http://colil.dbcls.jp:18052/sparql?query=define%20sql%3Adescribe-mode%20%22CBD%22%20%20DESCRIBE%20%3Chttp%3A%2F%2Fpurl.jp%2Fbio%2F10%2Fcolil%2Fid%2F21666802%3E&output=application%2Fatom%2Bxml2024-03-29T00:23:00.260156ZOData Service and Descriptor DocumentnodeID://b4650550182024-03-29T00:23:00.260156Z5nodeID://b114057062024-03-29T00:23:00.260156ZTo enhance the strength of RNAi, the screening was done in flies expressing Dicer2 from a UAS-dcr2 transgene [>>19<<]. One hundred and forty-eight lines exhibiting either altered tissue growth or lethality were then re-screened for possible effects on Fat-Hippo signaling by assaying the expression of downstream targets of the pathway, Wingless (Wg) andnodeID://b4650551182024-03-29T00:23:00.260156Z3nodeID://b4650552182024-03-29T00:23:00.260156Z2nodeID://b4650550152024-03-29T00:23:00.260156Z6nodeID://b114057162024-03-29T00:23:00.260156Zdco encodes a kinase that phosphorylates the Fat cytoplasmic domain and participates in Fat-Hippo signaling [6],[>>28<<],[29]. The requirement for Dco within Fat signaling is uncovered by expression of an antimorphic isoform, Dco3. Expression of Dco3 induces wing overgrowth (Figure 1L) [29]. This overgrowth is suppressed by Zyx RNAi, suggesting that ZyxnodeID://b114057262024-03-29T00:23:00.260156ZIn addition to its effects on Wts, fat mutation also decreases the levels of Ex at the sub-apical membrane [30]–[>>33<<]. Zyx RNAi was not able to reverse this effect of fat on Ex levels (Figure S5G–N). Depletion of Zyx in the wing disc also did not have visible effects on F-actin (Figure S5O,P).nodeID://b4650553182024-03-29T00:23:00.260156Z2nodeID://b4650551152024-03-29T00:23:00.260156Z3nodeID://b4650552152024-03-29T00:23:00.260156Z2nodeID://b114057362024-03-29T00:23:00.260156ZHowever, in addition to their role in Hippo signaling, functions for LATS proteins have also been identified in mitosis, and LATS1 has been localized to the mitotic apparatus [36],[>>37<<]. In the context of a study of mitotic functions of LATS1, it was reported that the C-terminus of human Zyxin, including the LIM domains, could bind to human LATS1, even though full-length Zyxin did not bind [36]. When we expressed anodeID://b4650553152024-03-29T00:23:00.260156Z2nodeID://b114057462024-03-29T00:23:00.260156Zcan also be post-translationally regulated, as the ability of the LIM domains of human LATS1 to bind Zyxin is masked within full-length Zyxin, but uncovered by Cdc2-mediated phosphorylation, presumably due to conformational change [>>36<<]. We hypothesize that the ability of Dachs to bind to both the N-terminus and the LIM domains of Zyx enables it to effect a conformational change in Zyx, resulting in an open configuration that can bind to Wts (Figure 8B).nodeID://b114057562024-03-29T00:23:00.260156Zkinase activity, it has been observed that Ex can bind directly to Yki and that when Ex is over-expressed it can repress Yki through a mechanism that involves direct sequestration of Yki, rather than regulation of Yki phosphorylation [>>43<<],[44]. Because this direct repression mechanism was based on over-expression experiments, the extent to which it contributes to normal Yki regulation in vivo remained uncertain.nodeID://b114057662024-03-29T00:23:00.260156ZFinally, we note that theoretical models of growth control in developing tissues have proposed that growth should be controlled by mechanical tension [47],[>>48<<], and direct evidence for mechanical effects on growth has been obtained in cultured cell models [49].nodeID://b4650549682024-03-29T00:23:00.260156Z11nodeID://b4650550232024-03-29T00:23:00.260156Z5nodeID://b4650551232024-03-29T00:23:00.260156Z3nodeID://b4650550202024-03-29T00:23:00.260156Z5nodeID://b4650552232024-03-29T00:23:00.260156Z2nodeID://b4650553232024-03-29T00:23:00.260156Z2nodeID://b4650551202024-03-29T00:23:00.260156Z3nodeID://b4650552202024-03-29T00:23:00.260156Z2nodeID://b4650553202024-03-29T00:23:00.260156Z2nodeID://b4650550572024-03-29T00:23:00.260156Z4nodeID://b4650551572024-03-29T00:23:00.260156Z2nodeID://b4650549762024-03-29T00:23:00.260156Z9nodeID://b4650550542024-03-29T00:23:00.260156Z4nodeID://b4650552572024-03-29T00:23:00.260156Z2nodeID://b4650551542024-03-29T00:23:00.260156Z2nodeID://b4650549732024-03-29T00:23:00.260156Z10nodeID://b4650552542024-03-29T00:23:00.260156Z2nodeID://b4650549702024-03-29T00:23:00.260156Z11nodeID://b4650550882024-03-29T00:23:00.260156Z3nodeID://b4650551882024-03-29T00:23:00.260156Z2nodeID://b4650552882024-03-29T00:23:00.260156Z2nodeID://b4650550962024-03-29T00:23:00.260156Z3nodeID://b4650551962024-03-29T00:23:00.260156Z2nodeID://b4650552962024-03-29T00:23:00.260156Z2nodeID://b4650550932024-03-29T00:23:00.260156Z3nodeID://b4650551932024-03-29T00:23:00.260156Z2nodeID://b4650550902024-03-29T00:23:00.260156Z3nodeID://b4650552932024-03-29T00:23:00.260156Z2nodeID://b4650551902024-03-29T00:23:00.260156Z2nodeID://b4650552902024-03-29T00:23:00.260156Z2nodeID://b4650550052024-03-29T00:23:00.260156Z6nodeID://b4650551052024-03-29T00:23:00.260156Z3nodeID://b4650552052024-03-29T00:23:00.260156Z2nodeID://b4650550022024-03-29T00:23:00.260156Z7nodeID://b4650551022024-03-29T00:23:00.260156Z3nodeID://b4650553052024-03-29T00:23:00.260156Z2nodeID://b4650552022024-03-29T00:23:00.260156Z2nodeID://b4650553022024-03-29T00:23:00.260156Z2nodeID://b4650550392024-03-29T00:23:00.260156Z4nodeID://b4650551392024-03-29T00:23:00.260156Z3nodeID://b4650552392024-03-29T00:23:00.260156Z2nodeID://b4650553392024-03-29T00:23:00.260156Z2nodeID://b4650550102024-03-29T00:23:00.260156Z6nodeID://b4650551102024-03-29T00:23:00.260156Z3nodeID://b4650552102024-03-29T00:23:00.260156Z2nodeID://b4650550442024-03-29T00:23:00.260156Z4nodeID://b4650551442024-03-29T00:23:00.260156Z2nodeID://b4650549632024-03-29T00:23:00.260156Z17nodeID://b4650552442024-03-29T00:23:00.260156Z2nodeID://b4650550412024-03-29T00:23:00.260156Z4nodeID://b4650551412024-03-29T00:23:00.260156Z3nodeID://b4650553442024-03-29T00:23:00.260156Z2nodeID://b4650552412024-03-29T00:23:00.260156Z2nodeID://b4650553412024-03-29T00:23:00.260156Z2nodeID://b4650550782024-03-29T00:23:00.260156Z3nodeID://b4650549972024-03-29T00:23:00.260156Z7nodeID://b4650551782024-03-29T00:23:00.260156Z2nodeID://b4650550752024-03-29T00:23:00.260156Z3nodeID://b4650552782024-03-29T00:23:00.260156Z2nodeID://b4650549942024-03-29T00:23:00.260156Z8nodeID://b4650551752024-03-29T00:23:00.260156Z2nodeID://b4650552752024-03-29T00:23:00.260156Z2nodeID://b4650550802024-03-29T00:23:00.260156Z3nodeID://b4650551802024-03-29T00:23:00.260156Z2nodeID://b4650552802024-03-29T00:23:00.260156Z2nodeID://b4650550292024-03-29T00:23:00.260156Z5nodeID://b4650551292024-03-29T00:23:00.260156Z3nodeID://b4650552292024-03-29T00:23:00.260156Z2nodeID://b4650550262024-03-29T00:23:00.260156Z5nodeID://b4650551262024-03-29T00:23:00.260156Z3nodeID://b4650553292024-03-29T00:23:00.260156Z2nodeID://b4650552262024-03-29T00:23:00.260156Z2nodeID://b4650553262024-03-29T00:23:00.260156Z2nodeID://b4650549792024-03-29T00:23:00.260156Z9nodeID://b4650550312024-03-29T00:23:00.260156Z5nodeID://b4650551312024-03-29T00:23:00.260156Z3nodeID://b4650552312024-03-29T00:23:00.260156Z2nodeID://b4650553312024-03-29T00:23:00.260156Z2nodeID://b4650550652024-03-29T00:23:00.260156Z4nodeID://b114056732024-03-29T00:23:00.260156ZFat