Active Merlin Binds RalB to Regulate Exocytosis

Active Merlin Binds RalB to Regulate Exocytosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Active Merlin Binds RalB to Regulate Exocytosis Robert Hennigan, Kaley McLaughlin, Nancy Ratner This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6890466/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Loss of NF2 tumor suppressor activity causes NF2-related schwannomatosis. Proximity biotinylation identified proteins proximal to Merlin isoform 1 and isoform 2 at confluence, when Merlin is active, but not in sub-confluent, growing cells. These data confirmed Merlin involvement in cell-cell and cell-substrate junctions, identified new signal transduction pathways, and highlighted a role for Merlin in intracellular transport. Direct binding assays identified the small GTPases RalA and RalB as high affinity PIP 2 -dependent Merlin binding proteins that co-localized with RalA/B on the plasma membrane. Merlin loss resulted in aberrant activation of RalA and RalB at high cell density. Merlin competitively inhibited RalB binding to its exocyst effectors Sec5 and Exo84 and regulated the kinetics of exocytosis in a RalB dependent manner. Thus, RalB is a novel binding partner for active Merlin, and the RalA/B pathway is a possible therapeutic target to treat NF2-related schwannoma. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Cell biology/Membrane trafficking/Exocytosis Biological sciences/Cancer/Tumour-suppressor proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction NF2-related schwannomatosis, formerly called Neurofibromatosis type 2, is an inherited disease characterized by benign peripheral nerve tumors known as schwannomas, slow growing tumors that cause significant morbidity and are resistant to chemotherapy 1 . NF2 -mutant schwannomas also arise in the general population, in which they represent 8% of all intracranial tumors 2 , 3 . NF2 loss is also common in meningiomas, mesotheliomas, and other types of sporadic tumors. Targeted deletion of the Nf2 gene in mouse Schwann cells leads to schwannoma 4 , c onfirming that NF2 is a bona-fide tumor suppressor gene, with loss of function causing tumorigenesis. The major phenotype of Nf2 -null cells in vitro is impaired contact inhibition of growth 5 , a fundamental hallmark of cancer and a key feature of NF2 pathogenesis. The NF2 gene encodes Merlin, a 70-kDa member of the Ezrin-Radixin-Moesin (ERM) branch of the band 4.1 superfamily 6 . Merlin secondary structure consists of an N-terminal FERM domain followed by a central a-helical region that positions the C-terminal domain (CTD) for intramolecular interaction with the FERM domain 7 . Upon the release of the CTD from the FERM domain, Merlin transitions to a more open “active” conformation that allows Merlin dimerization and FERM domain interaction with critical binding proteins 8 . A Merlin mutant that stabilizes the FERM-CTD interaction adopts a closed conformation that has impaired tumor suppressor activity 8 . Conversely, a more open, FERM-accessible conformation mutant retains tumor suppressor activity 8 . Merlin is active when cells are contact inhibited at confluence and inactive in growing, sub-confluent cells 5 . Merlin has two major splice variants, isoform 1 and isoform 2, that differ in their extreme C-terminal amino acids 9 . The isoform 2 C-terminus has a lower affinity for the FERM domain 10 , a more open conformation 8 and increased ability to dimerize 11 . However, both Merlin isoform 1 or isoform 2 are tumor suppressors 12 and mutations in both isoforms are detected in tumors 9 . Merlin is localized predominately to the inner face of the plasma membrane 13 where it is associated with a variety of cell to cell and cell to substrate junctional complexes, including integrin-based focal adhesions and cadherin-based adherens junctions 14 . This localization is facilitated by three pairs of basic amnio acids in the FERM domain that are necessary for association with lipid rafts and for growth suppression 15 . Membrane localization positions Merlin for binding the lipid second messenger phosphotydylinositol-4,5-biphosphate (PIP 2 ). PIP 2 binding causes an allosteric change in the Merlin central a-helical domain that forces the CTD and FERM domains apart 16 , allowing Merlin to assume it’s open, FERM accessible conformation 17 . PIP 2 -mediated transition to an open conformation leads to Merlin dimerization and increased affinity for target proteins including Lats1, YAP1 and ASPP2 11,16 . These data support a model in which Merlin is activated at high cell density by dimerization in response to transient, localized increases in PIP 2 levels, leading to contact inhibition of growth. In mammalian cells, contact inhibition of growth is mediated by the Hippo pathway, a growth inhibitory kinase cascade that responds to mechanosensory cues transmitted via cell junctional signaling complexes 18 . Merlin regulates the HIPPO pathway by binding to and activating the core HIPPO kinase, Lats1/2. Lats1/2 then phosphorylates the transcriptional co-activator YAP1, leading to its ubiquitination and degradation, thereby preventing YAP1 nuclear localization, leading to growth arrest 19 . While impaired Hippo signaling due to Merlin loss is considered a primary mechanism contributing to NF2 schwannomas development, other mechanisms likely also contribute to schwannoma development. For example, the constitutive activation of a variety other oncogenic signaling networks such as Ras-ERK, Rac1, src, b-catenin, and the mTOR protein kinase complex have been described in Merlin-deficient cells 20 . Additionally, reports in the literature implicate Merlin in establishing or maintaining cellular polarity 21 and in regulating intracellular vesicular trafficking and the endocytic process, micropinocytosis 22 , 23 . Our objective was to develop a more complete understanding of Merlin function in cell polarity, intracellular trafficking, and signal transduction. The challenge was in identifying those target proteins and pathways that are important for Merlin tumor suppressor function specifically when Merlin is active. We used a proximity biotinylation strategy coupled with a robust Merlin binding screening system to identify proteins that are proximal to both Merlin isoform 1 and isoform 2 at confluence, in contact inhibited cells when Merlin is active, but not in sub-confluent, growing cells when Merlin is inactive. We identified many novel Merlin proximal proteins that confirmed Merlin involvement in specific cell-cell and cell-substrate junctions, expanded the number of signal transduction pathways that Merlin may influence and highlight a critical role for Merlin in intracellular transport. We identified the small GTPases RalA and RalB as high affinity PIP 2 dependent Merlin binding proteins and demonstrate that Merlin competitively inhibits RalB binding to its exocyst effectors Sec5 and Exo84. Merlin loss increased Ral activity in confluent Schwann cells and regulates the kinetics of exocytosis in a RalB dependent manner. Our results identify RalB as a novel, critical binding partner for active Merlin and raise the possibility that the RalA/B pathway may be targeted therapeutically to treat NF2-related schwannomatosis. Results Contact Inhibition Parameters We defined the cell densities at which Merlin is active in the immortalized human Schwann cell line iHSC-1l 24 by comparing the growth parameters of Merlin knockout and scrambled gRNA control cell lines (Supplemental Fig. 1). YAP1 staining confirmed a significant increase in the proportion of cytosolic YAP1 in control cells 48 hours after seeding at high density (10–20,000 cells per cm 2 ) relative to low cell density (2,500 to 5000 cells per cm 2 that was not apparent in the Merlin knockout cells (Fig. 1 A, B, Supplemental Fig. 2). We generated iHSC-1l cell lines expressing Merlin isoform1 or isoform 2 with a recombinant ascorbate peroxidase (APEX2) 25 fused to their C-terminus (Fig. 1 A). We measured APEX activity in lysates from these cell lines. Mer1-APEX and Mer2-APEX activity was roughly equivalent and four-fold less than control cell lysates expressing APEX alone (Fig. 1 B). Merlin iso1 and iso2-APEX proteins were expressed ~ 1.6 and 2.0-fold over endogenous levels, as measured by densitometry (Fig. 1 C). There was no significant difference in the subcellular localization of endogenous Merlin (Figs. 1 D., E.), Merlin isoform 1-APEX (Figs. 1 F., G.) and Merlin isoform 2-APEX (Figs. 1 H., I.), ruling out artifacts caused by gross differences in subcellular localization. Proximity Biotinylation iHSC-1l cells expressing either Merlin iso1-APEX or Merlin iso2-APEX were plated at either 5,000 cells per cm 2 or 15,000 cells per cm 2 and incubated for 48 hours to produce either a sub-confluent, growing cell population or a confluent, contact inhibited cell population (Fig. 2 A). We initiated a 1-minute APEX mediated biotinylation reaction, then harvested cell lysates. Biotinylated proteins were purified by streptavidin affinity chromatography, washed extensively then processed for MALDI-TOF mass spectroscopy. These experiments were performed in triplicate. Western blots of aliquots showed significant biotinylated proteins with a range of molecular weights for both sub-confluent and confluent cells, with a noticeable increase in the confluent samples (Fig. 2 B). Merlin proximal proteins were identified from the mass spectroscopy data by the normalized total number of peptides mapping to each protein in the Merlin-expressing samples that were significantly greater than in the APEX control samples, as judged by T-test (p-value of < 0.05 with a 5% false discovery rate, Supplemental Spreadsheet 1). The mass spectroscopy data for isoform 1 and isoform 2 proximal proteins is displayed in Volcano plots showing the mean normalized peptide difference between Merlin samples and APEX only control and the statistical significance (Q= \(\:-{\text{log}}_{10}p\) ) (Fig. 2 C). We identified a total of 542 proteins across the four experimental samples that met the selection criteria. Of these, 81 proteins were also identified in our published proximity biotinylation dataset (indicated by boxed datapoints in Fig. 2 C) 14 . Furthermore, we identified multiple proteins reported to bind Merlin in the literature, including angiomotin 26 , Erbin 27 , YAP1 28 , TP53BP2 14 , PPP1R12A (Mypt1) 29 , Ezrin 30 and Moesin 10 (Table I), validating the new dataset. There were 466 proteins identified by isoform 1 and 410 by isoform 2 (Fig. 2 D). A total of 322 proteins were present in sub-confluent cells and 410 in confluent cells, with 200 proteins that overlapped. The number of proteins proximal to Merlin isoform 1 significantly increased in confluent cells, from 211 to 394 with 138 proteins identified at both cell densities but 256 identified exclusively in confluent cells. In contrast, 195 of the 210 proteins identified by Merlin isoform 2 were in sub-confluent cells, with only 57 in confluent cells. This suggests