1. Volume 154, Issue 6, Pages 1342-1355 (September 2013)
Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2 
Feng Yin, Jianzhong Yu, Yonggang Zheng, Qian Chen, Nailing Zhang, Duojia Pan 
Cell 
Volume 154, Issue 6, Pages (September 2013)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Merlin Promotes Hippo Signaling without Stimulating the Intrinsic Kinase Activity of Hpo/Mst
(A–C) Mer/Mer1–600 promotes Wts and Yki phosphorylation without affecting Hpo phosphorylation. S2R+ cell lysates expressing the indicated constructs were probed for P-Wts (A), P-Yki (B), or P-Hpo (C). Relative P-Wts and Wts levels were quantified in Figure S1B.
(D) In vitro kinase assay. Myc-Hpo was expressed alone or together with HA-Mer1–600 in S2R+ cells, immunoprecipitated, and incubated with GST-Wts, and the reaction products were probed for P-Wts.
(E) S2R+ cells transfected with the indicated plasmids along with control or hpo double-stranded RNA (dsRNA) were probed with the indicated antibodies. Note reduction of P-Wts levels by hpo RNAi.
(F) RNAi of mer leads to decreased P-Yki and P-Wts but increased P-Hpo levels in S2R+ cells. Also note comparable levels of total Yki, Wts, or Hpo in control and RNAimer cells.
(G) NF2 promotes Lats1 phosphorylation without affecting Mst1 phosphorylation. HEK293 cells expressing the indicated constructs were probed for P-Lats and P-Mst.
(H) Control (Nf2flox/flox) and Nf2 (Alb-Cre; Nf2flox/flox) livers from 2-month-old littermates were probed with the indicated antibodies. Note the comparable levels of Lats1 and YAP but diminished P-Lats and P-YAP levels in Nf2 mutant livers. Also note the increased P-Mst levels in Nf2 mutants.
See also Figure S1.
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4. Figure 2
Merlin Promotes Plasma-Membrane Association of Wts/Lats in Drosophila and Mammalian Cells
(A) Mer1–600, but not Mer or Ex, induces Wts membrane translocation. S2R+ cells expressing the indicated constructs were fractioned into membrane (M) and cytosolic (C) fraction and probed with the indicated antibodies. T: total cell lysates.
(B) Mer1–600 induces membrane association of Wts and WtsT1077A in S2R+ cells. Subcellular fractions were analyzed as in (A).
(C) RNAi of mer results in a significant increase of endogenous Wts in the cytoplasmic fraction relative to the membrane fraction. Subcellular fractions were probed with α-Wts antibody. Nervana, a plasma-membrane-localized Na+/K+ ATPase, serves as a control for fractionation.
(D) NF2 promotes membrane association of Lats1 and Lats1T1079A in HEK293 cells, as revealed by subcellular fractionation.
(E) Loss of NF2 results in increased cytosolic accumulation of endogenous Lats1 and Lats2. Subcellular fractions of confluent FH-912 and FC-912 cells were probed.
(F) FC-912 cells were transfected with empty vector (pcDNA), NF2, or NF2 mutants along with Myc-Lats1. Subcellular fractions were probed for Myc-Lats1. FC-912 cells expressing empty vector or NF2 mutants had higher Myc-Lats1 levels in cytosolic than membrane fraction, but FC-912 cells expressing WT NF2 showed similar cytosolic and membrane Myc-Lats1 levels. The cell lysates were also probed for the expression of NF2 mutants (bottom gel).
(G) Liver sections from 8-day-old control and Nf2-deficient littermates, stained with Lats2 (red) and the plasma-membrane marker Pan-cadherin (green). Lats2 is concentrated at the cell cortex immediately on the inner face of the plasma membrane in WT hepatocytes but shows a uniform cytoplasmic localization in Nf2-deficient hepatocytes.
See also Figure S2.