is large cadherin that acts as a transmembrane receptor for one branch of Hippo signaling [>>1<<]–[3],[5]. Fat-Hippo signaling influences the levels of Wts protein [6].nodeID://b4650551652024-03-29T00:23:00.260156Z2nodeID://b4650549842024-03-29T00:23:00.260156Z9nodeID://b114056832024-03-29T00:23:00.260156ZZyx is a Drosophila homologue of the vertebrate Zyxin, Lipoma preferred partner (LPP), and Thyroid-receptor interacting protein 6 (TRIP6) proteins [>>11<<],[12]. These proteins have three conserved LIM domains at their C-terminus, and they have been implicated in both cytoskeletal and transcriptional regulation [13]–[15].nodeID://b114056932024-03-29T00:23:00.260156Zmaterials and methodsnodeID://b4650552652024-03-29T00:23:00.260156Z2nodeID://b4650550622024-03-29T00:23:00.260156Z4nodeID://b4650551622024-03-29T00:23:00.260156Z2nodeID://b4650549812024-03-29T00:23:00.260156Z9nodeID://b4650552622024-03-29T00:23:00.260156Z2nodeID://b4650550992024-03-29T00:23:00.260156Z3nodeID://b4650551992024-03-29T00:23:00.260156Z2nodeID://b4650552992024-03-29T00:23:00.260156Z2nodeID://b4650550082024-03-29T00:23:00.260156Z6nodeID://b114057052024-03-29T00:23:00.260156ZresultsnodeID://b4650551082024-03-29T00:23:00.260156Z3nodeID://b4650552082024-03-29T00:23:00.260156Z2nodeID://b114057152024-03-29T00:23:00.260156Zdco encodes a kinase that phosphorylates the Fat cytoplasmic domain and participates in Fat-Hippo signaling [>>6<<],[28],[29].nodeID://b4650553082024-03-29T00:23:00.260156Z2nodeID://b114057252024-03-29T00:23:00.260156ZIn addition to its effects on Wts, fat mutation also decreases the levels of Ex at the sub-apical membrane [>>30<<]–[33]. Zyx RNAi was not able to reverse this effect of fat on Ex levels (Figure S5G–N). Depletion of Zyx in the wing disc also did not have visible effects on F-actin (Figure S5O,P).nodeID://b114057352024-03-29T00:23:00.260156ZHowever, in addition to their role in Hippo signaling, functions for LATS proteins have also been identified in mitosis, and LATS1 has been localized to the mitotic apparatus [>>36<<],[37]. In the context of a study of mitotic functions of LATS1, it was reported that the C-terminus of human Zyxin, including the LIM domains, could bind to human LATS1, even though full-length Zyxin did not bind [36]. When we expressed anodeID://b114057452024-03-29T00:23:00.260156ZSince Fat regulates the localization of Dachs [>>7<<], this regulated localization provides a mechanism by which Fat could modulate the interaction of Dachs with Zyx (although we note that Fat might affect the activity of Dachs in addition to affecting its localization).nodeID://b114057552024-03-29T00:23:00.260156ZHowever, as Wts appears to function within multi-protein complexes, including some components that can localize preferentially to the sub-apical membrane [41],[>>42<<], we hypothesize that Fat signaling affects a discrete pool of Wts within a complex at the membrane that is crucial for Hippo signaling, whereas there might be additional pools of Wts within the cell that are unaffected.nodeID://b114057652024-03-29T00:23:00.260156ZFinally, we note that theoretical models of growth control in developing tissues have proposed that growth should be controlled by mechanical tension [>>47<<],[48], and direct evidence for mechanical effects on growth has been obtained in cultured cell models [49].nodeID://b4650550162024-03-29T00:23:00.260156Z5nodeID://b4650551162024-03-29T00:23:00.260156Z3nodeID://b4650552162024-03-29T00:23:00.260156Z2nodeID://b4650550132024-03-29T00:23:00.260156Z6nodeID://b4650553162024-03-29T00:23:00.260156Z2nodeID://b4650551132024-03-29T00:23:00.260156Z3nodeID://b4650552132024-03-29T00:23:00.260156Z2nodeID://b4650553132024-03-29T00:23:00.260156Z2nodeID://b4650549692024-03-29T00:23:00.260156Z11nodeID://b4650553102024-03-29T00:23:00.260156Z2nodeID://b4650550472024-03-29T00:23:00.260156Z4nodeID://b4650549662024-03-29T00:23:00.260156Z12nodeID://b4650551472024-03-29T00:23:00.260156Z2nodeID://b4650552472024-03-29T00:23:00.260156Z2nodeID://b114056722024-03-29T00:23:00.260156ZThree interconnected, upstream branches of Hippo signaling have been characterized in Drosophila: Fat-dependent, Expanded-dependent, and Merlin-dependent [1]–[>>3<<]. These upstream branches converge on the kinase Warts (Wts), which can phosphorylate Yki. Phosphorylated Yki is retained in the cytoplasm, whereas unphosphorylated Yki can enter the nucleus and, in conjunction with DNA-binding partners,nodeID://b114056822024-03-29T00:23:00.260156Zdiscs and optic neuroepithelia, Dachs membrane localization is polarized within the plane of the tissue; this polarization reflects the graded expression of the Fat ligand Dachsous and the Fat pathway modulator Four-jointed [7],[9],[>>10<<]. The correlation of Dachs localization with Fat activity implicates Dachs regulation as a key step in Fat signaling, but how Dachs localization influences downstream events is unknown.nodeID://b114056922024-03-29T00:23:00.260156ZNotably, Zyxin has been implicated as playing a key role in mechanotransduction, as its localization to focal adhesions can be influenced by the application of mechanical tension to cells in culture [>>18<<].nodeID://b4650550522024-03-29T00:23:00.260156Z4nodeID://b4650551522024-03-29T00:23:00.260156Z2nodeID://b4650549712024-03-29T00:23:00.260156Z10nodeID://b4650552522024-03-29T00:23:00.260156Z2nodeID://b4650550892024-03-29T00:23:00.260156Z3nodeID://b4650551892024-03-29T00:23:00.260156Z2nodeID://b4650550862024-03-29T00:23:00.260156Z3nodeID://b4650552892024-03-29T00:23:00.260156Z2nodeID://b4650551862024-03-29T00:23:00.260156Z2nodeID://b4650552862024-03-29T00:23:00.260156Z2nodeID://b4650550832024-03-29T00:23:00.260156Z3nodeID://b4650551832024-03-29T00:23:00.260156Z2nodeID://b4650552832024-03-29T00:23:00.260156Z2nodeID://b114057042024-03-29T00:23:00.260156ZWts was detected using a published Wts anti-sera [>>6<<] at 1∶4,000. Protein bands were detected using anti-mouse IRdye680 and goat anti-rabbit IRdye800 (1∶10,000, LiCor) and scanning on a LiCor Odyssey.nodeID://b114057142024-03-29T00:23:00.260156ZIn