that the isoforms might have different functions in addition to their shared tumor suppressor function, and that isoform 1 may be more active in confluent cells while isoform 2 is more active in sub-confluent cells. Functional Enrichment Analysis To begin to understand the potential functional significance of these proteins we performed a STRING based functional enrichment analysis on the Merlin proximal proteins 31 , displayed as a proximity network 32 (Fig. 3 A). The proximal proteins were color coded for gene ontogeny terms to indicate proteins functioning in cell junctional complexes, intracellular trafficking and signal transduction (Table I). We identified 206 proteins associated with cell junctions, including 40 adherens junction proteins, 30 focal adhesions proteins and 28 tight junction proteins (Table I). These included N-cadherin, catenin-a, catenin-b, catenin-d, integrin-b1, integrin-a2 and the ephrin receptors EPHA2, EPHB2 and EPHB4 (Table II, Supplemental Spreadsheet 1). We also identified 137 proteins involved in intracellular transport or membrane trafficking, (87 and 80 proteins respectively), and 107 proteins more specifically mapping to endocytosis or exocytosis (Table I). These included 10 members of the Rab family of small GTPases, most prominently the endocytic Rab7a and the recycling/exocytic Ral11b (Table II, Supplemental Spreadsheet 1). Other trafficking proteins include the AP-2 complex subunits AP2A1 and AP2A2 and proteins critical for exocytosis, including 3 out of the 8 components of the exocyst, EXOC2, EXOC4 and EXOC7 (Table II, Supplemental Spreadsheet 1). Significantly, we identified 173 signal transduction proteins (Table I), with 48 mapping to the HIPPO pathway, including angionmotin, ASPP2, and YAP1 (Table II, Supplemental Spreadsheet 1). Surprisingly, neither of the HIPPO core kinases, Lats1/2 or Mst1/2, were identified. There were 40 members of the receptor tyrosine kinase pathway and 25 members of the PI3K/Akt3 pathways (Table I); a total of 54 proteins were kinases or had kinase activity. These included growth factor receptors (EGFR, MET, and PDGFRb), and protein kinases (PAK2, JAK1, integrin linked kinase, ILK1 and myosin light chain kinase MYLK) (Table II, Supplemental Spreadsheet 1). There were 25 phosphatidylinositol binding proteins and 36 molecular adapters (Table I). There were also 19 small GTPases, including 10 Rab family members plus CDC42, NRAS, RHOA, RRAS2, RAN, RAP1A, RAP2A, RALA and RALB (Table II, Supplemental Spreadsheet 1). A subset of this network consisted of 41 proteins biotinylated by both isoform 1 and isoform 2 in confluent cells. We hypothesized that this cluster would contain the proteins most likely to interact with active Merlin. Hierarchical cluster analysis revealed four subgroups (Fig. 3 B). There were 14 proximal to both isoform 1 and isoform 2 in sub-confluent and confluent cells, including the Merlin binding protein angiomotin and upstream cell junctional complexes like integrin-b1 and catenin-d (Fig. 3 B, Table II). Two sets totaling 20 proteins were proximal to both isoform 1 and isoform 2 in confluent cells and either isoform 1 or isoform 2 in sub-confluent cells. These included the molecular adapter proteins DOK1 and EPS8, the calcium binding protein calponin-2 and the small GTPases cdc42, Rap2 and RalA (Fig. 3 B, Table II). Finally, there was a subset of 7 proteins are proximal to both isoform 1 and isoform 2 exclusively in confluent cells. These included the small GTPases RalB and Rap1a, the v-SNARE protein SNAP-29, the large GPCR subunits GNAQ and GNAI2, the facilitated glucose transporter SLC2A1 and the endocytosis associated protein NECAP2 (Fig. 3 B, Table II). Merlin Binding We hypothesized that Merlin proximal proteins that are critical to its tumor suppressor function are likely to be signaling proteins that physically bind to active Merlin. We therefore focused screening experiments on a selection of Merlin-proximal cell junctional complex proteins, ERM proteins, kinases, phosphatases and small GTPases. Merlin is activated by PIP 2 binding 15 which induces a conformational change 16 that allows dimerization 11 and increases its affinity for binding 33 . Therefore, we screened for direct binding to Merlin binding using purified proteins in the presence and absence of PIP 2 11,14 (Fig. 4 A-C). Figure 4 D presents the results of 35 of these assays performed in triplicate, expressed relative to the GFP negative control. We evaluated known Merlin binding proteins angiomotin, Lats1, ASPP2 and YAP1. Merlin consistently showed the highest relative affinity for angiomotin, > 65-fold above control, in a PIP 2 independent manner. Merlin also showed relatively high affinity for Lats1, but significantly less for ASPP2 and YAP1. There was a marked PIP 2 -dependent increase in affinity for YAP1 (Fig. 4 D). In contrast, the cell adhesion complex proteins N-cadherin, b1-integrin, catenin-a, catenin-b and catenin-d showed low relative affinity binding, (2-4-fold) and minimal PIP 2 effect. Similarly, Merlin showed low relative affinity binding and small increases in the presence of PIP 2 to the ERM proteins Ezrin, Moesin, and the adapter protein Erbin. The same pattern was seen for kinases (PTK2, CHUK, ILK, MYLK, AKT1 and AKT2), and phosphatases (PP1A and MYPT) tested. All these proteins bound Merlin greater than they bound control, but with relatively low affinity and little increase in the presence of PIP 2 . In contrast, the small GTPases, N-Ras, cdc42, RhoA, Rab7a, Rab11b, RalA and RalB showed significant PIP 2 -dependent binding. Most striking was the high relative affinity and the significant increase in binding in the presence of PIP 2 that Merlin showed for RalA and RalB. Given the strong binding between Merlin and RalA and RalB, we searched for Ral-GAP, -GEF and effector proteins in our Merlin-proximal dataset. We identified Exo70, Sec8 and the Ral effector Sec5, three components of exocyst complex (Table II, Supplemental Spreadsheet 1). The exocyst is a conserved 8 protein complex that mediates the initial tethering of the vesicle to the plasma membrane 34 , stimulates SNARE complex assembly 35 and functions in both constitutive 36 and regulated exocytosis 37 . Merlin binding to Exo70, Sec8 and to the Ral effectors Sec5 and Exo84 was detectable and showed a slight increase in the presence of PIP 2 . We conclude that RalA and RalB are good candidates for critical signal transduction proteins that interact directly with active, PIP 2 -bound Merlin. Merlin Ral Interactions We took advantage of the NanoLuc-GFP binding assay to perform bioluminescence resonance energy transfer (BRET) to confirm close interaction between Merlin-NanoLuc and GFP RalA or GFP-RalB in complex. There was significant excitation of the GFP fluorescence, peaking at 510 nm in both GFP-RalA and GFP-RalB proteins when in complex with Merlin-NanoLuc, indicating that Merlin forms close complexes with both RalA and RalB (Fig. 5 A). Given the relatively high affinity that Merlin has for RalA and RalB we tested whether Merlin affects Ral activity. We assayed for both RalA and RalB activity in control and Merlin knockout iHSC-1l cells, at either sub-confluent or confluent cell densities. In sub-confluent cells, both RalA-GTP and RalB-GTP levels was equal between Merlin knockout and control cells, but in confluent cells RalA/B-GTP levels were significantly increased in Merlin knockout cells relative to control cells (Fig. 5 B). Increased RalA activity is consistent with reports in the literature 38 . Since our proximity biotinylation data identified RalB as proximal to both Merlin isoforms only confluent cells (Fig. 3 ), we chose to focus on RalB going forward. The Merlin-RalB interaction was impaired by deletion of Merlin’s unique 20 N-terminus (Merlin-DN18, Fig. 5 C) and lost in an in-frame deletion within the FERM domain (Merlin-D39-121, Fig. 5 B), suggesting that RalB binds to the Merlin FERM domain and that the Merlin N-terminus is necessary for this interaction. RalB binding was also impaired to the closed conformation mutant Merlin-AR 8 (Fig. 5 C). In contrast, the open conformation mutant Merlin-DEL 8 was unaffected (Fig. 5 C). These results suggest that RalB preferentially binds to Merlin’s open conformation and are consistent with the PIP 2 -dependent nature of this interaction. RalA and RalB are small G-proteins, which cycle between a GTP-bound active form and a GDP-bound inactive form. To determine if Merlin specifically binds to active or inactive RalB we performed Merlin-binding assays using 2 constitutively active mutants (RalB G23V and RalB Q72L ) and a dominant negative mutant (RalB D28N ). All 3 RalB proteins bound to Merlin in the presence of PIP 2 , with slight differences in relative binding efficiencies but no correlation to RalB activation status (Fig. 5 D). These results suggest that Merlin binds RalB in the presence of PIP 2 , suppresses RalB activity during contact inhibition of growth, and that in the absence of Merlin this negative regulation is lost. Next, to determine if Merlin binding either facilitates or inhibits RalB interaction with its exocyst effector proteins, we performed binding assays between RalB and Sec5 or Exo84 in the presence or absence of Merlin. These experiments showed a significant, 50%, reduction of RalB binding to Sec5 and Exo84 in the presence of Merlin (Fig. 5 E). This suggests that Merlin may function to competitively inhibit RalB binding to its exocyst effectors, Sec5 and Exo84. To determine if endogenous Merlin co-localizes with the Ral GTPases, iHSC-1l cells were co-stained with antibodies that detect Merlin and pan-RalA/B. Merlin was distributed on the ventral surface of the plasma membrane in a distinct punctate pattern and co-localized, in part, to cell junctions that can contain N-cadherin (Fig. 6 A). The exocyst marker Exo70 was also localized to the ventral plasma membrane, where it showed a punctate pattern with significant co-localization with endogenous Merlin (Fig. 6 B). Antibodies detecting RalA/B showed a similar ventral punctate staining pattern, and significant overlap with Merlin staining (Fig. 6 C). To better verify overlap, we acquired super-resolution microscopic images of Merlin and Ral A/B staining. This confirmed significant co-localization between Merlin and RalA/B, co-localized in ventral puncta (Fig. 6 D). This subcellular localization data is consistent with Merlin-Ral interaction and a role for Merlin and RalA/B in the process of exocytosis. Merlin Regulates Exocytosis via RalB The ability of Merlin to bind to RalB in a PIP 2 -dependent manner, to competitively inhibit RalB binding to its exocyst effectors Sec5 and Exo84 and the co-localization of endogenous Merlin with RalA/B punctate structures on the ventral plasma membrane strongly suggested that Merlin might regulate exocytosis via RalB at the exocyst. To test this idea, we used a vesicular membrane marker, VAMP2, fused to the pH sensitive GFP mutant, pHluorin, to measure exocytosis 39 . Using this marker, endocytic