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5. Figure 3 Active Merlin Binds to Wts/Lats through Its FERM Domain
(A) Wts associates with Mer1–600, but not Mer or Ex, as revealed by immunoprecipitation in S2R+ cells as indicated.
(B) Mer1–600 exhibits an open conformation. S2R+ cells expressing Mer-N fragment (1–375) and WT (375–635) or truncated ( ) Mer-C fragment were subjected to immunoprecipitation.
(C) Lats1 associates with the N-terminal half of NF2, as revealed by immunoprecipitation in HEK293 cells.
(D) Coimmunoprecipitation between endogenous NF2 and Lats1/2 in HEK293 cells. Endogenous Lats1 or Lats2 was detected in α-NF2, but not control, immunoprecipitates.
(E) NF2-Lats1 binding in vitro. HEK293 cell lysates containing Myc-Lats1 or Myc-Lats13m were incubated with glutathione Sepharose beads containing GST (as a control) or GST-NF2. GST-NF2 bound to Myc-Lats1 but not Myc-Lats13m.
(F–H) C-terminal truncation of NF2 results in a more open conformation and enhances Lats1 binding and phosphorylation. HEK293 cells expressing the indicated constructs were probed for association between NF2-N and NF2-C fragments (F), NF2-Lats1 association (G), and Lats1 hydrophobic motif phosphorylation (H).
(I) WT NF2, but not disease-associated missense mutants, associates with Lats1, as revealed by immunoprecipitation in HEK293 cells.
(J) WT NF2, but not disease-associated missense mutants, promotes membrane association of Lats1 in HEK293 cells, as revealed by subcellular fractionation.
See also Figure S3.
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6. Figure 4
Identification of a Conserved N-Terminal Motif in Wts/Lats that Mediates Merlin-Wts/Lats Interactions
(A) HEK293 cells expressing HA-NF2 and various truncations of Lats1 were subjected to immunoprecipitation as indicated.
(B) Alignment of the CNM in Wts/Lats protein from different species.
(C) Myc-Lats1, but not Myc-Lats13m, was coimmunoprecipitated with HA-NF2 in HEK293 cells.
(D and E) The CNM is required for NF2-induced membrane association of Lats1 in HEK293 cells. Subcellular fractionation (D) and immunofluorescent staining (E) of Myc-Lats1 or Myc-Lats13m in the presence or absence of NF2 are shown.
(F and G) The CNM is required for Mer1–600-mediated membrane association of Wts in Drosophila S2R+ cells. Subcellular fractionation (F) and immunofluorescent staining (G) of V5-Wts or V5-Wts3m in the presence or absence of Mer1–600 are shown.
(H) UAS-Wts and UAS-Wts3m transgenes inserted at identical chromosomal loci were crossed to nub-Gal4 driver. Note the small-wing phenotype induced by Wts but not Wts3m.
See also Figure S4.
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7. Figure 5
Constitutive Membrane Targeting Promotes Hpo-Mediated Hydrophobic Motif Phosphorylation and Tumor-Suppressor Activity of Wts
(A) S2R+ cells expressing the indicated constructs were probed for P-Yki and P-Wts.
(B) Enhanced growth-suppressive activity of membrane-targeted Wts and dominant-negative activity of membrane-targeted WtsT1077A. All UAS-Wts transgenes were inserted at the same locus. Note the complete loss of adult wings in nub: Myr-Wts flies and the suppression of the small-wing phenotype of nub:Hpo in flies that coexpressed Myr-WtsT1077A.
(C–E) Partial rescue of hpo mutant clones by Myr-Wts. Eye discs containing clones of the indicated genotype (GFP+, arrowheads) were stained for Diap1 (red). Note elevated Diap1 levels in hpo clones without or with Myr-Wts expression. Also note the smaller clone size and less rounded shape of the latter genotype. Clone size is quantified in (E). Values are mean ± standard error of the mean (SEM); n = 12 for each genotype.
See also Figure S5.