the developing wing, fat and ex make substantial contributions to Yki regulation, whereas Mer has a lesser role [6],[24]–[>>27<<]. Thus, we investigated the relationship between the requirement for Zyx and those for fat and ex. Expression of fat or ex RNAi throughout the wing, under nub-Gal4 control, results in overgrown wings (Figure 1E,I,S). Strikingly, the wingnodeID://b4650550912024-03-29T00:23:00.260156Z3nodeID://b114057242024-03-29T00:23:00.260156ZThe influence of fat on Warts levels also requires dachs [>>6<<]. Zyx RNAi did not detectably affect Dachs localization (Figure S4D,E), nor did Zyx RNAi affect Fat localization (Figure S5E,F).nodeID://b4650551912024-03-29T00:23:00.260156Z2nodeID://b4650552912024-03-29T00:23:00.260156Z2nodeID://b114057342024-03-29T00:23:00.260156ZAs Dachs can also associate with Warts in co-immunoprecipitation assays [>>6<<], and both Zyx and dachs are required for the fat-dependent regulation of Wts levels, we also investigated binding between Zyx and Wts.nodeID://b114057442024-03-29T00:23:00.260156ZdiscussionnodeID://b114057542024-03-29T00:23:00.260156ZHowever, as Wts appears to function within multi-protein complexes, including some components that can localize preferentially to the sub-apical membrane [>>41<<],[42], we hypothesize that Fat signaling affects a discrete pool of Wts within a complex at the membrane that is crucial for Hippo signaling, whereas there might be additional pools of Wts within the cell that are unaffected.nodeID://b114056692024-03-29T00:23:00.260156ZThe Hippo pathway has emerged as an important regulator of growth during metazoan development, and its dysregulation is implicated in diverse cancers [1]–[>>3<<]. Hippo signaling is effected by transcriptional co-activator proteins, Yorkie (Yki) in Drosophila and YAP and TAZ in mammals [4].nodeID://b114057642024-03-29T00:23:00.260156ZAs the association of unconventional myosins with F-actin can also be influenced by external force [>>46<<], our discovery of binding between a myosin protein (Dachs) and Zyx raises the possibility that other myosins might also interact with Zyxin family proteins, which could potentially influence either their tension-based recruitment or theirnodeID://b114056792024-03-29T00:23:00.260156ZFat regulates the localization of Dachs to the sub-apical membrane: when fat is mutant, Dachs accumulates on the membrane around the entire circumference of the cell, and when Fat is over-expressed, Dachs is mostly cytoplasmic [>>7<<]. In imaginal discs and optic neuroepithelia, Dachs membrane localization is polarized within the plane of the tissue; this polarization reflects the graded expression of the Fat ligand Dachsous and the Fat pathway modulator Four-jointednodeID://b114056892024-03-29T00:23:00.260156ZIn cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesions and adherens junctions [>>13<<],[15],[18].nodeID://b114056992024-03-29T00:23:00.260156ZUAS lines employed include UAS-dco3 [29],[48], UAS-d:V5[9F] and UAS-d:V5 [50] [7], UAS-d:citrine [28] (B.K. Staley, unpublished), UAS-Zyx:V5, and UAS-Ypet:Zyx [>>35<<]. Gal4 lines employed include Dll-Gal4, ex-lacZ en-Gal4 UAS-GFP/CyO;UAS-dcr2/TM6b, en-Gal4/CyO; th-lacZ UAS-dcr2/TM6b, ci-Gal4 UAS-dcr2 [3] /TM6b, w UAS-dcr2[X]; nub-Gal4[ac-62], w; AyGal4 UAS-GFP/C yO;UAS-dcr2/TM6b, y w hs-FLP[122];nodeID://b4650550032024-03-29T00:23:00.260156Z6nodeID://b4650551032024-03-29T00:23:00.260156Z3nodeID://b4650552032024-03-29T00:23:00.260156Z2nodeID://b4650550002024-03-29T00:23:00.260156Z7nodeID://b4650551002024-03-29T00:23:00.260156Z3nodeID://b4650553032024-03-29T00:23:00.260156Z2nodeID://b4650552002024-03-29T00:23:00.260156Z2nodeID://b4650553002024-03-29T00:23:00.260156Z2nodeID://b4650550372024-03-29T00:23:00.260156Z5nodeID://b4650551372024-03-29T00:23:00.260156Z3nodeID://b4650550342024-03-29T00:23:00.260156Z5nodeID://b4650552372024-03-29T00:23:00.260156Z2nodeID://b4650551342024-03-29T00:23:00.260156Z3nodeID://b4650553372024-03-29T00:23:00.260156Z2nodeID://b4650552342024-03-29T00:23:00.260156Z2nodeID://b4650553342024-03-29T00:23:00.260156Z2nodeID://b4650550682024-03-29T00:23:00.260156Z4nodeID://b4650549872024-03-29T00:23:00.260156Z8nodeID://b4650551682024-03-29T00:23:00.260156Z2nodeID://b4650552682024-03-29T00:23:00.260156Z2nodeID://b4650550762024-03-29T00:23:00.260156Z3nodeID://b4650551762024-03-29T00:23:00.260156Z2nodeID://b4650550732024-03-29T00:23:00.260156Z3nodeID://b4650552762024-03-29T00:23:00.260156Z2nodeID://b4650549922024-03-29T00:23:00.260156Z8nodeID://b4650551732024-03-29T00:23:00.260156Z2nodeID://b4650550702024-03-29T00:23:00.260156Z4nodeID://b4650552732024-03-29T00:23:00.260156Z2nodeID://b4650551702024-03-29T00:23:00.260156Z2nodeID://b4650552702024-03-29T00:23:00.260156Z2nodeID://b114056682024-03-29T00:23:00.260156ZThe Hippo pathway has emerged as an important regulator of growth during metazoan development, and its dysregulation is implicated in diverse cancers [>>1<<]–[3]. Hippo signaling is effected by transcriptional co-activator proteins, Yorkie (Yki) in Drosophila and YAP and TAZ in mammals [4].nodeID://b4650550192024-03-29T00:23:00.260156Z5nodeID://b114056782024-03-29T00:23:00.260156ZThe molecular mechanism by which this is achieved is not understood, but dachs is genetically required for the influence of Fat on Wts levels, downstream gene expression, and organ growth [6]–[>>8<<]. Fat regulates the localization of Dachs to the sub-apical membrane: when fat is mutant, Dachs accumulates on the membrane around the entire circumference of the cell, and when Fat is over-expressed, Dachs is mostly cytoplasmic [7]. InnodeID://b114056882024-03-29T00:23:00.260156ZTranslocations involving LPP identified it as an oncogene involved in lipomas and other cancers [>>13<<]. In cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesions and adherens junctions [13],[15],[18]. Notably, Zyxin has been implicated as playing a key role innodeID://b4650551192024-03-29T00:23:00.260156Z3nodeID://b4650552192024-03-29T00:23:00.260156Z2nodeID://b114056982024-03-29T00:23:00.260156ZUAS lines employed include UAS-dco3 [29],[48], UAS-d:V5[9F] and UAS-d:V5 [50] [7], UAS-d:citrine [>>28<<] (B.K. Staley, unpublished), UAS-Zyx:nodeID://b4650553192024-03-29T00:23:00.260156Z2nodeID://b4650550242024-03-29T00:23:00.260156Z5nodeID://b4650551242024-03-29T00:23:00.260156Z3nodeID://b4650552242024-03-29T00:23:00.260156Z2nodeID://b4650550212024-03-29T00:23:00.260156Z5nodeID://b4650553242024-03-29T00:23:00.260156Z2nodeID://b4650551212024-03-29T00:23:00.260156Z3nodeID://b4650552212024-03-29T00:23:00.260156Z2nodeID://b4650553212024-03-29T00:23:00.260156Z2nodeID://b4650550582024-03-29T00:23:00.260156Z4nodeID://b4650549772024-03-29T00:23:00.260156Z9nodeID://b4650551582024-03-29T00:23:00.260156Z2nodeID://b4650550552024-03-29T00:23:00.260156Z4nodeID://b4650552582024-03-29T00:23:00.260156Z2nodeID://b4650549742024-03-29T00:23:00.260156Z10nodeID://b4650551552024-03-29T00:23:00.260156Z2nodeID://b4650552552024-03-29T00:23:00.260156Z2nodeID://b4650550632024-03-29T00:23:00.260156Z4nodeID://b4650551632024-03-29T00:23:00.260156Z2nodeID://b4650550602024-03-29T00:23:00.260156Z4nodeID://b4650551602024-03-29T00:23:00.260156Z2nodeID://b4650552602024-03-29T00:23:00.260156Z2nodeID://b4650550972024-03-29T00:23:00.260156Z3nodeID://b4650551972024-03-29T00:23:00.260156Z2nodeID://b4650550942024-03-29T00:23:00.260156Z3nodeID://b4650552972024-03-29T00:23:00.260156Z2nodeID://b4650551942024-03-29T00:23:00.260156Z2nodeID://b4650552942024-03-29T00:23:00.260156Z2nodeID://b4650550092024-03-29T00:23:00.260156Z6nodeID://b4650551092024-03-29T00:23:00.260156Z3nodeID://b4650550062024-03-29T00:23:00.260156Z6nodeID://b4650552092024-03-29T00:23:00.260156Z2nodeID://b4650553092024-03-29T00:23:00.260156Z2nodeID://b4650551062024-03-29T00:23:00.260156Z3nodeID://b4650552062024-03-29T00:23:00.260156Z2nodeID://b4650553062024-03-29T00:23:00.260156Z2nodeID://b4650550112024-03-29T00:23:00.260156Z6nodeID://b4650551112024-03-29T00:23:00.260156Z3nodeID://b4650552112024-03-29T00:23:00.260156Z2nodeID://b4650553112024-03-29T00:23:00.260156Z2nodeID://b114056712024-03-29T00:23:00.260156ZThree interconnected, upstream branches of Hippo signaling have been characterized in Drosophila: Fat-dependent, Expanded-dependent, and Merlin-dependent [>>1<<]–[3]. These upstream branches converge on the kinase Warts (Wts), which can phosphorylate Yki. Phosphorylated Yki is retained in the cytoplasm, whereas unphosphorylated Yki can enter the nucleus and, in conjunction with DNA-bindingnodeID://b4650550452024-03-29T00:23:00.260156Z4nodeID://b114056812024-03-29T00:23:00.260156Zdiscs and optic neuroepithelia, Dachs membrane localization is polarized within the plane of the tissue; this polarization reflects the graded expression of the Fat ligand Dachsous and the Fat pathway modulator Four-jointed [7],[>>9<<],[10]. The correlation of Dachs localization with Fat activity implicates Dachs regulation as a key step in Fat signaling, but how Dachs localization influences downstream events is unknown.nodeID://b4650551452024-03-29T00:23:00.260156Z2nodeID://b4650549642024-03-29T00:23:00.260156Z14nodeID://b4650552452024-03-29T00:23:00.260156Z2nodeID://b4650550422024-03-29T00:23:00.260156Z4nodeID://b114056912024-03-29T00:23:00.260156ZIn cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesions and adherens junctions [13],[15],[>>18<<]. Notably, Zyxin has been implicated as playing a key role in mechanotransduction, as its localization to focal adhesions can be influenced by the application of mechanical tension to cells in culture [18].nodeID://b4650551422024-03-29T00:23:00.260156Z2nodeID://b4650553452024-03-29T00:23:00.260156Z2nodeID://b4650552422024-03-29T00:23:00.260156Z2nodeID://b4650553422024-03-29T00:23:00.260156Z2nodeID://b4650550792024-03-29T00:23:00.260156Z3nodeID://b4650549982024-03-29T00:23:00.260156Z7nodeID://b4650551792024-03-29T00:23:00.260156Z2nodeID://b4650552792024-03-29T00:23:00.260156Z2nodeID://b4650549952024-03-29T00:23:00.260156Z8nodeID://b4650550842024-03-29T00:23:00.260156Z3nodeID://b4650551842024-03-29T00:23:00.260156Z2nodeID://b114057032024-03-29T00:23:00.260156ZCo-immunoprecipitation assays were performed as described previously [>>6<<]. Cell lysates were cleared using protein G beads (Sigma). Anti-V5 or anti-FLAG M2 beads (Sigma) were incubated with cell lysates overnight at 4°C, then washed six times with RIPA buffer and boiled in SDS-PAGE loading buffer.nodeID://b114057132024-03-29T00:23:00.260156ZIn the developing wing, fat and ex make substantial contributions to Yki regulation, whereas Mer has a lesser role [6],[>>24<<]–[27]. Thus, we investigated the relationship between the requirement for Zyx and those for fat and ex. Expression of fat or ex RNAi throughout the wing, under nub-Gal4 control, results in overgrown wings (Figure 1E,I,S). Strikingly, thenodeID://b4650552842024-03-29T00:23:00.260156Z2nodeID://b4650550812024-03-29T00:23:00.260156Z3nodeID://b114057232024-03-29T00:23:00.260156ZFat exerts a post-transcriptional influence on the levels of Wts protein [>>6<<]. The genetic placement of Zyx upstream of wts and within the Fat branch of the pathway suggested that Zyx might also affect Wts levels.nodeID://b4650551812024-03-29T00:23:00.260156Z2nodeID://b114057332024-03-29T00:23:00.260156ZA distinguishing feature of Dachs localization is its polarization within the plane of the epithelium, which occurs in response to the Fj and Ds gradients (Figure 6J) [7],[>>9<<]. Zyx, by contrast, is not planar-polarized (Figure 6I); hence, Zyx and Dachs are expected to overlap on only one side of wing disc cells. A distinguishing feature of Zyx staining is that it often displays puncta of larger, more intensenodeID://b4650552812024-03-29T00:23:00.260156Z2nodeID://b114057432024-03-29T00:23:00.260156ZBy contrast to the crucial role of Dachs in stimulating binding between full-length Zyx and Wts, full-length Jub binds efficiently to Wts, and full-length vertebrate homologues of Jub bind to LATS proteins [39],[>>40<<]. Moreover, Jub bound only very weakly Dachs (Figure S6B). Thus, although Zyx and Jub share the ability to associate with Wts through their LIM domains, both genetic and biochemical studies indicate that the regulation and consequences ofnodeID://b114057532024-03-29T00:23:00.260156ZAs Jub is equally required for both Fat-Hippo and Ex-Hippo signaling and acts genetically between hippo and wts [>>40<<], Jub appears to inhibit Wts activation.nodeID://b114057632024-03-29T00:23:00.260156ZZyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension [15],[18],[>>45<<]. The observation that increasing tension on stress fibers stimulates Zyxin accumulation at focal adhesions is intriguing in light of our observation that Zyx tends to accumulate at higher levels at intercellular vertices in imaginalnodeID://b4650550272024-03-29T00:23:00.260156Z5nodeID://b4650551272024-03-29T00:23:00.260156Z3nodeID://b4650552272024-03-29T00:23:00.260156Z2nodeID://b4650553272024-03-29T00:23:00.260156Z2nodeID://b114056702024-03-29T00:23:00.260156ZHippo signaling is effected by transcriptional co-activator proteins, Yorkie (Yki) in Drosophila and YAP and TAZ in mammals [>>4<<]. Three interconnected, upstream branches of Hippo signaling have been characterized in Drosophila: Fat-dependent, Expanded-dependent, and Merlin-dependent [1]–[3]. These upstream