1. Cell 107, 843–854. (10.1016/S0092-8674(01)00614-6) PubMed DOI

McGreevy EM, Vijayraghavan D, Davidson LA, Hildebrand JD. 2015. Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure. Biol. Open 4, 186–196. (10.1242/bio.20149589) PubMed DOI PMC

Nishimura T, Honda H, Takeichi M. 2012. Planar cell polarity links axes of spatial dynamics in neural-tube closure. Cell 149, 1084–1097. (10.1016/j.cell.2012.04.021) PubMed DOI

Strutt DI, Weber U, Mlodzik M. 1997. The role of RhoA in tissue polarity and frizzled signalling. Nature 387, 292–295. (10.1038/387292a0) PubMed DOI

Winter CG, Wang B, Ballew A, Royou A, Karess R, Axelrod JD, Luo L. 2001. Drosophila Rho-associated kinase (Drok) links frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105, 81–91. (10.1016/S0092-8674(01)00298-7) PubMed DOI

Butler MT, Wallingford JB. 2018. Spatial and temporal analysis of PCP protein dynamics during neural tube closure. eLife 7, e36456. (10.7554/eLife.36456) PubMed DOI PMC

Newman-Smith E, Kourakis MJ, Reeves W, Veeman M, Smith WC. 2015. Reciprocal and dynamic polarization of planar cell polarity core components and myosin. eLife 4, e05361. (10.7554/eLife.05361) PubMed DOI PMC

Wallingford JB. 2019. We are all developmental biologists. Dev. Cell 50, 132–137. (10.1016/j.devcel.2019.07.006) PubMed DOI

Wallingford JB, Niswander LA, Shaw GM, Finnell RH. 2013. The continuing challenge of understanding, preventing, and treating neural tube defects. Science 339, 1222002. (10.1126/science.1222002) PubMed DOI PMC

Wang Y. 2012. Protein-protein interaction techniques: dissect PCP signaling in Xenopus. Methods Mol. Biol. 839, 27–41. (10.1007/978-1-61779-510-7_3) PubMed DOI

Haigo SL, Hildebrand JD, Harland RM, Wallingford JB. 2003. Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr. Biol. 13, 2125–2137. (10.1016/j.cub.2003.11.054) PubMed DOI

Shindo A, Wallingford JB. 2014. PCP and septins compartmentalize cortical actomyosin to direct collective cell movement. Science 343, 649–652. (10.1126/science.1243126) PubMed DOI PMC

Bryja V, Červenka I, Čajánek L. 2017. The connections of Wnt pathway components with cell cycle and centrosome: side effects or a hidden logic? Crit. Rev. Biochem. Mol. Biol. 52, 614–637. (10.1080/10409238.2017.1350135) PubMed DOI PMC

Gao C, Chen YG. 2010. Dishevelled: the hub of Wnt signaling. Cell. Signal. 22, 717–727. (10.1016/j.cellsig.2009.11.021) PubMed DOI

Kelly LK, Wu J, Yanfeng WA, Mlodzik M. 2016. Frizzled-induced Van Gogh phosphorylation by CK1ε promotes asymmetric localization of core PCP factors in Drosophila. Cell Rep. 16, 344–356. (10.1016/j.celrep.2016.06.010) PubMed DOI PMC

Wallingford JB, Habas R. 2005. The developmental biology of dishevelled: an enigmatic protein governing cell fate and cell polarity. Development 132, 4421–4436. (10.1242/dev.02068) PubMed DOI

Andreeva A, Lee J, Lohia M, Wu X, Macara IG, Lu X. 2014. PTK7-Src signaling at epithelial cell contacts mediates spatial organization of actomyosin and planar cell polarity. Dev. Cell 29, 20–33. (10.1016/j.devcel.2014.02.008) PubMed DOI PMC

Zehnder SM, Suaris M, Bellaire MM, Angelini TE. 2015. Cell volume fluctuations in MDCK monolayers. Biophys. J. 108, 247–250. (10.1016/j.bpj.2014.11.1856) PubMed DOI PMC