events are indicated by transient increases in fluorescence in small areas on the ventral cell surface, which can be visualized in live cells by TIRF microscopy over for 1–2 min., in images taken at 100 msec. intervals (Fig. 7 A). We compared exocytosis between control and Merlin knockout iHSC-1l cells and tabulated the number, size, intensity and duration of events. Examples mapping exocytic events in control and Merlin knockout cells are presented in Fig. 7 B and C. There was no significant difference in the number, size or intensity of the exocytic events between control and Merlin knockout. However, we identified a significant difference in event duration. This result is apparent in a frequency histogram of these data (Fig. 7 D). In control cells ~ 59% of the events lasted between 1000 and 2000 msec., ~ 33% from 2000 to 3000 msec. and ~ 8% lasted longer than 3000 msec. In Merlin knockout cells the figures are ~ 39%, 41% and 20% respectively. The median duration for control and Merlin knockout is 1300 msec. vs 1600 msec with mean values of 1612 vs 1974 respectively (Fig. 7 E). These data show that loss of Merlin affects the kinetics of exocytosis. To test if RalB activity is necessary for this effect on the kinetics of exocytosis, we generated control and Merlin knockout cell lines co-expressing VAMP2-pHluorin with mCherry fused to active RalB G23V , dominant negative RalB D28N or an mCherry control. The control cells expressing mCherry alone had a mean exocytic duration of 1664 msec (Fig. 6 H). Expression of active RalB G23V significantly increased this value to 1874 msec., phenocopying the effect of Merlin loss (Fig. 7 F). In contrast, Merlin knockout cells expressing mCherry alone had a mean exocytic duration of 1928 msec and expression of active RalB G23V did not significantly change this value (1979 msec). In contrast, Merlin knockout cells expressing dominant negative RalB D28N had a mean exocytic event duration of 1608 msec, reverting to control event duration. We conclude that Merlin regulates exocytic kinetics by inhibiting RalB activity. Discussion Merlin Proximal Proteins We identified 542 Merlin-proximal proteins in Schwann cells, including known Merlin proteins and proteins identified in a previous Merlin proximity biotinylation study 14 , validating the dataset. Overall, we confirmed and extended earlier data by identifying the cell surface receptors for cell to cell and cell to substrate cell junctions (Table I). A striking new result was the strong representation of Merlin-proximal proteins involved in intracellular vesicular trafficking, an observation that is consistent with data from the literature 22 , 23 , 40 – 42 . Finally, we identified 165 proteins involved in general signal transduction, the majority of which are kinases, molecular adapters or phosphatidylinositol binding proteins, including 48 proteins associated with the HIPPO pathway and 19 small GTPases, including 10 members of the Rab subfamily. The other small GTPases were, CDC42, NRAS, RRAS2, RALA, RALB, RAN, RAP1A, RAP2A, and RHOA. Notable by its absence was RAC1, the small GTPase that was most often associated with Merlin in the early literature 43 . The absence of a protein like Rac1 and a known Merlin binding protein like Lats1/2 does cannot exclude these proteins as Merlin interactors because of biases inherent in the proximity biotinylation system 44 . Merlin Binding Proteins Our goal was to identify proteins that interact with Merlin when it is activated by PIP 2 -mediated dimerization 11 . Direct binding assays allowed us to test for interaction in the presence and absence of PIP 2 , that is, to test for binding to active Merlin 11 . Since PIP 2 signaling is transient and spatially restricted to membranes, interactions that are increased when PIP 2 is present are likely to represent a small but critical proportion of Merlin molecules within cells. Because the assay is quantitative and, although we did not formally measure K d , we were able to compare the relative affinity between Merlin and its binding proteins and to reveal a hierarchy of Merlin binding affinities. Anigomotin showed the highest affinity for Merlin. Binding was independent of PIP 2 , suggesting a distinct function for the Merlin-angiomotin complex. The HIPPO pathway proteins Lats1, ASPP2 and YAP1 showed strong Merlin binding, albeit with lower affinity than angiomotin; of these proteins only YAP1 showed significantly increased binding in the presence of PIP 2 . The small GTPases RalA and RalB had a higher relative affinity for Merlin, roughly 7-fold over the GFP control, which increased dramatically to more than 50-fold over control in the presence of PIP 2 . This level of binding is significantly higher than any other tested proximal proteins and on par with that of angiomotin. RalA and RalB are proximal to both isoform 1 and isoform 2 in confluent cells, with RalB being one of the seven proteins that are proximal to both isoforms exclusively in confluent cells. Endogenous Merlin colocalizes with RalA/B on the ventral surface of cells, in a punctate pattern similar to that of the exocyst component, Exo70, and consistent with a subcellular localization where exocytosis occurs. RalA/B activity is significantly increased in confluent Merlin knockout cells, but not in sub-confluent cells. Increased RalA activity upon Merlin loss was described more than 20 years ago 38 , although the mechanism described in that paper, Merlin binding to and inhibiting the Ral activator, RalGDS, is not supported by our data since RalGDS is not among the Merlin proximal proteins we identified. However, we cannot rule this out as a mechanism by which RalA/B is activated in confluent cells upon Merlin loss. Merlin competitively inhibited RalB binding to the exocyst effectors Sec5 and Exo84. Since both RalA and RalB bind to Sec5 and Exo84 it is likely that Merlin competitively inhibits RalA binding to the exocyst as well. Loss of Merlin caused a RalB dependent change in exocytosis kinetics that is phenocopied by active RalB in wild type cells. Taken together these data identify RalA and RalB as a key binding partner for active Merlin and show that Merlin regulates exocytosis in a RalB dependent manner (Fig. 3 B). RalA and RalB Both RalA and RalB play critical roles a diverse set of cellular functions including proliferation, cell survival, differentiation motility and cellular polarization 45 . Mechanistically, many of these biological effects are dependent upon RalA/B regulation of endocytosis 46 and exocytosis 47 . Ral regulation of endocytosis is achieved via the Ral effector RalBP which binds to the endocytic machinery via AP-2 to mediate EGF receptor mediated endocytosis 48 . RalBP was not among the Merlin proximal proteins we identified. Rather, the three exocyst components, Exo70, Sec8 and the Ral effector protein Sec5 were identified as Merlin proximal. These exocyst components plus the other Ral effector, Exo84, are all low affinity Merlin binding proteins (Fig. 4 ), suggesting that the Merlin-Ral interaction may affect exocytosis rather than endocytosis, although the close coordination between endocytosis and exocytosis suggests that we cannot exclude a role for Merlin-Ral interaction in endocytosis as well as exocytosis. The exocyst is responsible for tethering the exocytic vesicle to the inner face of the plasma membrane. It is a critical point of regulation via its interactions with vesicular and plasma membrane proteins, phosphoinositides, kinases, and GTPases 49 , including cdc42, RhoA 50 , RalA and RalB 51 . The exocyst is a Ral effector 51 , and active, GTP bound forms of both RalA and RalB can bind to Sec5 and Exo84 52 and mediate the assembly of mature exocyst complexes from 4-protein precursor subcomplexes I and II 53 . We note that other proteins involved in exocytic vesicle fusion were identified as Merlin-proximal, including Rab11b and its effector WDR44, the v-SNAREs SNAP-29 and YKT6 and the syntaxin binding proteins STXBP1 and STXBP2, key regulators of the SNARE complex that is essential for exocytic vesicle fusion to the plasma membrane (Table II, Supplemental Spreadsheet 1). Indeed, SNAP-29 is one of the 7 proteins that, like RalB, is proximal to both Merlin isoforms only at confluence. Exocytosis in Cancer Biology Fusion of exocytic vesicles with the plasma membrane is the mechanism by which integral membrane proteins and membrane lipids are incorporated into the plasma membrane and intra-vesicular contents are released into extracellular space 54 . The RalB-dependent changes in exocytosis kinetics seen upon Merlin loss may reflect distinct subtypes of exocytic events that can be visualized by the VAMP2-pHluorin reporter. Exocytic events with a longer duration are characteristic of “full fusion” exocytosis in which the vesicle membrane completely integrates with the plasma membrane, along with new membrane components and integral membrane proteins including cell junctional proteins and growth factor receptors 55 . Short duration exocytic events include “kiss-and-run” exocytosis, in which the vesicle opens and closes transiently, leading to the release of soluble intra-vesicular contents into the extracellular space, without incorporation of the vesicular membrane and associated integral membrane proteins into the plasma membrane 56 . The shift from shorter to longer duration events upon loss of Merlin is consistent with a shift from the “kiss and run” to the “full fusion” exocytic events that require the exocyst. Loss of exocyst function impairs “full fusion” and increases the proportion of “kiss and run” events 57 . Therefore, the changes in exocyst kinetics that we observed may reflect a shift to more “full fusion” events mediated by the exocyst in response to the increased RalB activity seen in Merlin KO cells at confluence or by the active RalB G23V mutant in control cells. Ral-regulation of exocytosis is crucial for cell growth, cell-cell communication, and establishment of cell polarity 58 . During development of highly polarized Schwann cells, Ral GTPases promote radial axonal sorting in peripheral nerves through the exocyst complex 59 . NF2 mutant schwannoma are epithelioid cells characterized by dysregulated polarity, caused by loss of Merlin function 60 . A key mechanism by which cell polarity is established and maintained is asymmetric membrane trafficking controlled by the exocyst 37 and Ral activity is implicated in this process 61 . We hypothesize that upon contact-inhibition, cells are not actively growing and do not require plasma membrane expansion, full fusion exocytosis is down regulated by active-PIP 2 -bound Merlin dimers that bind to RalA/B and to prevent association with the exocyst and inhibit full fusion exocytosis. In NF2-schwannomatosis the absence of Merlin the failure to inhibit the RalA/B interaction with the exocyst causes increased “full fusion” exocytosis, leading to changes in the cell surface proteome that affects cell polarity in tumor cells. Therapeutic Implications The identification of