bioRxiv preprint doi: https://doi.org/10.1101/770735; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1 Mechanical Roles of Vinculin/β-catenin interaction in Adherens Junction 2 3 Cristina Bertocchi1, Andrea Ravasio1, Hui Ting Ong2, Yusuke Toyama2,3, Pakorn Kanchanawong2,4 4 1 5 Pontificia Universidad Católica de Chile, Santiago Chile, 8330025. 6 2 Mechanobiology Institute, Singapore. National University of Singapore, Republic of Singapore, 7 117411. 8 3Department of Biological Sciences, National University of Singapore, Singapore, Republic of 9 Singapore, 117543. 10 4Department of Biomedical Engineering. National University of Singapore, Republic of Singapore, 11 117411. 12 13 Correspondence: [email protected] or [email protected] 14 15 Summary 16 Mechanical force transmission through the adherens junctions (AJs) are highly regulated 17 processes essential for multicellular organization of tissues. AJ proteins such as E-cadherin, α- 18 catenin, and vinculin have been shown to be sensing or bearing mechanical forces being transmitted 19 between the actin cytoskeleton and the intercellular contacts. However, the molecular organization 20 and connectivity of these components remains not well understood. Using a super-resolution 21 microscopy approach, we report that vinculin, once activated, could form a direct structural 22 connection with β-catenin, which can bypass α-catenin, one of the main mechanotransducers in AJs. 23 Direct vinculin/β-catenin interaction is capable of supporting mechanical tension and contributes to 24 the stabilization of the cadherin-catenin complexes. These findings suggest a multi-step model for 25 the force-dependent reinforcement of AJs whereby α-catenin may serve as the initial catalytic 26 activator of vinculin, followed by vinculin translocation to form a direct link between E-cadherin- 27 bound β-catenin and the actin cytoskeleton. 28 29 Keywords: vinculin, β-catenin, α-catenin, adherens junctions, mechanotransduction, super- 30 resolution microscopy 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 bioRxiv preprint doi: https://doi.org/10.1101/770735; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 46 Introduction 47 Within epithelial tissues, forces between cells are borne by specialized cell-cell contact 48 structures. The propagation of such forces are essential for developmental morphogenesis, tissue 49 homeostasis, and collective cell migration [1-4], and thus is subject to complex cell-type- and 50 context- specific spatiotemporal regulations. The Adherens Junctions (AJs) are prominent adhesive 51 structures in epithelia that physically couple neighbouring cells via Ca2+-dependent homophilic 52 interaction between the extracellular domains of classical cadherins [5-7]. The cadherin receptors in 53 AJs are, in turn, coupled with the actomyosin cytoskeleton via an extensive ensemble of cytoplasmic 54 proteins [8], thereby integrating the mechanobiological processes of cell-cell adhesions with many 55 important cellular pathways[9]. In recent years, the functions of AJs are increasingly better 56 understood across multiple length scales, particularly at the tissues and molecular levels[2]. 57 However, several aspects of AJ functions, especially at the nanoscale regime where AJ proteins are 58 collectively organized into supramolecular assembly, have been largely unexplored until recently 59 [10]. Hence, processes occurring at this length scale, including how mechanical force transmission 60 are mediated and regulated through AJs, remain less well understood. 61 The ternary complex of epithelial (E-) cadherin, β-catenin, and α-catenin, is widely 62 considered to be the main conduit of mechanical force in AJs [11]. The coupling of actomyosin 63 contractility into intramolecular tension in E-cadherin and α-catenin has been demonstrated using 64 Forster Resonance Energy Transfer (FRET) probes [12-14]. Moreover, cell biological and single- 65 molecule force spectroscopy studies have revealed a tension-dependent conformational changes in 66 α-catenin which gates the recruitment of vinculin, an adaptor protein that reinforces the connection 67 between AJs and the actin cytoskeleton[15, 16]. These proteins were observed by super-resolution 68 microscopy to form a compact cadherin-catenin compartment proximal to the plasma membrane, 69 with vinculin being centrally located between the cadherin-catenin compartment and the actin 70 cytoskeletons [17]. Furthermore, vinculin was found to be conformationally extended upon 71 mechanical and biochemical signals to modulate the AJ nanostructure as well as mechanical tension, 72 consistent with the molecular clutch model [17]. However, given the multi-functional context- 73 dependent roles of many AJ components [18, 19], the wealth of intra-AJ interaction partners[4], and 74 the high-density organization and dynamic plasticity of AJs, these findings could still represented 75 only the most prominent modes of action. Furthermore, the understanding of how these principal 76 AJ components function as an integrated system is still obscure. 