Humphries AC, Molina-Pelayo C, Sil P, Hazelett CC, Devenport D, Mlodzik M. 2023. A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core planar cell polarity factors prickle and dishevelled. PLoS Genet. 19, e1010849. (10.1371/journal.pgen.1010849) PubMed DOI PMC

Jenny A, Darken RS, Wilson PA, Mlodzik M. 2003. Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling. EMBO J. 22, 4409–4420. (10.1093/emboj/cdg424) PubMed DOI PMC

Kunimoto K, Weiner AT, Axelrod JD, Vladar EK. 2022. Distinct overlapping functions for prickle1 and prickle2 in the polarization of the airway epithelium. Front. Cell Dev. Biol. 10, 976182. (10.3389/fcell.2022.976182) PubMed DOI PMC

Strutt H, Gamage J, Strutt D. 2016. Robust asymmetric localization of planar polarity proteins is associated with organization into signalosome-like domains of variable stoichiometry. Cell Rep. 17, 2660–2671. (10.1016/j.celrep.2016.11.021) PubMed DOI PMC

Mentink RA, Rella L, Radaszkiewicz TW, Gybel T, Betist MC, Bryja V, Korswagen HC. 2018. The planar cell polarity protein VANG-1/Vangl negatively regulates Wnt/β-catenin signaling through a Dvl dependent mechanism. PLoS Genet. 14, e1007840. (10.1371/journal.pgen.1007840) PubMed DOI PMC

Schihada H, Kowalski-Jahn M, Turku A, Schulte G. 2021. Deconvolution of WNT-induced frizzled conformational dynamics with fluorescent biosensors. Biosens. Bioelectron. 177, 112948. (10.1016/j.bios.2020.112948) PubMed DOI

Kimura K, et al. . 1996. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245–248. (10.1126/science.273.5272.245) PubMed DOI

Martin AC, Goldstein B. 2014. Apical constriction: themes and variations on a cellular mechanism driving morphogenesis. Development 141, 1987–1998. (10.1242/dev.102228) PubMed DOI PMC

Hoffmeister M, Riha P, Neumüller O, Danielewski O, Schultess J, Smolenski AP. 2008. Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets. J. Biol. Chem. 283, 2297–2306. (10.1074/jbc.M706825200) PubMed DOI

Tsai IC, Amack JD, Gao ZH, Band V, Yost HJ, Virshup DM. 2007. A Wnt-CKIvarepsilon-Rap1 pathway regulates gastrulation by modulating SIPA1L1, A Rap GTPase activating protein. Dev. Cell 12, 335–347. (10.1016/j.devcel.2007.02.009) PubMed DOI PMC

Strutt H, Gamage J, Strutt D. 2019. Reciprocal action of casein kinase Iε on core planar polarity proteins regulates clustering and asymmetric localisation. eLife 8, e45107. (10.7554/eLife.45107) PubMed DOI PMC

Ossipova O, Kim K, Sokol SY. 2015. Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and myosin II signaling. Biol. Open 4, 722–730. (10.1242/bio.201511676) PubMed DOI PMC

Ossipova O, Chuykin I, Chu CW, Sokol SY. 2015. Vangl2 cooperates with Rab11 and myosin V to regulate apical constriction during vertebrate gastrulation. Development 142, 99–107. (10.1242/dev.111161) PubMed DOI PMC

Yoon J, Sun J, Lee M, Hwang YS, Daar IO. 2023. Wnt4 and ephrinB2 instruct apical constriction via dishevelled and non-canonical signaling. Nat. Commun. 14, 337. (10.1038/s41467-023-35991-6) PubMed DOI PMC

Tsuji T, Ohta Y, Kanno Y, Hirose K, Ohashi K, Mizuno K. 2010. Involvement of p114-RhoGEF and Lfc in Wnt-3a- and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells. Mol. Biol. Cell 21, 3590–3600. (10.1091/mbc.e10-02-0095) PubMed DOI PMC