branches converge on the kinase Warts (Wts), which cannodeID://b114056802024-03-29T00:23:00.260156ZIn imaginal discs and optic neuroepithelia, Dachs membrane localization is polarized within the plane of the tissue; this polarization reflects the graded expression of the Fat ligand Dachsous and the Fat pathway modulator Four-jointed [>>7<<],[9],[10]. The correlation of Dachs localization with Fat activity implicates Dachs regulation as a key step in Fat signaling, but how Dachs localization influences downstream events is unknown.nodeID://b114056902024-03-29T00:23:00.260156ZIn cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesions and adherens junctions [13],[>>15<<],[18]. Notably, Zyxin has been implicated as playing a key role in mechanotransduction, as its localization to focal adhesions can be influenced by the application of mechanical tension to cells in culture [18].nodeID://b4650550322024-03-29T00:23:00.260156Z5nodeID://b4650551322024-03-29T00:23:00.260156Z3nodeID://b4650552322024-03-29T00:23:00.260156Z2nodeID://b4650553322024-03-29T00:23:00.260156Z2nodeID://b4650550692024-03-29T00:23:00.260156Z4nodeID://b4650551692024-03-29T00:23:00.260156Z2nodeID://b4650550662024-03-29T00:23:00.260156Z4nodeID://b4650552692024-03-29T00:23:00.260156Z2nodeID://b4650549852024-03-29T00:23:00.260156Z9nodeID://b4650551662024-03-29T00:23:00.260156Z2nodeID://b4650552662024-03-29T00:23:00.260156Z2nodeID://b4650549822024-03-29T00:23:00.260156Z9nodeID://b4650552632024-03-29T00:23:00.260156Z2nodeID://b114057022024-03-29T00:23:00.260156ZFehon), rat anti-Fat (1∶400) [29], mouse anti-V5 (1∶400, Invitrogen), mouse anti-Wg (1∶400, DSHB), and rabbit anti-Yki (1∶400) [>>22<<]. F-actin was stained using Alexa Fluor 546 phalloidin (1∶100, Invitrogen), and DNA was stained using Hoechst (Invitrogen).nodeID://b114057122024-03-29T00:23:00.260156ZIn the developing wing, fat and ex make substantial contributions to Yki regulation, whereas Mer has a lesser role [>>6<<],[24]–[27].nodeID://b4650550712024-03-29T00:23:00.260156Z4nodeID://b4650551712024-03-29T00:23:00.260156Z2nodeID://b114057222024-03-29T00:23:00.260156ZHowever, the observation that Zyx depletion could enhance the small wing phenotype of a putative null allele of dachs (Figures 1S, S1E,F) [>>7<<] implies that Zyx also has some Dachs-independent influence on growth.nodeID://b114057322024-03-29T00:23:00.260156ZA distinguishing feature of Dachs localization is its polarization within the plane of the epithelium, which occurs in response to the Fj and Ds gradients (Figure 6J) [>>7<<],[9]. Zyx, by contrast, is not planar-polarized (Figure 6I); hence, Zyx and Dachs are expected to overlap on only one side of wing disc cells. A distinguishing feature of Zyx staining is that it often displays puncta of larger, morenodeID://b4650552712024-03-29T00:23:00.260156Z2nodeID://b114057422024-03-29T00:23:00.260156ZBy contrast to the crucial role of Dachs in stimulating binding between full-length Zyx and Wts, full-length Jub binds efficiently to Wts, and full-length vertebrate homologues of Jub bind to LATS proteins [>>39<<],[40]. Moreover, Jub bound only very weakly Dachs (Figure S6B). Thus, although Zyx and Jub share the ability to associate with Wts through their LIM domains, both genetic and biochemical studies indicate that the regulation andnodeID://b114057522024-03-29T00:23:00.260156Zeffect on Wts levels, by contrast to the Hippo-pathway-mediated effect on Wts kinase activity, established the concept of distinct mechanisms for regulating Wts—one that affects Wts levels and another that affects Wts activity [>>6<<]. Our identification of distinct genetic requirements for Zyx and Jub provide further support for this concept.nodeID://b114056672024-03-29T00:23:00.260156ZintroductionnodeID://b114057622024-03-29T00:23:00.260156ZZyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension [15],[>>18<<],[45]. The observation that increasing tension on stress fibers stimulates Zyxin accumulation at focal adhesions is intriguing in light of our observation that Zyx tends to accumulate at higher levels at intercellular vertices in imaginalnodeID://b114056772024-03-29T00:23:00.260156ZThe molecular mechanism by which this is achieved is not understood, but dachs is genetically required for the influence of Fat on Wts levels, downstream gene expression, and organ growth [>>6<<]–[8]. Fat regulates the localization of Dachs to the sub-apical membrane: when fat is mutant, Dachs accumulates on the membrane around the entire circumference of the cell, and when Fat is over-expressed, Dachs is mostly cytoplasmic [7].nodeID://b114056872024-03-29T00:23:00.260156ZGene-targeted mutations in murine Zyxin or Lpp have no significant effect on mouse development, presumably due to redundancy among family members [16],[>>17<<]. Translocations involving LPP identified it as an oncogene involved in lipomas and other cancers [13]. In cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focal adhesionsnodeID://b114056972024-03-29T00:23:00.260156ZUAS lines employed include UAS-dco3 [29],[48], UAS-d:V5[9F] and UAS-d:V5 [50] [>>7<<], UAS-d:citrine [28] (B.K. Staley, unpublished), UAS-Zyx:V5, and UAS-Ypet:Zyx [35]. Gal4 lines employed include Dll-Gal4, ex-lacZ en-Gal4 UAS-GFP/CyO;UAS-dcr2/TM6b, en-Gal4/CyO; th-lacZ UAS-dcr2/TM6b, ci-Gal4 UAS-dcr2 [3] /TM6b, wnodeID://b4650550172024-03-29T00:23:00.260156Z5nodeID://b4650551172024-03-29T00:23:00.260156Z3nodeID://b4650552172024-03-29T00:23:00.260156Z2nodeID://b4650550142024-03-29T00:23:00.260156Z6nodeID://b4650553172024-03-29T00:23:00.260156Z2nodeID://b4650551142024-03-29T00:23:00.260156Z3nodeID://b4650552142024-03-29T00:23:00.260156Z2nodeID://b4650553142024-03-29T00:23:00.260156Z2nodeID://b4650550482024-03-29T00:23:00.260156Z4nodeID://b4650551482024-03-29T00:23:00.260156Z2nodeID://b4650549672024-03-29T00:23:00.260156Z11nodeID://b114057092024-03-29T00:23:00.260156ZBased on these experiments, a single gene, Zyx102 (Zyx) [>>11<<],[12], which is located at 102F7 near the tip of the fourth chromosome, was identified as a novel component of the Fat-Hippo signaling pathway.nodeID://b114057192024-03-29T00:23:00.260156ZLike Zyx, dachs is required for normal wing and leg growth and acts genetically downstream of fat and dco but upstream of warts [>>6<<]–[8]. To examine the genetic relationship between Zyx and dachs, we took advantage of the observation that over-expression of Dachs can promote wing overgrowth (Figure 1Q) [7].nodeID://b4650552482024-03-29T00:23:00.260156Z2nodeID://b114057292024-03-29T00:23:00.260156ZPCP phenotypes have also been described in this region of dachs mutant wings [>>34<<]. Zyx RNAi, by contrast, had no