RalA and RalB as binding partners for active, PIP 2 -bound Merlin define a mechanism underlying previously described trafficking and cell polarity defects in NF2 null cells. The coordinated nature of exocytosis and endocytosis and the correlation of increased exocytic duration with “full fusion” exocytosis 57 raises the possibility that Merlin loss may cause differential expression of key proteins on the cell surface. The cell surface is the critical interface between tumor cells and the extracellular environment and is the point of interaction with pharmacological agents. Increased trafficking of differentially expressed cell surface proteins may also make these cells vulnerable to endocytic nanoparticle-based drug delivery strategies 62 . Constitutive RalB activity has been shown to be critical for survival in other human tumor cells 63 . This effect is mediated by Sec5-dependent activation of the IkB kinase family member TBK1 64 , raising the possibility that pharmacological inhibition of the Ral itself, or of its downstream effector pathways, may also induce death in schwannoma cells. The observation that loss of Merlin leads to increased RalA/B activity in confluent Schwann cells also identifies them as potential therapeutic target in NF2 schwannoma. As Ral-specific inhibitors for in vivo use become available, it will be of great interest to test their effects in this tumor type. That Ral small GTPases represent a new signal transduction pathway that may be exploited, either alone or in combination with agents targeting the HIPPO pathway, to treat NF2-related schwannomatosis. Materials and Methods Recombinant DNA All recombinant DNA subcloning was performed using NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs, Ipswich, MA, Cat# E5520s). DNA fragments were PCR amplified using Q5 High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, Cat# M0492S) and purified Monarch® DNA Cleanup Columns (New England Biolabs, Ipswich, MA, Cat# T1034L). Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, Iowa). The products of recombinant DNA manipulations were confirmed by sequencing (CCHMC Genomics Sequencing Facility, Cincinnati, OH). The pLNh-APEX vector was derived from plasmids lentiCRISPR, APEX2-GBP and pLenti-CMV-GFP using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Cat# E5520s). The cDNAs for Merlin isoform1 and isoform 2 were cloned in pLNh-APEX in frame N-terminal to APEX2. pLenti-CMVie-XX-mCh was constructed by cloning the mCherry ORF from pcDNA3-FlipGFP(Casp3 cleavage seq) T2A mCherry into pLenti CMVie-IRES-BlastR. RalB was subcloned into the vector pLenti-CMVie-XX-mCh-BlastR in frame, C-terminal to mCherry to generate the lentiviral expression vector pLnCMV-Ch-RalB. The RalB mutants G23V, Q72L and D28N were constructed by PCR mediated site directed mutagenesis using primer containing the appropriate mutant. The vector pLentiCMV-VAMP2-pHluorin-Hygro was constructed by subcloning the VAMP2-pHluorin ORF in place of GFP in pLenti-CMV-GFP. The plasmids expressing GFP fusions for a-Catenin, d-catenin, PPP1R12A, RhoA, RALA and RALB cDNA ORF Clones cDNA ORF Clone, were purchased from Sino Biological US, Inc. (Wayne, PA, Cat#: HG-12388-ACG, HG-16488-ACG, HG-13990-ACG, HG12110-ANG, HG52476-ANG, HG15052-ANG). The plasmids pEGFP-C3-Exo70, pEGFP-C3-Sec8, pEGFP-C3-Exo84 and pEGFP-C3-Sec5 were a gift from Channing Der. (Addgene plasmids # 53761, # 53758, #53762, #53756. pClneoMyc human ERBIN was a gift from Yutaka Hata (Addgene plasmid # 40214. Full-length MLCK-GFP was a gift from Anne Bresnick (Addgene plasmid # 46316. pcDNA3-EGFP-Cdc42(wt) was a gift from Klaus Hahn (Addgene plasmid # 12599). Cadherin-EGFP was a gift from Valeri Vasioukhin (Addgene plasmid # 18870. Integrin b1-GFP was a gift from Martin Humphries (Addgene plasmid # 69804). EGFP-Rab7A was a gift from Qing Zhong (Addgene plasmid # 28047). Human b-catenin GFP was a gift from Alpha Yap (Addgene plasmid # 71367). pEGFP-Akt1(WT) and pEGFP-Akt2 were a gift from Thomas Leonard & Ivan Yudushkin (Addgene plasmids # 86637, #86593. pLVX-EF1a-EGFP-RAB11B-IRES-Puromycin was a gift from David Andrews (Addgene plasmid # 134860). pLenti-PGK-RAP1A-WT-neo was a gift from Roland Friedel (Addgene plasmid # 156173). pCMV-Vamp2-pHluorin was a gift from Brad Zuchero (Addgene plasmid # 190151). The plasmids pEGFP-C3-Exo70, pEGFP-C3-Sec8, pEGFP-C3-Exo84 and pEGFP-C3-Sec5 were a gift from Channing Der. (Addgene plasmids # 53761, # 53758, #53762, #53756). pLenti CMVie-IRES-BlastR was a gift from Ghassan Mouneimne (Addgene plasmid # 119863). pcDNA3-FlipGFP(Casp3 cleavage seq) T2A mCherry was a gift from Xiaokun Shu (Addgene plasmid # 124428. The plasmid lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961. APEX2-GBP was a gift from Rob Parton (Addgene plasmid # 67651). pLenti-CMV-GFP Hygro (656-4) was a gift from Eric Campeau & Paul Kaufman (Addgene plasmid # 17446. IKK1-EYFP was a gift from Johannes A. Schmid (Addgene plasmid # 111206). The plasmids mEmerald-Ezrin-N-14, mEmerald-Moesin-N-14, mEmerald-PTK2-C-10, mEmerald-ILK-N-17 were a gift from Michael Davidson (Addgene plasmid #54090, #54185, #54240, #54127). IKK1-EYFP was a gift from Johannes A. Schmid (Addgene plasmid # 111206). R777-E187 Hs.PPP1CA was a gift from Dominic Esposito (Addgene plasmid # 70471). Lenti-CAG-GFP-hKRAS4B-Zeocin was a gift from Dr. Liang Hu, (Cincinnati, Children’s Hospital). The lentiviral packaging plasmids psPAX2 and pMD2.G were a gift from Didier Trono (Addgene plasmids #12260, #12259) Cell Culture The immortalized human Schwann cell line, iHSC1-l, was gift from Dr. M. R. Wallace 24 . HEK-293T cells were purchased from ATCC (CRL-2316). Cells were grown in DMEM supplemented with 10% FBS and Pen/Strep at 37ºC 5% CO 2 . 293T cells were transfected with 25000 MW, linear polyethylemimine (PEI, Polysciences, Warrington, PA. (cat# 23966-1). For lentiviral production, HEK-293T cells were co-transfected with the lentiviral expression plasmid and the packaging plasmids psPAX2 and pMD2.G using PEI (1.62 pmol:1.32 pmol:1.32 pmol ratio). After 24 hours, the media was changed and cells were grown in a minimal volume of fresh media for 48 hours. Conditioned media was collected, passed through a 0.22µ filter, aliquoted and stored at -80ºC. iHSC-1l cells were infected with a titration of lentiviral conditioned media in the presence of 8 µg/ml polybrene (MilliporeSigma, St. Louis, MO). After 48 hours, the cells were washed 3x with sterile PBS and grown in selection media, with either 2 µg/ml puromycin, 100 µg/ml hygromycin, or 5 µg/ml blasticidin. Immunofluorescence Cells were plated in 8-well chamber slides (Cat# 80806-90, Ibidi, Fitchburg, WI). Cells were fixed in 4% paraformaldehyde, permeabilized, blocked and probed with primary antibodies, washed in PBS probed with Alexa 488 or Alexa 555 conjugated secondary antibodies (Cat# A32731, A32727, ThermoFisher Waltham, MA) and counterstained with DAPI and Alexa-647 conjugated-phalloidin (Cat# 8940S, Cell Signaling Technology, Danvers, MA). Images were acquired on a Nikon C2plus Confocal microscope using either a 20x, 0.75 NA, Nikon PlanApo l or a 60x 1.4 NA, Apo lS Oil objective. Super-resolution microscopy was performed in the CCHMC Bio-Imaging and Analysis Facility (RRID:SCR_022628). Images were acquired on a Nikon Ti2, AX confocal equipped with an NSPARC ISM spatial array confocal detector in SR mode using a 100x, 1.45 NA, PLAN APO λD, Oil objective. The antibodies used were: Merlin (Cat# 6995, Cell Signaling Technology, Davners, MA), RalA/B (Cat# sc-374582, Santa Cruz Biotechnology, Dallas, TX), Active Ral (Cat# 26913, NewEast Biosciences, Glenmoore, PA), RalA (Cat# 21034, NewEast Biosciences, Glenmoore, PA), RalB (Cat# 21033, NewEast Biosciences, Glenmoore, PA), YAP1 (Cat# PA1-46189, ThermoFisher, Waltham, MA), N-Cadherin (Cat# 14215, Cell Signaling Technology, Davners, MA), Exo70 (Cat# MABT186, Millipore Sigma, St Louis, MA), Exo70 (Cat# sc-365825, Santa Cruz Biotechnology, Dallas, TX), APEX (Cat# CAC09177, Biomatik, Kitchener, ON, Canada), Sec5 (Cat# sc-393230, Santa Cruz Biotechnology, Dallas, TX). Proximity Biotinylation Three cell lines were generated by infecting iHSC-1l cells with lentivirus expressing the fusion proteins Merlin isoform 1-APEX2, Merlin isoform 2-APEX2 or control APEX2. Hygromycin resistant cell populations were then selected and pooled. APEX activity was measured in lysates using ECL Chemiluminescent HRP substrate (Cat# WBKLS0050, Millipore Sigma, St Louis, MO). Proximity biotinylation was performed in triplicate in 15 cm dishes. For sub-confluent samples cells were plated at 5000 cells per cm 2 , confluent samples were plated at 15,000 cells per cm 2 . Cells were incubated for 48 hours then processed for biotinylation. Proximity biotinylation was performed as described in the literature 25 . Briefly, media was changed to fresh media supplemented with 500 µM biotin-tyramide (Cat#. SML2135-50MG, MilliporeSigma, St. Lousi, MO) and incubated for 30 min @ 37ºC. To initiate biotinylation, fresh 100 mM H 2 O 2 in PBS was added to 1 mM final concentration, cells were incubated at 37ºC for 1 minute. The reaction was then quenched in final concentration of 5 mM Trolox (Cat# 238813-5g, MilliporeSigma, St Louis, MO), 10 mM sodium ascorbate and 10 mM sodium azide. Cells were then rinsed with ice cold PBS and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (HALT, Pierce) and Benzonase (Sigma-Aldrich). Biotinylated proteins were isolated from cleared lysates by incubating with Streptavidin-Mag agarose beads (Cat# GE28-9857-99, MilliporeSigma, St Louis, MO) overnight at 4ºC with agitation. Beads were captured in a magnetic stand, washed 3x with RIPA, 3x with 2M urea in TBS pH 7.4, 3x with TBS pH 7.4 and 5% aliquots were resuspended in a fluorescent protein staining loading buffer (Instant Bands, Cat# PFS001P, Thomas Scientific, Swedesboro, NJ) run on 4–20% gradient SDS–polyacrylamide gel electrophoresis (PAGE) (Bio-Rad). Fluorescent total proteins were visualized in an Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Gels were then blotted and probed with streptavidin-IRDye680 and antibodies to Merlin. The remaining beads were washed 10x with 50 mM ammonium bicarbonate then sent to the Taplin Biological Mass Spectrometry Facility at Harvard Medical School and processed for matrix-assisted laser desorption/ionization mass spectroscopy. Mass spec data consisting of peptide number mapping to specific proteins were normalized to the total number peptides per sample, expressed per 10,000 peptides. Data was displayed as a proximity network using Cytoscape 65 . Gene ontogeny and functional enrichment was performed within Cytoscape using the String database 312 . Hierarchical cluster analysis was performed using the web application Morpheus (Broad Institute, MIT, https://software.broadinstitute.org/morpheus ). Statistics Collated mass spec data was analyzed for statistically significant increases over the APEX only control by multiple unpaired T-test and a 5% false discovery rate using the two-stage step-up