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8. Figure 6
Sav-Mediated Membrane Association of Hpo and Regulation of Merlin-Wts Binding by the Actin Cytoskeleton
(A) S2R+ cells expressing Myc-Hpo together with HA-Mer1–600 or FLAG-Sav were fractioned into membrane (M) and cytosolic (C) fraction. T: total cell lysates. Sav, but not Mer1–600, promoted Hpo membrane association.
(B) S2R+ cells expressing V5-Wts together with HA-Mer1–600 or FLAG-Sav were analyzed by subcellular fractionation. Mer1–600, but not Sav, promoted Wts membrane association.
(C) RNAi of sav results in decreased membrane localization of endogenous Hpo. Subcellular fractions were probed with α-Hpo antibody.
(D) Savshrp6 mimics a sav allele that deletes just the SARAH domain (Kango-Singh et al., 2002). Unlike WT Sav, Savshrp6 failed to induce membrane recruitment of Hpo.
(E) S2R+ cells expressing the indicated constructs were analyzed for P-Wts. WT Sav, but not Savshrp6 (Kango-Singh et al., 2002) or SavΔFBM (Yu et al., 2010), greatly potentiated Mer1–600-induced Wts phosphorylation.
(F and G) S2R+ cells expressing the indicated constructs were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2), or Fosk (30 μM, lane 3) for 1 hr, followed by immunoprecipitation. LatB, but not Fosk, promoted Mer-Wts interaction (F). Neither drug affected Sav-Hpo interaction (G).
(H) S2R+ cells expressing the indicated constructs were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2), or C3 (2 μg/ml, lane 3), followed by immunoprecipitation.
(I) S2R+ cells were treated with DMSO (0.1%, lane 1), LatB (1 μg/ml, lane 2), or C3 (2 μg/ml, lane 3), followed by immunoprecipitation with anti-Mer antibody and detection with anti-Wts antibody.
(J) S2R+ cells expressing HA-Mer1–600 and V5-Wts were treated with DMSO, LatB, or C3 as in (H) and (I), followed by immunoprecipitation. Neither treatment affected interactions between Mer1–600 and Wts.
(K) Schematic models of Merlin function in Hippo signaling. For simplicity, only proteins relevant to the current study are illustrated in the schematic diagrams. Left: the prevailing linear model placing Merlin upstream of Hpo activation. Right: a new model based on the current study highlighting a dual mechanism in which Merlin and Sav promote the membrane association of Wts and Hpo, respectively.
See also Figure S6.
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9. Figure 7
Loss of Mer/NF2 and Sav/Sav1 Leads to Synergistic Defects in Hippo Signaling in Drosophila and Mice
(A) Synergistic effect of mer and sav mutations in Drosophila. Mid-pupal retina of the indicated genotypes was stained for Discs-Large to highlight cell outlines. Note the significantly increased number of interommatidial cells in mer; sav double-mutant eyes.
(B–E) Synergistic effect of Nf2 and Sav1 mutations in mice. Livers from 8-day-old pups of the following genotypes were analyzed: control (Alb-Cre), Nf2 mutant (Alb-Cre; Nf2flox2/flox2), Sav1 mutant (Alb-Cre; Sav1flox/flox), and double mutant (Alb-Cre; Nf2flox2/flox2; Sav1flox/flox). Whole amount liver images (B), H&E staining (C), and Pan-CK staining (D) are shown, along with quantification of liver weight (E). Values are mean ± standard deviation (SD); n = 5 for each genotype. Whereas Nf2 and Sav1 livers displayed mild or no overproliferation of the CK-positive BECs, Nf2; Sav1 double-mutant livers were predominantly occupied by the BECs. Also note the unique external appearance and overgrowth of Nf2; Sav1 double-mutant livers.
(F) Western blot analysis of liver lysates from mouse livers analyzed in (B)–(E). Liver lysates from three mice of each genotype were probed. Note the further decrease in YAP and Lats phosphorylation in Nf2; Sav1 double-mutant livers compared to the single mutant. Also note the comparable level of Lats1 in control and Nf2 mutant livers.