detectable effect on wing PCP (Figure S1N), and a PCP phenotype was also still detected in fat Zyx double RNAi wings (Figure S1O). Genes previously identified as influencing Fat-PCP signaling (i.e., fat, ds,nodeID://b114057392024-03-29T00:23:00.260156ZZyx is a Drosophila member of a group of cytoskeletal-associated proteins with three C-terminal LIM domains [>>38<<]. These comprise two families:nodeID://b114057492024-03-29T00:23:00.260156ZPrior studies identified two mechanisms by which Fat signaling could influence Yki activity, as fat mutation reduces both the levels of Wts protein [6] and the amount of Ex at the sub-apical membrane [31]–[>>33<<]. It has not been possible to completely uncouple these two pathways for Fat-Hippo signaling, although the observation that over-expression of Wts can efficiently suppress fat overgrowth phenotypes, but only partially suppresses exnodeID://b114057592024-03-29T00:23:00.260156ZAlthough the functional significance of this interaction in vertebrates has not yet been established, our observations raise the possibility that the oncogenic effects of human LPP mutations [>>13<<] could be due to an ability of these aberrant LPP fusion proteins to negatively regulate LATS proteins, resulting in inappropriate activation of YAP or TAZ.nodeID://b4650550562024-03-29T00:23:00.260156Z4nodeID://b4650551562024-03-29T00:23:00.260156Z2nodeID://b4650550532024-03-29T00:23:00.260156Z4nodeID://b4650552562024-03-29T00:23:00.260156Z2nodeID://b4650549722024-03-29T00:23:00.260156Z10nodeID://b4650551532024-03-29T00:23:00.260156Z2nodeID://b4650552532024-03-29T00:23:00.260156Z2nodeID://b4650550502024-03-29T00:23:00.260156Z4nodeID://b4650551502024-03-29T00:23:00.260156Z2nodeID://b4650552502024-03-29T00:23:00.260156Z2nodeID://b4650550872024-03-29T00:23:00.260156Z3nodeID://b4650551872024-03-29T00:23:00.260156Z2nodeID://b4650552872024-03-29T00:23:00.260156Z2nodeID://b114056762024-03-29T00:23:00.260156ZFat-Hippo signaling influences the levels of Wts protein [>>6<<]. The molecular mechanism by which this is achieved is not understood, but dachs is genetically required for the influence of Fat on Wts levels, downstream gene expression, and organ growth [6]–[8].nodeID://b114056862024-03-29T00:23:00.260156ZGene-targeted mutations in murine Zyxin or Lpp have no significant effect on mouse development, presumably due to redundancy among family members [>>16<<],[17]. Translocations involving LPP identified it as an oncogene involved in lipomas and other cancers [13]. In cultured cell assays, Zyxin and its paralogues can affect cell motility and actin polymerization and can localize to focalnodeID://b114056962024-03-29T00:23:00.260156ZUAS lines employed include UAS-dco3 [29],[48], UAS-d:V5[9F] and UAS-d:V5 [>>50<<] [7], UAS-d:nodeID://b4650550922024-03-29T00:23:00.260156Z3nodeID://b4650551922024-03-29T00:23:00.260156Z2nodeID://b4650552922024-03-29T00:23:00.260156Z2nodeID://b4650550042024-03-29T00:23:00.260156Z6nodeID://b4650551042024-03-29T00:23:00.260156Z3nodeID://b4650550012024-03-29T00:23:00.260156Z7nodeID://b4650552042024-03-29T00:23:00.260156Z2nodeID://b4650553042024-03-29T00:23:00.260156Z2nodeID://b4650551012024-03-29T00:23:00.260156Z3nodeID://b4650552012024-03-29T00:23:00.260156Z2nodeID://b4650553012024-03-29T00:23:00.260156Z2nodeID://b4650550382024-03-29T00:23:00.260156Z4nodeID://b4650551382024-03-29T00:23:00.260156Z3nodeID://b114057082024-03-29T00:23:00.260156Ztissue growth or lethality were then re-screened for possible effects on Fat-Hippo signaling by assaying the expression of downstream targets of the pathway, Wingless (Wg) and thread (th, more commonly referred to as Diap1) [20],[>>21<<], in wing discs in which RNAi lines were expressed in anterior cells under ci-Gal4 control (Table S1).nodeID://b4650552382024-03-29T00:23:00.260156Z2nodeID://b4650550352024-03-29T00:23:00.260156Z5nodeID://b114057182024-03-29T00:23:00.260156ZExpression of Dco3 induces wing overgrowth (Figure 1L) [>>29<<]. This overgrowth is suppressed by Zyx RNAi, suggesting that Zyx acts downstream of dco (Figure 1P,S).nodeID://b4650551352024-03-29T00:23:00.260156Z3nodeID://b114057282024-03-29T00:23:00.260156ZIn addition to regulating transcription, Fat also regulates planar cell polarity (PCP) (reviewed in [1],[>>5<<]). PCP in the adult wing is manifest in the orientation of wing hairs, which point distally.nodeID://b4650553382024-03-29T00:23:00.260156Z2nodeID://b114057382024-03-29T00:23:00.260156ZThe discovery of this latent ability of Zyx to bind Wts, together with our discovery of Zyx-Dachs binding, and previous identification of Dachs-Wts binding [>>6<<], indicates that Dachs, Zyx, and Wts each have the ability to bind to one another.nodeID://b4650552352024-03-29T00:23:00.260156Z2nodeID://b4650553352024-03-29T00:23:00.260156Z2nodeID://b114057482024-03-29T00:23:00.260156ZPrior studies identified two mechanisms by which Fat signaling could influence Yki activity, as fat mutation reduces both the levels of Wts protein [6] and the amount of Ex at the sub-apical membrane [>>31<<]–[33]. It has not been possible to completely uncouple these two pathways for Fat-Hippo signaling, although the observation that over-expression of Wts can efficiently suppress fat overgrowth phenotypes, but only partially suppresses exnodeID://b4650549882024-03-29T00:23:00.260156Z8nodeID://b4650550432024-03-29T00:23:00.260156Z4nodeID://b4650551432024-03-29T00:23:00.260156Z2nodeID://b4650550402024-03-29T00:23:00.260156Z4nodeID://b4650552432024-03-29T00:23:00.260156Z2nodeID://b4650551402024-03-29T00:23:00.260156Z3nodeID://b4650553432024-03-29T00:23:00.260156Z2nodeID://b4650552402024-03-29T00:23:00.260156Z2nodeID://b4650553402024-03-29T00:23:00.260156Z2nodeID://b4650550772024-03-29T00:23:00.260156Z3nodeID://b4650549962024-03-29T00:23:00.260156Z7nodeID://b4650551772024-03-29T00:23:00.260156Z2nodeID://b4650550742024-03-29T00:23:00.260156Z3nodeID://b4650552772024-03-29T00:23:00.260156Z2nodeID://b4650549932024-03-29T00:23:00.260156Z8nodeID://b4650551742024-03-29T00:23:00.260156Z2nodeID://b4650552742024-03-29T00:23:00.260156Z2nodeID://b4650549902024-03-29T00:23:00.260156Z8nodeID://b4650550252024-03-29T00:23:00.260156Z5nodeID://b4650551252024-03-29T00:23:00.260156Z3nodeID://b4650550222024-03-29T00:23:00.260156Z5nodeID://b4650552252024-03-29T00:23:00.260156Z2nodeID://b4650553252024-03-29T00:23:00.260156Z2nodeID://b4650551222024-03-29T00:23:00.260156Z3nodeID://b4650552222024-03-29T00:23:00.260156Z2nodeID://b4650553222024-03-29T00:23:00.260156Z2nodeID://b4650550592024-03-29T00:23:00.260156Z4nodeID://b4650551592024-03-29T00:23:00.260156Z2nodeID://b4650549782024-03-29T00:23:00.260156Z9nodeID://b4650552592024-03-29T00:23:00.260156Z2nodeID://b4650549752024-03-29T00:23:00.260156Z10nodeID://b4650550642024-03-29T00:23:00.260156Z4nodeID://b114057012024-03-29T00:23:00.260156ZFehon), rat