method 66 with Graphpad Prism 10. For binding assays, unpaired T-test were used to test significance. Merlin Binding Assays Direct Merlin binding assays were performed, with modifications, as previously described 11 , 14 . Assays were performed in triplicate, by incubating a probe consisting of purified Merlin-NanoLuc fusion protein with magnetic beads coated with GFP-fused bait proteins, isolated from transiently transfected 293T cells, in the presence or absence of 200 µM PI(4,5)P 2 diC8 (Cat# P-4058-2, Echelon Biosciences, Salt Lake City, UT). Merlin NanoLuc Probe Purification Twelve 15 cm dishes with 1.3 x 10 7 293T cells per plate were transfected with plasmid expressing Merlin fused N-terminal to NanoLuc with a dual Streptag affinity tag at its C-terminus (pMerNLst2) using 1 mg/ml PEI, at a 5:1 µl PEI to µg DNA ratio (250 µl PEI to 50 µg plasmid, per plate). Cells were incubated for 48 hours. Cell lysates were harvested by washing with ice cold PBS, then lysing in a high salt lysis buffer (20 mM TrisCl pH 8.0, 500 mM NaCl, 10 mM MgCl 2 , 0.5% NP-40 supplemented with 1x HALT protease and phosphatase inhibitor, Cat# 78430, ThermoFisher, Waltham, MA) and 1:5000 Benzonase (Cat# E1014-5KU, Millipore Sigma, St Louis, MA). Lysates were cleared by centrifugation at 21,000 xg for 10 min. at 4ºC. Merlin-NanoLucST2 protein was affinity purified by gravity flow over a 0.2 ml bed volume Strep-TactinXT 4Flow high-capacity resin column (Cat# 2-5030-002, ThermoFisher, Waltham, MA). Columns were washed with 15 column volumes (CV) High Salt Wash Buffer (500 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.1% Triton X-100, 0.1% Tween-20 + HALT), then 15 CV Normal Salt Wash Buffer (150 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.1% Triton X-100, 0.1% Tween-20 + HALT) followed by 15 CV of TBS (150 mM NaCl, 20 mM Tris-Cl pH 8.0). Merlin-NanoLucST2 was eluted stepwise with 0.3 ml of Elution Buffer (100 mM Tris-Cl pH 8.0, 150 mM NaCl, 50 mM Biotin + HALT) per step for a total of 8 fractions. Purification was monitored by NanoLuc luciferase assays on all fractions (Cat# N1110, Promega, Madison WI), measured using white round bottom 96-well plates in a FlexStation 3 plate reader (Molecular Devices, San Jose, CA). Positive fractions were pooled, passed through a 0.22 µ filter then loaded onto a NCG Quest plus FPLC for gel filtration over a 10x300 Enrich SEC 650 size exclusion column (Bio-Rad, Hercules, CA) equilbtated in TBS (20 mM Tris-Cl pH 7.4, 150 mM NaCl). Collected fractions were assayed for NanoLuc activity. High activity fractions corresponding to the closed conformation 11 eluting at 13–14 ml were pooled. Concentration was determined by A 280 in a Nanodrop 2000c (ThermoFisher, Waltham, MA). Purity was determined by SDS-PAGE using Instant Bands loading dye imaged on the Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Preps were adjusted 0.5 mg/ml BSA and 0.1% Tween-20 and stored at 4ºC for no more than two weeks. GFP-Bait Protein Purification GFP-bait expressing plasmids were transfected into 5 x 10 6 HEK 293T cells in a 10 cm dish and incubated for 48 hours. Cells were rinsed with cold PBS, harvested in Lysis Buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 2 mM, MgCl 2, 0.5 mM EDTA, 0.5% NP-40 + 1x HALT and 1:5000 Bensonase), cleared by centrifugation at 21,000 xg for 10 min. at 4ºC. Lystates were incubated with 30 µl washed GFP-Trap_MA magnetic agarose beads (Cat# GTMA020, Bulldog Bio Inc. Portsmouth, NH) overnight at 4ºC with agitation. The beads were captured, washed three times with High Salt Wash Buffer (20 mM Tris-Cl pH 7.4, 0.5 M NaCl, 0.1% Triton X-100, 0.1% Tween 20), followed by three washes with Low Salt Wash Buffer (20 mM Tris-Cl pH 7.4, 50 mM NaCl, 10% Glycerol, 0.2% NP-40) then three washes with Normal Salt Wash Buffer (20 mM Tris-Cl pH 7.4,150 mM NaCl, 0.1% Triton X-100, 0.1% Tween 20) and finally resuspended in Blocking Buffer (20 mM Tris-Cl pH, 7.4, 150 mM NaCl, 2.5 mg/ml BSA, 0.05% Tween-20). Final bead fluorescence was measured on a FlexStation 3 plate reader (Molecular Devices, San Jose, CA). Direct Binding Assays Each preparation GFP-bait-bound was divided into six wells of a white round bottom 96- well plates. The beads were captured on a 96-well magnetic stand (EpiMag HT (96-Well) Magnetic Separator, Cat# Q10002-1, EpiGenTek, Farmingdale, NY), resuspended in a 30-µl Blocking Buffer and incubated for 1 hour at room temperature with agitation. The beads were recovered magnetically then resuspended in 100 nM Merlin-NLuc protein in Blocking Buffer with or without 200 µM PI(4,5)P 2 diC8 and incubated at room temperature for 1 hour with agitation. Beads were then captured magnetically, washed four times with TBST, resuspended in 25 µl TBST and NanoLuc luciferase activity was measured using NanoGlo Luciferase Substrate Buffer (Promega, Madison, WI). NanoLuc activity, GFP fluorescence and BRET spectra were measured on a FlexStation 3 (Molecular Devices, San Jose, CA). For display purposes the plates were also imaged on that Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Data was expressed a % NanoLuc activity bound to the beads relative to the input Merlin-NanoLuc probe, normalized to the GFP-only negative control. Ral Activity and Exocytosis Assays Ral activity was assayed in sub-confluent and confluent Control and Merlin knockout iHSC-1l cells using the RalB activity assay (Cat# 81501, NewEast Biosciences, Glenmoore, PA) by immunoprecipitation with antibodies to active Ral and then probes with antibodies specific for RalA and RalB, (Cat # 26913, 21034, 21033, NewEast Biosciences, Glenmoore, PA). For exocytosis assays, Control and Merlin knockout iHSC-1λ cells were infected with lentivirus expressing VAMP2-pHluorin and selected with 100 µg/ml hygromycin. Subsequently, both resulting cell lines were infected with lentivirus expressing mCherry-RalB wt , mCherry-RalB G23V , mCherry-RalB D28N and control mCherry and expressing clones were selected with 5 µg/ml blasticidin. Exocytosis was measured in live VAMP2-pHluorin expressing cells in the CCHMC Bio-Imaging and Analysis Facility (RRID:SCR_022628). Imaging was performed on a TIRF equipped Nikon TiE microscope equipped with a Tokai Hit stage top incubation system and an Andor DU-897 C-6347 EMCCD camera using a Nikon Apo TIRF 100x Oil DIC, 1.49 NA. Time lapse images of live VAMP2-pHluorin expressing cells were acquired using TIRF illumination for 2 min. at 100 msec intervals. Exocytosis was analyzed using the ExoJ plugin 67 on FIJI/Image J software run on the CCHMC High-Performance Computing cluster(RRID:SCR_022622). Declarations Acknowlegements This work was supported by the Department of Defense, Congressionally Directed Medical Research Program Neurofibromatosis Research Program Awards NF120118, NF160078 and NF190083 and the University of Cincinnati Brain Tumor Center Jejurikar Fellowship Program to RFH. We thank Dr. Shyra Tedesco and Dr. Brad Ozanne for critically reading the manuscript. 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RalA and the exocyst complex influence neuronal polarity through PAR-3 and aPKC. Journal of cell science 122 , 1499-1506 (2009). https://doi.org/10.1242/jcs.044339 Cong, V. T. et al. How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells? Chem Soc Rev 51 , 7531-7559 (2022). https://doi.org/10.1039/d1cs00707f Chien, Y. & White, M. A. RAL GTPases are linchpin modulators of human tumour-cell proliferation and survival. EMBO reports 4 , 800-806 (2003). https://doi.org/10.1038/sj.embor.embor899 Chien, Y. & White, M. A. Characterization of RalB-Sec5-TBK1 function in human oncogenesis. Methods Enzymol 438 , 321-329 (2008). https://doi.org/10.1016/S0076-6879(07)38022-1 Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13 , 2498-2504 (2003). https://doi.org/10.1101/gr.1239303 Benjamini, Y., Krieger, A. M. & Yekutieli, D. Adaptive linear step-up procedures that control the false. Biometrika 93 , 491-507 (2006). Liu, J. et al. ExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection and analysis of exocytosis (Cold Spring Harbor Laboratory, 2022). Tables Tables I and II are available in the Supplementary Files section Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalSpreadsheet1.xlsx Dataset 1 SupplementalSpreadsheet2.xlsx Dataset 2 SupplementalFigures.docx Supplemental Figures 1-3 Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6890466","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483689574,"identity":"0e953900-cc0c-4d46-a761-f20249501e71","order_by":0,"name":"Robert Hennigan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBAC+wYeMJ3YACQOALEcAwMPfi0GB0AKEhBajEnTArWOkJbjvQcfV/5gSOyX7j148EvNnfT+9rNHN/xgsMmXd8Dhl55zyYZnEhjqZ845l3BY5tiz3Bln8tJu9jCkWW48gF2LnUSOmWQD0GEbbuQYHJZgO5y7QYLH7DYDw2EDwwbsWowlcsx/grTsB2v5dzjdgJAWwxk5ZoxgWyRyDA5+bDucANcij8v7Z84lSzakSSTOANnC2HfYcMaZHLObPQZpBgZ4Quxjg41NYv+MHOOPP74dludvP2N240eFjYE8DodBgQSYZEbEiAEoyvBqgQDGH8g8AraMglEwCkbByAEAuGdlL4qRABkAAAAASUVORK5CYII=","orcid":"","institution":"Cincinnati Children’s Hospital Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Robert","middleName":"","lastName":"Hennigan","suffix":""},{"id":483689575,"identity":"d088af01-6bbb-4325-a76e-1177895a0d1e","order_by":1,"name":"Kaley McLaughlin","email":"","orcid":"","institution":"Cincinnati Children’s Hospital Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Kaley","middleName":"","lastName":"McLaughlin","suffix":""},{"id":483689576,"identity":"0742f3cb-d170-43c7-8e0f-4773b5950e82","order_by":2,"name":"Nancy Ratner","email":"","orcid":"https://orcid.org/0000-0001-5030-9354","institution":"CCHMC","correspondingAuthor":false,"prefix":"","firstName":"Nancy","middleName":"","lastName":"Ratner","suffix":""}],"badges":[],"createdAt":"2025-06-13 19:30:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6890466/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6890466/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86634436,"identity":"7664aa13-0f52-4f0d-b389-c6cd2d3d9513","added_by":"auto","created_at":"2025-07-14 07:06:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6245474,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin-APEX Cell Lines\u003c/p\u003e\n\u003cp\u003eA.\tControl and Merlin knockout cells plated at low cell density (2,500 cells per cm2) and high cell density (20,000 cells per cm2), incubated for 48 hours then fixed and stained with anti-YAP1 antibodies and counterstained for nuclei and actin with DAPI and phalloidin and confocal images were acquired with 60x objective. \u0026nbsp;Scale bar = 50 µ.\u003c/p\u003e\n\u003cp\u003eB.\tQuantitation of the nuclear to cytoplasmic ratio of YAP1 at different cell densities. \u0026nbsp;Control and Merlin knockout cells were plated at 20,000, 10,000, 5,000 and 2,500 cells per cm2, incubated for 48 hours, stained with antibodies to YAP1 then confocal images were acquired with 20x objective, 4 fields per point. \u0026nbsp;Acquisition parameters were constant for all images. \u0026nbsp;To assess the proportion of YAP1 in the nucleus, the DAPI stained images were used to make a binary mask that was applied to the YAP1 image to measure the integrated density of the YAP1 signal specific to the nucleus. \u0026nbsp;The proportion of nuclear staining was expressed as: \u0026nbsp;% nuclear YAP1 = nuclear signal ÷ (total signal-nuclear signal).