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10. Figure S1
Gain-of-Function Activity of Mer1–600 in Drosophila, Related to Figure 1
(A) The wing-specific nub-Gal4 driver was used to drive the expression of Mer or Mer1–600. Note the small-wing phenotype induced by overexpression of Mer1–600, but not Mer.
(B) Densitometric quantification of relative P-Wts/Wts in experiments presented in Figure 1A. Data in the bar graph represent mean ± SD of three independent experiments. Note the enhanced Wts phosphorylation induced by Mer1–600 compared to WT Mer.
(C) NF2 promotes Lats2 phosphorylation. HEK293 cells expressing the indicated constructs were probed for P-Lats.
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11. Figure S2
Further Characterization of Merlin-Induced Membrane Association of Wts/Lats, Related to Figure 2
(A–C) Mutation of the Mats-binding site in Wts did not affect Mer1–600–induced membrane translocation. (A and B) Coimmunoprecipitation assays confirming that WtsR708A is defective in Mats binding (A) but is normal in Mer1-600 binding (B). (C) Subcellular fractionation assay showing normal translocation of WtsR708A by Mer1–600.
(D) NF2 promotes membrane association of Lats2 in HEK293 cells, as revealed by subcellular fractionation.
(E) The Nf2−/− FC-912 cells are defective in endogenous Lats1/2 phosphorylation. Protein lysates from FH-912 and FC-912 cells were probed with antibodies against P-Lats, Lats1, NF2, and tubulin. Note the comparable level of Lats1 but diminished P-Lats in FC-912 cells.
(F) The Nf2−/− FC-912 cells are defective in Mst1/Sav1/Mob1-promoted Lats phosphorylation. FC-912 cells expressing the indicated constructs were probed for P-Lats. Expression of Mst1/Sav1/Mob1 resulted in weak activation of Lats phosphorylation, which was greatly enhanced by coexpression of NF2.
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12. Figure S3
Further Characterization of Merlin-Wts/Lats Interactions, Related to Figure 3
(A) Mer1–600 and Wts synergistically promote tissue undergrowth. Overexpression of Mer or Mer1–600 by the GMR-Gal4 driver has no visible effects on eye size. Flies expressing a GMR-Wts transgene show a slight reduction in eye size. This phenotype is enhanced by co-overexpression of Mer1–600, but not full-length Mer.
(B) NF2 associates with Lats2 in HEK293 cells as revealed by coimmunoprecipitation.
(C and D) Enhanced Lats1 binding and phosphorylation by NF2S518A compared to NF2S518D. HEK293 cells expressing the indicated constructs were probed for NF2-Lats1 association (C) and Lats1 hydrophobic motif phosphorylation (D).
(E) Wts associates with N-terminal half of Mer. V5-Wts was cotransfected with HA-Mer1–375 or HA-Mer376–635 in S2R+ cells and subjected to immunoprecipitation.
(F) Wts associates with the N-terminal half of Mer. V5-Wts, HA-Mer1–375, and HA-Mer were cotransfected into S2R+ cells. Mer1–375, but not full-length Mer, was coimmunoprecipitated by V5-Wts.
(G) An intact FERM domain is required for Mer-Wts binding. S2R+ cells expressing HA-Wts and various truncations of Mer were subjected to immunoprecipitation as indicated. Also shown is schematic diagram of Mer constructs tested, with the FERM domain shown in gray color. Any further truncation of the minimal FERM domain (1–350) abolished Wts binding.
(H) Structure of the FERM domain of human NF2 protein, showing the three subdomains (A/B/C) and the position of disease-associated NF2 mutations tested in this study.
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13. Figure S4
Further Characterization of the CNM in Wts/Lats, Related to Figure 4
(A) NF2 associates with Lats21–150. HEK293 cells expressing HA-NF2 and full length Lats2 or Lats21–150 were subjected to immunoprecipitation as indicated.