anti-Fat (1∶400) [>>29<<], mouse anti-V5 (1∶400, Invitrogen), mouse anti-Wg (1∶400, DSHB), and rabbit anti-Yki (1∶400) [22].nodeID://b4650551642024-03-29T00:23:00.260156Z2nodeID://b114057112024-03-29T00:23:00.260156Zthe sub-cellular localization of Yki: activation of Hippo signaling promotes cytoplasmic localization of Yki, whereas inactivation of Hippo signaling allows nuclear localization of Yki, which corresponds to Yki activation [22],[>>23<<]. Zyx RNAi reduced nuclear Yki. This effect was subtle at late third instar, when levels of Yki in the nucleus are already low, but was evident in younger wing discs, which have higher levels of nuclear Yki (Figure 2C,D).nodeID://b4650552642024-03-29T00:23:00.260156Z2nodeID://b4650550612024-03-29T00:23:00.260156Z4nodeID://b4650549802024-03-29T00:23:00.260156Z9nodeID://b4650551612024-03-29T00:23:00.260156Z2nodeID://b114057212024-03-29T00:23:00.260156ZTo examine the genetic relationship between Zyx and dachs, we took advantage of the observation that over-expression of Dachs can promote wing overgrowth (Figure 1Q) [>>7<<]. This overgrowth was completely suppressed by Zyx RNAi (Figures 1S, S1G), as was the influence of Dachs over-expression on ex-lacZ expression (Figure S4A,B). Thus, Zyx is required for Dachs-promoted activation of Yki. Over-expression ofnodeID://b114057312024-03-29T00:23:00.260156ZThis is similar to the membrane localization of Dachs [>>7<<]. Indeed, when we compared Zyx and Dachs localization, using epitope-tagged constructs, we observed that the membrane staining is at the same apical-basal position and that they partially co-localize (Figure 6G,H).nodeID://b4650552612024-03-29T00:23:00.260156Z2nodeID://b114057412024-03-29T00:23:00.260156Zet al. (2010) recently reported that mutation or RNAi-mediated depletion of Jub reduces growth through interactions with the Hippo pathway, and through genetic and protein interaction experiments positioned Jub as a regulator of Wts [>>40<<]. In agreement with this, we found that RNAi-mediated depletion of Jub reduces wing growth (Figure 1M,N,S), expression of Hippo pathway target genes, and nuclear Yki (Figure S7), and that wts is epistatic to Jub (Figure 3C).nodeID://b114057512024-03-29T00:23:00.260156ZIntriguingly, mutation of dachs did suppress the influence of fat on Ex levels [>>30<<]. Although it is possible that this difference between dachs and Zyx results from technical differences in the experimental paradigms (e.g., mutant clones versus RNAi), it is also possible that dachs can influence Ex levels independentlynodeID://b4650550982024-03-29T00:23:00.260156Z3nodeID://b4650551982024-03-29T00:23:00.260156Z2nodeID://b114057612024-03-29T00:23:00.260156ZZyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension [>>15<<],[18],[45].nodeID://b4650550952024-03-29T00:23:00.260156Z3nodeID://b4650552982024-03-29T00:23:00.260156Z2nodeID://b4650551952024-03-29T00:23:00.260156Z2nodeID://b4650552952024-03-29T00:23:00.260156Z2nodeID://b4650550072024-03-29T00:23:00.260156Z6nodeID://b4650551072024-03-29T00:23:00.260156Z3nodeID://b4650552072024-03-29T00:23:00.260156Z2nodeID://b4650553072024-03-29T00:23:00.260156Z2nodeID://b4650550122024-03-29T00:23:00.260156Z6nodeID://b4650551122024-03-29T00:23:00.260156Z3nodeID://b4650552122024-03-29T00:23:00.260156Z2nodeID://b4650553122024-03-29T00:23:00.260156Z2nodeID://b114057582024-03-29T00:23:00.260156ZThe ability of Zyx LIM domains to interact with Wts is conserved in their human homologues [>>36<<]. Although the functional significance of this interaction in vertebrates has not yet been established, our observations raise the possibility that the oncogenic effects of human LPP mutations [13] could be due to an ability of thesenodeID://b4650550492024-03-29T00:23:00.260156Z4nodeID://b4650551492024-03-29T00:23:00.260156Z2nodeID://b4650552492024-03-29T00:23:00.260156Z2nodeID://b4650550462024-03-29T00:23:00.260156Z4nodeID://b4650551462024-03-29T00:23:00.260156Z2nodeID://b4650549652024-03-29T00:23:00.260156Z13nodeID://b4650552462024-03-29T00:23:00.260156Z2nodeID://b4650553462024-03-29T00:23:00.260156Z2nodeID://b4650549622024-03-29T00:23:00.260156Z18nodeID://b4650549992024-03-29T00:23:00.260156Z7nodeID://b114057002024-03-29T00:23:00.260156ZGal4 lines employed include Dll-Gal4, ex-lacZ en-Gal4 UAS-GFP/CyO;UAS-dcr2/TM6b, en-Gal4/CyO; th-lacZ UAS-dcr2/TM6b, ci-Gal4 UAS-dcr2 [>>3<<] /TM6b, w UAS-dcr2[X]; nub-Gal4[ac-62], w; AyGal4 UAS-GFP/C yO;UAS-dcr2/TM6b, y w hs-FLP[122]; AyGal4 UAS-GFP/CyO, tub-Gal80ts/CyO,Act-GFP; tub-Gal4 UAS-dcr2/ TM6b, w; tub-Gal4/CyO-GFP.nodeID://b114057102024-03-29T00:23:00.260156Zcontrolling the sub-cellular localization of Yki: activation of Hippo signaling promotes cytoplasmic localization of Yki, whereas inactivation of Hippo signaling allows nuclear localization of Yki, which corresponds to Yki activation [>>22<<],[23]. Zyx RNAi reduced nuclear Yki. This effect was subtle at late third instar, when levels of Yki in the nucleus are already low, but was evident in younger wing discs, which have higher levels of nuclear Yki (Figure 2C,D).nodeID://b4650550512024-03-29T00:23:00.260156Z4nodeID://b4650551512024-03-29T00:23:00.260156Z2nodeID://b114057202024-03-29T00:23:00.260156ZLike Zyx, dachs is required for normal wing and leg growth and acts genetically downstream of fat and dco but upstream of warts [6]–[>>8<<]. To examine the genetic relationship between Zyx and dachs, we took advantage of the observation that over-expression of Dachs can promote wing overgrowth (Figure 1Q) [7].nodeID://b114057302024-03-29T00:23:00.260156ZWe also examined a UAS-Ypet:Zyx transgene [>>35<<]. Although our localization studies are subject to the caveat that Zyx protein was over-expressed, the two different tagged Zyx proteins have similar localization profiles, and similar localization profiles were observed using differentnodeID://b4650552512024-03-29T00:23:00.260156Z2nodeID://b114057402024-03-29T00:23:00.260156ZDrosophila have a single member of each family; Zyx is a member of the Zyxin family, and Ajuba LIM protein (Jub) is a member of the Ajuba family. Ajuba has been reported to interact with a human homologue of Warts, LATS2 [>>39<<], and Das Thakur et al. (2010) recently reported that mutation or RNAi-mediated depletion of Jub reduces growth through interactions with the Hippo pathway, and through genetic and protein interaction experiments positioned Jub as anodeID://b114057502024-03-29T00:23:00.260156Zto completely uncouple these two pathways for Fat-Hippo