\u003c/p\u003e\n\u003cp\u003eC.\tA schematic diagram representing Merlin with the proximity biotinylation enzyme APEX2 fused to its C-terminus. \u0026nbsp;Below: the amino acid sequences for the Merlin isoform 1 and isoform 2 C-terminus. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD.\tNormalized APEX activity in APEX control, Merlin isoform 1-APEX and Merlin isoform 2-APEX\u003c/p\u003e\n\u003cp\u003eE.\tImmunoblot analysis of cell lysate from parental iHSC-1λ, and Merlin isoform 1-APEX and Merlin isoform 2-APEX expressing cells probed with antibodies to Merlin and β-actin as a loading control.\u003c/p\u003e\n\u003cp\u003eF-K Subcellular location of Merlin in confluent monolayers (F., H., J.) and individual cells (G., I., K.) for endogenous Merlin (F., G.), Merlin isoform 1-APEX (H., I.) and Merlin isoform 2-APEX (J., K.).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/5c9cb4a5c4ffca75f8a8746e.png"},{"id":86633901,"identity":"279c8074-60cc-463c-9568-8dd61b0de5b1","added_by":"auto","created_at":"2025-07-14 06:58:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3751752,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin Proximity Biotinylation\u003c/p\u003e\n\u003cp\u003eA. Typical subconfluent (left) and confluent (right) cell densities for Merlin isoform 1-APEX proximity biotinylation experiments.\u003c/p\u003e\n\u003cp\u003eB. Western blots of biotinylated proteins from Merlin isoform 1-APEX from subconfluent and confluent cells probed with antibodies to Merlin (top) and streptavidin to visualize biotinylated proteins (bottom).\u003c/p\u003e\n\u003cp\u003eC. Merlin isoform 1 proximal proteins, displayed as Volcano plots showing statistical significance plotted against the peptide difference between the Merlin and control. The Y-axis plots the Q value (Q= -log\u003csub\u003e10\u003c/sub\u003e p). The dotted line indicates the statistical significance threshold (p=0.05, Q=1.30). The X-axis plots the mean difference of normalized peptide counts per identified protein between Merlin-APEX expressing cells and APEX expressing control cells. Data from subconfluent cells is plotted in blue and from confluent cells is plotted in red. Proteins that had been identified in our published, BirA-based proximity biotinylation experiments are indicated by a surrounding square\u003c/p\u003e\n\u003cp\u003eD. Venn diagrams depicting the number of proteins biotinylated by Merlin isoform 1-APEX (left graph) and Merlin isoform 2-APEX (right graph) greater than APEX only control (5% FDR) in confluent and subconfluent cells. The Venn diagrams show the number of unique and common proteins comparing (left to right) isoform 1 vs isoform 2, confluent vs subconfluent for all identified proteins, confluent vs subconfluent for isoform 1 and confluent vs subconfluent for isoform 2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/34b51166ed227ea4a9e3b8f8.png"},{"id":86633285,"identity":"33ab260f-581c-4686-bede-c5aba260cb2e","added_by":"auto","created_at":"2025-07-14 06:50:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2986624,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin Proximity Network\u003c/p\u003e\n\u003cp\u003eA. Merlin proximal proteins displayed as a proximity network for Merlin isoform 1 (right) and isoform 2 (left) in subconfluent (bottom) and confluent (top) cells. The nodes are color coded based on gene ontogeny terms derived from STRING based functional enrichment analysis for cell junctional proteins (red), signal transduction proteins (green) and intracellular transport (blue). Proteins sets that are proximal to both isoform 1 and isoform 2 in confluent cells are highlighted.\u003c/p\u003e\n\u003cp\u003eB. Hierarchical cluster analysis showing four subsets of proteins proximal to both isoform 1 and isoform 2 in confluent cells are shown. The mean normalized peptide numbers are displayed within the box the relevant gene symbol and protein name are presented. The blue cells indicate no significant difference with APEX alone controls.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/5bea7515d813226e131f3a00.png"},{"id":86633296,"identity":"150237d4-d695-43c4-a90c-8b9a6555fc1f","added_by":"auto","created_at":"2025-07-14 06:50:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2529644,"visible":true,"origin":"","legend":"\u003cp\u003eDirect Merlin Binding Assays\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; A schematic diagram of the direct Merlin binding assay showing the Merlin-NanoLuc probe, the GFP fused bait protein bound to anti-GFP nanobody bound beads.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; Merlin-NanoLuc probe purification by gel filtration showing the fractionation of affinity purified Merlin-NanoLuc activity and A\u003csup\u003e280\u003c/sup\u003e, showing the pooled peak 1 used in the binding assays.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp; Aliquots of purified Merlin-NanoLuc proteins stained for total protein.\u0026nbsp; Left to right: Marker, BSA titration from 1000 to 125 ng, Merlin-NanoLuc from the initial affinity purification step, and Merlin-NanoLuc pooled from peak 1.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp; Luminescence images from 96 well plates containing triplicate Merlin binding assays in the presence and absence of di8-PIP\u003csub\u003e2\u003c/sub\u003e for 33 putative Merlin binding proteins.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp; A heat map showing luminometer data for Merlin binding activity in triplicate.\u0026nbsp; Data are normalized to the mean of the GFP control for the reactions shown in D.\u0026nbsp; The intensity of the blue color is proportional to the relative binding.\u0026nbsp; The data highlighted in bold indicates statistical significance (T-test, p \u0026lt;= 0.05).\u0026nbsp; The last column shows the fold increased binding in the presence of PIP\u003csub\u003e2\u003c/sub\u003e, bold numerals are statistically significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/a7af02495be69f838a2a87e6.png"},{"id":86633281,"identity":"4564ad0c-7579-41c0-8c12-5eaa48a7ef41","added_by":"auto","created_at":"2025-07-14 06:50:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":841150,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin-RalA/B Interaction\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; BRET assays of Merlin-NanoLuc:GFP-RalA (left) and Merlin-NanoLuc:GFP-RalB (right) showing excitation of the GFP-Ral proteins by the luminescence of Merlin-NanoLuc in complex.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; RalA and RalB activity in Control and Merlin KO cells in subconfluent and confluent conditions.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp; Co-immunoprecipitation of NanoLuc-RalB with wild type and mutant Merlin-GFPs.\u0026nbsp; Left to right: NanolLuc alone control, wild type Merlin, an N-terminal deletion mutant D18, the FERM domain deletion mutant D39-121, the closed conformation mutant AR and the open conformation mutant DEL.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp; Merlin binding for RalB wild type, the G23V and Q72L active mutants and the D28N dominant negative mutant showing an image of the luminiescence (left) and normalized Merlin binding activity (right) in the presence and absence of di8-PIP\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp; Co-immunoprecipitation of mCherry-RalB with the exocyst effectors Sec5-NanoLuc and Exo84-NanoLuc in the presence of either control GFP of Merlin-GFP.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/fc2b0f46faa8425cd043ca56.png"},{"id":86633909,"identity":"fb79238d-7ca3-4d5f-ac21-6f17031f21fe","added_by":"auto","created_at":"2025-07-14 06:58:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9300609,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of Endogenous Merlin, N-cadherin, Exo70 and RalA/B\u003c/p\u003e\n\u003cp\u003eA. Confocal images showing the pattern of co-localization of endogenous Merlin (green) and N-Cadherin (red) in the immortalized human Schwann cell line, iHSC-1l. Bar = 50 µ.\u003c/p\u003e\n\u003cp\u003eB. Confocal images showing the pattern of co-localization of endogenous Merlin (green) and Exo70 (red) in the immortalized human Schwann cell line, iHSC-1l. Bar = 50 µ.\u003c/p\u003e\n\u003cp\u003eC. Confocal images showing the pattern of co-localization of endogenous Merlin (green) and RalA/B (red) in the immortalized human Schwann cell line, iHSC-1l. Bar = 50 µ.\u003c/p\u003e\n\u003cp\u003eD. NSPARC super-resolution showing the co-localization of Merlin (green) and RalA/B (red) in iHSC-1l. Bar = 20 µ\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/3449606fc71f5114d4560be5.png"},{"id":86633301,"identity":"4f5a9de7-9079-4d93-b59a-9b57dff7720b","added_by":"auto","created_at":"2025-07-14 06:50:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1486732,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin Regulation of Exocytosis Kinetics\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; An image of showing a summation of VAMP2-pHlourin exocytic events over a 1-minute time lapse at 100 msec intervals for a total of 601 frames per cell in a live iHSCL-1l Schwann cell.\u0026nbsp; Exocytic events were defined by the appearance of an increase in green fluorescence of at least 4-fold above background in a 4-8 square pixel in area for up to 11 seconds with a R\u003csup\u003e2\u003c/sup\u003e of at least 0.8 for decay kinetics and 0.75 for Gaussian distribution using ExoJ plugin of Image J.\u0026nbsp; Inset: time course of a single, 4 second, exocytic event shown at 100 msec intervals.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; An example of the total number of exocytic events identified in a control iHSC-1l Schwann cell over a 1-minute time course at 100 msec. intervals. The red circles identify event durations greater than 1.5 sec. and the yellow squares identify event durations less than or equal to 1.5 sec.\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp; An example of the total number of exocytic events identified in a Merlin knockout iHSC-1l Schwann cell over a 1-minute time course at 100 msec. intervals. The red circles identify event durations greater than 1.5 sec. and the yellow squares identify event durations less than or equal to 1.5 sec.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp; Frequency histogram depicting the exocytic event frequency comparing iHSC-1l control and iHSC-1l Merlin knockout cells showing a shift to longer event duration events in Merlin knockout cells.\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean exocytic event duration plus SEM of iHSC-1l control and iHSC-1l Merlin knockout cells.