77 Biochemical evidences thus far indicate that the linkage between cadherins and the 78 contractile actin cytoskeleton is indirect [8]. While recent studies have focused largely on the force 79 transmission hierarchy of E-cadherin/β-catenin/α-catenin/vinculin/actin, we note that several 80 alternate pathways may also participate in connecting cadherin with actin, including myosin VI [20], 81 EPLIN [21], α-actinin [22], and afadin [23, 24]. Additionally, β-catenin may interact directly with 82 vinculin [25], which could potentially form an alternate bypass connection from cadherin to the actin 83 cytoskeleton. Collectively, these suggest that in AJs a mixture of different mechanical connectors, 84 rather than a single dominant tether, may couple the cadherin receptors and actomyosin 85 cytoskeletons. However, these alternate interactions have been comparatively under-studied. In 86 this report, we investigated the alternate force transmission pathways in AJs using an integrated 87 approach of super-resolution microscopy, laser nanoscissor, and live-cell microscopy. We found that 88 vinculin, once activated, could form a mechanical connection directly to β-catenin, bypassing α- 89 catenin. However, α-catenin may be required for vinculin activation. These data is suggestive of a 90 multi-step mechanochemical dynamics whereby α-catenin may catalyse vinculin activation in a bioRxiv preprint doi: https://doi.org/10.1101/770735; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 91 force-dependent manner, followed by the translocation of vinculin to β-catenin to form a more 92 direct mechanical connection, potentially underpinning AJ reinforcement. 93 94 Results and Discussion 95 96 Super-resolution microscopy reveals an alternate nanoscale organization of vinculin in the absence 97 of α-catenin 98 In a recent study, we have demonstrated the use of cadherin biomimetic substrate system 99 to facilitate ultra-high resolution imaging of cadherin-based adhesions [17], Fig. 1A. The use of such 100 platform, in conjunction with surface-generated structured illumination microscopy [26, 27], permits 101 sub-20 nm precision analysis along the vertical (z)-axis perpendicular to the plasma membrane. This 102 helped elucidate the nanoscale stratification of proteins into distinct layers, and permit the 103 investigation of conformation changes in proteins such as vinculin [17]. Using this strategy to 104 investigate MDCK (Madin-Derby Canine Kidney) epithelial cells, we investigated the dependence of 105 the nanoscale architecture of E-cadherin-based adhesions on α-catenin, comparing control cells with 106 a stable cell line expressing α-catenin shRNA[28]. As shown in Fig. 1B-C, the fluorophore z-position 107 relative to the substrate surface (z = 0Dnm) was analysed pixel-wise, with the median value, zcentre, 108 for adhesion regions of interest (ROIs) used as the representative protein z-position. In both of 109 these conditions, immunostaining showed that endogenous vinculin localizes to the cadherin-based 110 adhesions (Fig. 1E-F). Using fluorescent protein (FP) fusion as probes, we found that E-cadherin (C- 111 terminal GFP fusion) was located at similar z-positions. In contrast, a drastic difference was observed 112 in the z-position of vinculin. As shown in Fig, 1C-D and Supplementary Table S1, in control MDCK, 113 vinculin is positioned proximal to the cadherin-catenin compartment (z <60 nm), but upon α-catenin 114 depletion, vinculin localizes to a much higher z-position (z>80 nm), coinciding with the actomyosin 115 compartment, probably via the association with actin or actin-regulatory proteins such as VASP or α- 116 actinin [29, 30]. As we have shown earlier [17], we found that the central position of vinculin is 117 restored upon re-expression of α-catenin indicative of the role of α-catenin in the central placement 118 of vinculin. 119 Next we sought to investigate the alternate localization of vinculin. Since vinculin is able to 120 switch between the compact and the extended