signaling, although the observation that over-expression of Wts can efficiently suppress fat overgrowth phenotypes, but only partially suppresses ex overgrowth phenotypes [>>30<<], suggested that the influence of Fat on Wts levels might be more critical.nodeID://b114057602024-03-29T00:23:00.260156ZOne of the most intriguing aspects of Zyxin family proteins is their role in mediating effects of mechanical force on cell behavior [>>18<<]. Zyxin family proteins can localize to focal adhesions of cultured fibroblasts, and this localization is modulated by mechanical tension [15],[18],[45].nodeID://b4650550852024-03-29T00:23:00.260156Z3nodeID://b114056752024-03-29T00:23:00.260156ZFat is large cadherin that acts as a transmembrane receptor for one branch of Hippo signaling [1]–[3],[>>5<<]. Fat-Hippo signaling influences the levels of Wts protein [6].nodeID://b114056852024-03-29T00:23:00.260156ZThese proteins have three conserved LIM domains at their C-terminus, and they have been implicated in both cytoskeletal and transcriptional regulation [13]–[>>15<<]. Gene-targeted mutations in murine Zyxin or Lpp have no significant effect on mouse development, presumably due to redundancy among family members [16],[17]. Translocations involving LPP identified it as an oncogene involved in lipomasnodeID://b4650551852024-03-29T00:23:00.260156Z2nodeID://b4650552852024-03-29T00:23:00.260156Z2nodeID://b4650550822024-03-29T00:23:00.260156Z3nodeID://b114056952024-03-29T00:23:00.260156ZBoth Zyx RNAi lines gave similar effects on growth and gene expression in combination with multiple Gal4 lines and also behaved similarly in epistasis tests. UAS lines employed include UAS-dco3 [29],[>>48<<], UAS-d:V5[9F] and UAS-d:nodeID://b4650551822024-03-29T00:23:00.260156Z2nodeID://b4650552822024-03-29T00:23:00.260156Z2http://purl.jp/bio/10/colil/id/216668022024-03-29T00:23:00.260156ZPMC010.1371%2Fjournal.pbio.1000624nodeID://b4650550282024-03-29T00:23:00.260156Z5nodeID://b4650551282024-03-29T00:23:00.260156Z3nodeID://b114057072024-03-29T00:23:00.260156Zaltered tissue growth or lethality were then re-screened for possible effects on Fat-Hippo signaling by assaying the expression of downstream targets of the pathway, Wingless (Wg) and thread (th, more commonly referred to as Diap1) [>>20<<],[21], in wing discs in which RNAi lines were expressed in anterior cells under ci-Gal4 control (Table S1).nodeID://b4650552282024-03-29T00:23:00.260156Z2nodeID://b114057172024-03-29T00:23:00.260156Zdco encodes a kinase that phosphorylates the Fat cytoplasmic domain and participates in Fat-Hippo signaling [6],[28],[>>29<<]. The requirement for Dco within Fat signaling is uncovered by expression of an antimorphic isoform, Dco3. Expression of Dco3 induces wing overgrowth (Figure 1L) [29]. This overgrowth is suppressed by Zyx RNAi, suggesting that Zyx actsnodeID://b114057272024-03-29T00:23:00.260156ZIn addition to regulating transcription, Fat also regulates planar cell polarity (PCP) (reviewed in [>>1<<],[5]). PCP in the adult wing is manifest in the orientation of wing hairs, which point distally.nodeID://b4650553282024-03-29T00:23:00.260156Z2nodeID://b114057372024-03-29T00:23:00.260156ZIn the context of a study of mitotic functions of LATS1, it was reported that the C-terminus of human Zyxin, including the LIM domains, could bind to human LATS1, even though full-length Zyxin did not bind [>>36<<]. When we expressed a C-terminal polypeptide comprising the LIM domains of Zyx (Zyx-LD) in S2 cells, only very low levels of protein could be detected (Figure 7B–D). Nonetheless, this C-terminal polypeptide bound efficiently to Wts (FigurenodeID://b114057472024-03-29T00:23:00.260156ZPrior studies identified two mechanisms by which Fat signaling could influence Yki activity, as fat mutation reduces both the levels of Wts protein [>>6<<] and the amount of Ex at the sub-apical membrane [31]–[33].nodeID://b114057572024-03-29T00:23:00.260156Zactivity, it has been observed that Ex can bind directly to Yki and that when Ex is over-expressed it can repress Yki through a mechanism that involves direct sequestration of Yki, rather than regulation of Yki phosphorylation [43],[>>44<<]. Because this direct repression mechanism was based on over-expression experiments, the extent to which it contributes to normal Yki regulation in vivo remained uncertain.nodeID://b114057672024-03-29T00:23:00.260156Ztheoretical models of growth control in developing tissues have proposed that growth should be controlled by mechanical tension [47],[48], and direct evidence for mechanical effects on growth has been obtained in cultured cell models [>>49<<]. However, a mechanism for how this might be achieved has been lacking.nodeID://b4650550362024-03-29T00:23:00.260156Z5nodeID://b4650551362024-03-29T00:23:00.260156Z3nodeID://b4650552362024-03-29T00:23:00.260156Z2nodeID://b4650550332024-03-29T00:23:00.260156Z5nodeID://b4650551332024-03-29T00:23:00.260156Z3nodeID://b4650553362024-03-29T00:23:00.260156Z2nodeID://b4650552332024-03-29T00:23:00.260156Z2nodeID://b4650550302024-03-29T00:23:00.260156Z5nodeID://b4650551302024-03-29T00:23:00.260156Z3nodeID://b4650553332024-03-29T00:23:00.260156Z2nodeID://b4650552302024-03-29T00:23:00.260156Z2nodeID://b4650549892024-03-29T00:23:00.260156Z8nodeID://b4650553302024-03-29T00:23:00.260156Z2nodeID://b4650550672024-03-29T00:23:00.260156Z4nodeID://b4650549862024-03-29T00:23:00.260156Z8nodeID://b4650551672024-03-29T00:23:00.260156Z2nodeID://b4650552672024-03-29T00:23:00.260156Z2nodeID://b4650549832024-03-29T00:23:00.260156Z9nodeID://b114056742024-03-29T00:23:00.260156ZFat is large cadherin that acts as a transmembrane receptor for one branch of Hippo signaling [1]–[>>3<<],[5]. Fat-Hippo signaling influences the levels of Wts protein [6].nodeID://b114056842024-03-29T00:23:00.260156ZThese proteins have three conserved LIM domains at their C-terminus, and they have been implicated in both cytoskeletal and transcriptional regulation [>>13<<]–[15]. Gene-targeted mutations in murine Zyxin or Lpp have no significant effect on mouse development, presumably due to redundancy among family members [16],[17]. Translocations involving LPP identified it as an oncogene involved innodeID://b114056942024-03-29T00:23:00.260156ZBoth Zyx RNAi lines gave similar effects on growth and gene expression in combination with multiple Gal4 lines and also behaved similarly in epistasis tests. UAS lines employed include UAS-dco3 [>>29<<],[48], UAS-d:V5[9F] and UAS-d:nodeID://b4650550722024-03-29T00:23:00.260156Z4nodeID://b4650549912024-03-29T00:23:00.260156Z8nodeID://b4650551722024-03-29T00:23:00.260156Z2nodeID://b4650552722024-03-29T00:23:00.260156Z2