\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp; Mean exocytic duration of iHSC-1l control and iHSC-1l Merlin knockout cells expressing control mCherry, mCherry-RalB, the active mutant mCherry-RalB\u003csup\u003eG23V\u003c/sup\u003e and the dominant negative mutant mCherry-RalB\u003csup\u003eD28N\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/fc5999fc760b938fba90fbcd.png"},{"id":86633906,"identity":"776d9d9f-a43d-4315-8ff4-a32d0030817d","added_by":"auto","created_at":"2025-07-14 06:58:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":258430,"visible":true,"origin":"","legend":"\u003cp\u003eMerlin-Ral Regulation of Exocytosis\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;A schematic diagram of Merlin regulation of exocytosis by competitive inhibition of RalB.\u0026nbsp; In actively growing, sub-confluent cells (top), Merlin is inactive and RalB mediated exocytosis leads to the export of integral membrane proteins and lipid plasma membrane components to the cell surface, supporting the expansion of the plasma membrane that is necessary for active cell growth.\u0026nbsp; Contact-inhibited cells that are not actively growing (bottom) do not require plasma membrane expansion, Merlin is activated by PIP\u003csub\u003e2\u003c/sub\u003e mediated dimerization, binds to RalB, to prevent association with the exocyst and inhibit full fusion exocytosis.\u0026nbsp;\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/bcefc0e6cf7c9db290656c52.png"},{"id":86635844,"identity":"198f7b9b-f820-4330-b087-b29e6764ad5e","added_by":"auto","created_at":"2025-07-14 07:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25273874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/8731094f-4a43-4e8a-9cc3-23fdc43c9695.pdf"},{"id":86633278,"identity":"d93c35fa-f67e-41c0-aac1-1629759b3635","added_by":"auto","created_at":"2025-07-14 06:50:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":895514,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"SupplementalSpreadsheet1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/1b387b06e193025b395f1e06.xlsx"},{"id":86633292,"identity":"f86d85bb-9bac-4480-a19c-d44b3a1acedc","added_by":"auto","created_at":"2025-07-14 06:50:50","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":371750,"visible":true,"origin":"","legend":"Dataset 2","description":"","filename":"SupplementalSpreadsheet2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/41b646d9ac0cd49797c9f0f5.xlsx"},{"id":86633289,"identity":"a9a02ad9-92e3-46d1-9672-d749b373501c","added_by":"auto","created_at":"2025-07-14 06:50:50","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4616996,"visible":true,"origin":"","legend":"Supplemental Figures 1-3","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/fae1701371d38cb76a593b1c.docx"},{"id":86633287,"identity":"8be5ad5d-3be9-4d11-b457-501fe763d373","added_by":"auto","created_at":"2025-07-14 06:50:50","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":407180,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6890466/v1/fc5016e62b6911f721003c9f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Active Merlin Binds RalB to Regulate Exocytosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNF2-related schwannomatosis, formerly called Neurofibromatosis type 2, is an inherited disease characterized by benign peripheral nerve tumors known as schwannomas, slow growing tumors that cause significant morbidity and are resistant to chemotherapy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNF2\u003c/em\u003e-mutant schwannomas also arise in the general population, in which they represent 8% of all intracranial tumors\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNF2\u003c/em\u003e loss is also common in meningiomas, mesotheliomas, and other types of sporadic tumors. Targeted deletion of the \u003cem\u003eNf2\u003c/em\u003e gene in mouse Schwann cells leads to schwannoma\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ec\u003c/em\u003eonfirming that \u003cem\u003eNF2\u003c/em\u003e is a bona-fide tumor suppressor gene, with loss of function causing tumorigenesis. The major phenotype of \u003cem\u003eNf2\u003c/em\u003e-null cells \u003cem\u003ein vitro\u003c/em\u003e is impaired contact inhibition of growth\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, a fundamental hallmark of cancer and a key feature of NF2 pathogenesis.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eNF2\u003c/em\u003e gene encodes Merlin, a 70-kDa member of the Ezrin-Radixin-Moesin (ERM) branch of the band 4.1 superfamily\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Merlin secondary structure consists of an N-terminal FERM domain followed by a central a-helical region that positions the C-terminal domain (CTD) for intramolecular interaction with the FERM domain\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Upon the release of the CTD from the FERM domain, Merlin transitions to a more open \u0026ldquo;active\u0026rdquo; conformation that allows Merlin dimerization and FERM domain interaction with critical binding proteins\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A Merlin mutant that stabilizes the FERM-CTD interaction adopts a closed conformation that has impaired tumor suppressor activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Conversely, a more open, FERM-accessible conformation mutant retains tumor suppressor activity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Merlin is active when cells are contact inhibited at confluence and inactive in growing, sub-confluent cells\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Merlin has two major splice variants, isoform 1 and isoform 2, that differ in their extreme C-terminal amino acids\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The isoform 2 C-terminus has a lower affinity for the FERM domain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, a more open conformation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and increased ability to dimerize\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, both Merlin isoform 1 or isoform 2 are tumor suppressors\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and mutations in both isoforms are detected in tumors\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMerlin is localized predominately to the inner face of the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e where it is associated with a variety of cell to cell and cell to substrate junctional complexes, including integrin-based focal adhesions and cadherin-based adherens junctions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This localization is facilitated by three pairs of basic amnio acids in the FERM domain that are necessary for association with lipid rafts and for growth suppression\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Membrane localization positions Merlin for binding the lipid second messenger phosphotydylinositol-4,5-biphosphate (PIP\u003csub\u003e2\u003c/sub\u003e). PIP\u003csub\u003e2\u003c/sub\u003e binding causes an allosteric change in the Merlin central a-helical domain that forces the CTD and FERM domains apart\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, allowing Merlin to assume it\u0026rsquo;s open, FERM accessible conformation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. PIP\u003csub\u003e2\u003c/sub\u003e-mediated transition to an open conformation leads to Merlin dimerization and increased affinity for target proteins including Lats1, YAP1 and ASPP2\u003csup\u003e11,16\u003c/sup\u003e. These data support a model in which Merlin is activated at high cell density by dimerization in response to transient, localized increases in PIP\u003csub\u003e2\u003c/sub\u003e levels, leading to contact inhibition of growth.\u003c/p\u003e \u003cp\u003eIn mammalian cells, contact inhibition of growth is mediated by the Hippo pathway, a growth inhibitory kinase cascade that responds to mechanosensory cues transmitted via cell junctional signaling complexes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Merlin regulates the HIPPO pathway by binding to and activating the core HIPPO kinase, Lats1/2. Lats1/2 then phosphorylates the transcriptional co-activator YAP1, leading to its ubiquitination and degradation, thereby preventing YAP1 nuclear localization, leading to growth arrest\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. While impaired Hippo signaling due to Merlin loss is considered a primary mechanism contributing to NF2 schwannomas development, other mechanisms likely also contribute to schwannoma development. For example, the constitutive activation of a variety other oncogenic signaling networks such as Ras-ERK, Rac1, src, b-catenin, and the mTOR protein kinase complex have been described in Merlin-deficient cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Additionally, reports in the literature implicate Merlin in establishing or maintaining cellular polarity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and in regulating intracellular vesicular trafficking and the endocytic process, micropinocytosis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur objective was to develop a more complete understanding of Merlin function in cell polarity, intracellular trafficking, and signal transduction. The challenge was in identifying those target proteins and pathways that are important for Merlin tumor suppressor function specifically when Merlin is active. We used a proximity biotinylation strategy coupled with a robust Merlin binding screening system to identify proteins that are proximal to both Merlin isoform 1 and isoform 2 at confluence, in contact inhibited cells when Merlin is active, but not in sub-confluent, growing cells when Merlin is inactive. We identified many novel Merlin proximal proteins that confirmed Merlin involvement in specific cell-cell and cell-substrate junctions, expanded the number of signal transduction pathways that Merlin may influence and highlight a critical role for Merlin in intracellular transport. We identified the small GTPases RalA and RalB as high affinity PIP\u003csub\u003e2\u003c/sub\u003e dependent Merlin binding proteins and demonstrate that Merlin competitively inhibits RalB binding to its exocyst effectors Sec5 and Exo84. Merlin loss increased Ral activity in confluent Schwann cells and regulates the kinetics of exocytosis in a RalB dependent manner. Our results identify RalB as a novel, critical binding partner for active Merlin and raise the possibility that the RalA/B pathway may be targeted therapeutically to treat NF2-related schwannomatosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eContact Inhibition Parameters\u003c/h2\u003e \u003cp\u003eWe defined the cell densities at which Merlin is active in the immortalized human Schwann cell line iHSC-1l\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e by comparing the growth parameters of Merlin knockout and scrambled gRNA control cell lines (Supplemental Fig.