Kratzer MC, Becker SFS, Grund A, Merks A, Harnoš J, Bryja V, Giehl K, Kashef J, Borchers A. 2020. The Rho guanine nucleotide exchange factor Trio is required for neural crest cell migration and interacts with Dishevelled. Development 147, dev186338. (10.1242/dev.186338) PubMed DOI

Peradziryi H, Tolwinski NS, Borchers A. 2012. The many roles of PTK7: a versatile regulator of cell–cell communication. Arch. Biochem. Biophys. 524, 71–76. (10.1016/j.abb.2011.12.019) PubMed DOI

Nishimura T, Takeichi M. 2008. Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling. Development 135, 1493–1502. (10.1242/dev.019646) PubMed DOI

Grego-Bessa J, Hildebrand J, Anderson KV. 2015. Morphogenesis of the mouse neural plate depends on distinct roles of cofilin 1 in apical and basal epithelial domains. Development 142, 1305–1314. (10.1242/dev.115493) PubMed DOI PMC

Mancini P, Ossipova O, Sokol SY. 2021. The dorsal blastopore lip is a source of signals inducing planar cell polarity in the Xenopus neural plate. Biol. Open 10, bio058761. (10.1242/bio.058761) PubMed DOI PMC

Seitz K, Dürsch V, Harnoš J, Bryja V, Gentzel M, Schambony A. 2014. β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca²⁺ pathway in Xenopus gastrulation. PLoS ONE 9, e87132. (10.1371/journal.pone.0087132) PubMed DOI PMC

Harnoš J, et al. . 2019. Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1. Nat. Commun. 10, 1804. (10.1038/s41467-019-09651-7) PubMed DOI PMC

Lindqvist M, et al. . 2010. Vang-like protein 2 and Rac1 interact to regulate adherens junctions. J. Cell. Sci. 123, 472–483. (10.1242/jcs.048074) PubMed DOI PMC

Itoh K, Ossipova O, Sokol SY. 2014. GEF-H1 functions in apical constriction and cell intercalations and is essential for vertebrate neural tube closure. J. Cell. Sci. 127, 2542–2553. (10.1242/jcs.146811) PubMed DOI PMC

Hollenstein DM, Maurer-Granofszky M, Reiter W, Anrather D, Gossenreiter T, Babic R, Hartl N, Kraft C, Hartl M. 2023. Chemical aacetylation of ligands and two-step digestion protocol for reducing codigestion in affinity purification-mass spectrometry. J. Proteome Res. 22, 3383–3391. (10.1021/acs.jproteome.3c00424) PubMed DOI PMC

Stejskal K, Potěšil D, Zdráhal Z. 2013. Suppression of peptide sample losses in autosampler vials. J. Proteome Res. 12, 3057–3062. (10.1021/pr400183v) PubMed DOI

Zahn N, et al. . 2022. Normal table of Xenopus development: a new graphical resource. Development 149, 14. (10.1242/dev.200356) PubMed DOI PMC

Butler MT, Wallingford JB. 2015. Control of vertebrate core planar cell polarity protein localization and dynamics by prickle 2. Development 142, 3429–3439. (10.1242/dev.121384) PubMed DOI PMC

Tadjuidje E, Cha SW, Louza M, Wylie C, Heasman J. 2011. The functions of maternal dishevelled 2 and 3 in the early Xenopus embryo. Dev. Dyn. 240, 1727–1736. (10.1002/dvdy.22671) PubMed DOI

Cox J, Mann M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372. (10.1038/nbt.1511) PubMed DOI

Cox J, Neuhauser N, Michalski A, Scheltema RA, Olsen JV, Mann M. 2011. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805. (10.1021/pr101065j) PubMed DOI

Perez-Riverol Y, et al. . 2019. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450. (10.1093/nar/gky1106) PubMed DOI PMC

Novotná Š, Maia LA, Radaszkiewicz KA, Roudnicky P, Harnos J. 2024. Supplementary material from: Linking planar polarity signaling to actomyosin contractility during vertebrate neurulation. Figshare (10.6084/m9.figshare.c.7542548) PubMed DOI

Linking planar polarity signalling to actomyosin contractility during vertebrate neurulation