(B) A minimal fragment containing the CNM confers Mer1–600-induced membrane translocation on a heterologous protein. Wts1–50 (WT) or Wts1–50(3m) (with mutation of the CNM) was fused to GFP. GFP immunofluorescence of Wts1–50-GFP and Wts1–50 (3m)-GFP was examined in the presence or absence of Mer1–600 in S2R+ cells. Mer1–600 induced membrane association of Wts1–50-GFP, but not Wts1–50(3m)-GFP.
(C) Mer-C inhibits interactions between Mer-N and Wts1–50. S2R+ cells expressing Mer-N (HA-Mer1-375) and Wts1–50-GFP with or without Mer-C (Flag-Mer375–635) were subjected to immunoprecipitation as indicated.
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14. Figure S5
Dominant-Negative Activity of Membrane-Targeted WtsT1077A, Related to Figure 5
(A) Subcellular fractionation of Drosophila S2R+ cells expressing the indicated Wts constructs. Wts is localized in both membrane and cytosol, whereas Myr-Wts and Myr-Wts1077A show exclusive membrane association. Nervana, a plasma-membrane-localized Na+/K+ ATPase, serves as a control for fractionation. M: membrane; C: cytosol; T: total.
(B) Immunofluorescence of Drosophila S2R+ cells expressing the indicated Wts constructs. Wts is localized in both membrane and cytosol, whereas Myr-Wts and Myr-Wts1077A show exclusive membrane localization.
(C) The GMR-Gal4 driver was used to drive the expression of the indicated transgenes in the eye. Note the smaller eye size of GMR:Myr-Wts flies compared to GMR:Wts flies, and the suppression of small-eye phenotype of GMR-Hpo in flies that coexpressed Myr-WtsT1077A. Also note the increased eye size in GMR:Yki+ Myr-WtsT1077A flies compared to GMR:Yki flies.
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15. Figure S6
Sav and Mer Promote Membrane Association of Hpo and Wts, Respectively, Related to Figure 6
(A) Subcellular fractionation of Drosophila S2R+ cells expressing the indicated epitope-tagged proteins. Mer and Sav are predominantly localized in membrane fraction, whereas Hpo, Wts, and Yki show both membrane and cytoplasm localization and Mats is predominantly localized in the cytosol. Nervana, a transmembrane Na+/K+ ATPase, serves as a control for fractionation. M: membrane; C: cytosol; T: total.
(B) Subcellular fractionation of HEK293, probed for the indicated endogenous proteins. NF2 and Sav1 are predominantly localization in membrane, whereas Mst1, Lats1, Mob1, and YAP are more enriched in the cytosol. Pan-Cadherin serves as a control for fractionation. M: membrane; C: cytosol; T: total.
(C) S2R+ cells expressing Myc-Hpo together with HA-Ex or FLAG-Mats were analyzed by subcellular fractionation. Neither Ex nor Mats promoted Hpo membrane association.
(D) S2R+ cells expressing V5-Wts together with FLAG-Mats or Myc-Hpo were analyzed by subcellular fractionation. Neither Mats nor Hpo promoted Wts membrane association.
(E and F) HEK293 cells expressing the indicated constructs were analyzed by subcellular fractionation. Sav1, but not NF2, promoted Mst1 membrane association (E). In contrast, NF2, but not Sav1, promoted Lats1 membrane association (F).
(G and H) Immunofluorescence of Drosophila S2R+ cells (G) and HEK293 (H) expressing the indicated constructs. Note the translocation of both Hpo/Mst1 and Wts/Lats1 from the cytoplasm to the plasma membrane in the presence of both Sav/Sav1 and Mer1–600/NF2.
(I) RNAi knockdown of Mer attenuated LatB- or C3-induced Yki phosphorylation. S2R+ cells transfected with HA-Yki and dsRNA against GFP or Mer were treated with LatB or C3, followed by immunoblotting with the indicated antibodies. Note LatB- and C3-induced Yki phosphorylation, both of which were greatly reduced by RNAi knockdown of Mer.
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