\u0026nbsp;1). YAP1 staining confirmed a significant increase in the proportion of cytosolic YAP1 in control cells 48 hours after seeding at high density (10\u0026ndash;20,000 cells per cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e) relative to low cell density (2,500 to 5000 cells per cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e that was not apparent in the Merlin knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B, Supplemental Fig.\u0026nbsp;2). We generated iHSC-1l cell lines expressing Merlin isoform1 or isoform 2 with a recombinant ascorbate peroxidase (APEX2)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e fused to their C-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). We measured APEX activity in lysates from these cell lines. Mer1-APEX and Mer2-APEX activity was roughly equivalent and four-fold less than control cell lysates expressing APEX alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Merlin iso1 and iso2-APEX proteins were expressed\u0026thinsp;~\u0026thinsp;1.6 and 2.0-fold over endogenous levels, as measured by densitometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). There was no significant difference in the subcellular localization of endogenous Merlin (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD., E.), Merlin isoform 1-APEX (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF., G.) and Merlin isoform 2-APEX (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH., I.), ruling out artifacts caused by gross differences in subcellular localization.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProximity Biotinylation\u003c/h3\u003e\n\u003cp\u003eiHSC-1l cells expressing either Merlin iso1-APEX or Merlin iso2-APEX were plated at either 5,000 cells per cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e or 15,000 cells per cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and incubated for 48 hours to produce either a sub-confluent, growing cell population or a confluent, contact inhibited cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We initiated a 1-minute APEX mediated biotinylation reaction, then harvested cell lysates. Biotinylated proteins were purified by streptavidin affinity chromatography, washed extensively then processed for MALDI-TOF mass spectroscopy. These experiments were performed in triplicate. Western blots of aliquots showed significant biotinylated proteins with a range of molecular weights for both sub-confluent and confluent cells, with a noticeable increase in the confluent samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMerlin proximal proteins were identified from the mass spectroscopy data by the normalized total number of peptides mapping to each protein in the Merlin-expressing samples that were significantly greater than in the APEX control samples, as judged by T-test (p-value of \u0026lt;\u0026thinsp;0.05 with a 5% false discovery rate, Supplemental Spreadsheet 1). The mass spectroscopy data for isoform 1 and isoform 2 proximal proteins is displayed in Volcano plots showing the mean normalized peptide difference between Merlin samples and APEX only control and the statistical significance (Q= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:-{\\text{log}}_{10}p\\)\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We identified a total of 542 proteins across the four experimental samples that met the selection criteria. Of these, 81 proteins were also identified in our published proximity biotinylation dataset (indicated by boxed datapoints in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Furthermore, we identified multiple proteins reported to bind Merlin in the literature, including angiomotin\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, Erbin\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, YAP1\u003csup\u003e28\u003c/sup\u003e, TP53BP2\u003csup\u003e14\u003c/sup\u003e, PPP1R12A (Mypt1)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, Ezrin\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and Moesin\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e (Table I), validating the new dataset. There were 466 proteins identified by isoform 1 and 410 by isoform 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). A total of 322 proteins were present in sub-confluent cells and 410 in confluent cells, with 200 proteins that overlapped. The number of proteins proximal to Merlin isoform 1 significantly increased in confluent cells, from 211 to 394 with 138 proteins identified at both cell densities but 256 identified exclusively in confluent cells. In contrast, 195 of the 210 proteins identified by Merlin isoform 2 were in sub-confluent cells, with only 57 in confluent cells. This suggests that the isoforms might have different functions in addition to their shared tumor suppressor function, and that isoform 1 may be more active in confluent cells while isoform 2 is more active in sub-confluent cells.\u003c/p\u003e\n\u003ch3\u003eFunctional Enrichment Analysis\u003c/h3\u003e\n\u003cp\u003eTo begin to understand the potential functional significance of these proteins we performed a STRING based functional enrichment analysis on the Merlin proximal proteins\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, displayed as a proximity network\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The proximal proteins were color coded for gene ontogeny terms to indicate proteins functioning in cell junctional complexes, intracellular trafficking and signal transduction (Table I). We identified 206 proteins associated with cell junctions, including 40 adherens junction proteins, 30 focal adhesions proteins and 28 tight junction proteins (Table I). These included N-cadherin, catenin-a, catenin-b, catenin-d, integrin-b1, integrin-a2 and the ephrin receptors EPHA2, EPHB2 and EPHB4 (Table II, Supplemental Spreadsheet 1). We also identified 137 proteins involved in intracellular transport or membrane trafficking, (87 and 80 proteins respectively), and 107 proteins more specifically mapping to endocytosis or exocytosis (Table I). These included 10 members of the Rab family of small GTPases, most prominently the endocytic Rab7a and the recycling/exocytic Ral11b (Table II, Supplemental Spreadsheet 1). Other trafficking proteins include the AP-2 complex subunits AP2A1 and AP2A2 and proteins critical for exocytosis, including 3 out of the 8 components of the exocyst, EXOC2, EXOC4 and EXOC7 (Table II, Supplemental Spreadsheet 1). Significantly, we identified 173 signal transduction proteins (Table I), with 48 mapping to the HIPPO pathway, including angionmotin, ASPP2, and YAP1 (Table II, Supplemental Spreadsheet 1). Surprisingly, neither of the HIPPO core kinases, Lats1/2 or Mst1/2, were identified. There were 40 members of the receptor tyrosine kinase pathway and 25 members of the PI3K/Akt3 pathways (Table I); a total of 54 proteins were kinases or had kinase activity. These included growth factor receptors (EGFR, MET, and PDGFRb), and protein kinases (PAK2, JAK1, integrin linked kinase, ILK1 and myosin light chain kinase MYLK) (Table II, Supplemental Spreadsheet 1). There were 25 phosphatidylinositol binding proteins and 36 molecular adapters (Table I). There were also 19 small GTPases, including 10 Rab family members plus CDC42, NRAS, RHOA, RRAS2, RAN, RAP1A, RAP2A, RALA and RALB (Table II, Supplemental Spreadsheet 1).\u003c/p\u003e \u003cp\u003eA subset of this network consisted of 41 proteins biotinylated by both isoform 1 and isoform 2 in confluent cells. We hypothesized that this cluster would contain the proteins most likely to interact with active Merlin. Hierarchical cluster analysis revealed four subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). There were 14 proximal to both isoform 1 and isoform 2 in sub-confluent and confluent cells, including the Merlin binding protein angiomotin and upstream cell junctional complexes like integrin-b1 and catenin-d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table II). Two sets totaling 20 proteins were proximal to both isoform 1 and isoform 2 in confluent cells and either isoform 1 or isoform 2 in sub-confluent cells. These included the molecular adapter proteins DOK1 and EPS8, the calcium binding protein calponin-2 and the small GTPases cdc42, Rap2 and RalA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table II). Finally, there was a subset of 7 proteins are proximal to both isoform 1 and isoform 2 exclusively in confluent cells. These included the small GTPases RalB and Rap1a, the v-SNARE protein SNAP-29, the large GPCR subunits GNAQ and GNAI2, the facilitated glucose transporter SLC2A1 and the endocytosis associated protein NECAP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Table II).\u003c/p\u003e\n\u003ch3\u003eMerlin Binding\u003c/h3\u003e\n\u003cp\u003eWe hypothesized that Merlin proximal proteins that are critical to its tumor suppressor function are likely to be signaling proteins that physically bind to active Merlin. We therefore focused screening experiments on a selection of Merlin-proximal cell junctional complex proteins, ERM proteins, kinases, phosphatases and small GTPases. Merlin is activated by PIP\u003csub\u003e2\u003c/sub\u003e binding\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e which induces a conformational change\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e that allows dimerization\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and increases its affinity for binding\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Therefore, we screened for direct binding to Merlin binding using purified proteins in the presence and absence of PIP\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e11,14\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD presents the results of 35 of these assays performed in triplicate, expressed relative to the GFP negative control. We evaluated known Merlin binding proteins angiomotin, Lats1, ASPP2 and YAP1. Merlin consistently showed the highest relative affinity for angiomotin, \u0026gt; 65-fold above control, in a PIP\u003csub\u003e2\u003c/sub\u003e independent manner. Merlin also showed relatively high affinity for Lats1, but significantly less for ASPP2 and YAP1. There was a marked PIP\u003csub\u003e2\u003c/sub\u003e-dependent increase in affinity for YAP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, the cell adhesion complex proteins N-cadherin, b1-integrin, catenin-a, catenin-b and catenin-d showed low relative affinity binding, (2-4-fold) and minimal PIP\u003csub\u003e2\u003c/sub\u003e effect. Similarly, Merlin showed low relative affinity binding and small increases in the presence of PIP\u003csub\u003e2\u003c/sub\u003e to the ERM proteins Ezrin, Moesin, and the adapter protein Erbin. The same pattern was seen for kinases (PTK2, CHUK, ILK, MYLK, AKT1 and AKT2), and phosphatases (PP1A and MYPT) tested. All these proteins bound Merlin greater than they bound control, but with relatively low affinity and little increase in the presence of PIP\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, the small GTPases, N-Ras, cdc42, RhoA, Rab7a, Rab11b, RalA and RalB showed significant PIP\u003csub\u003e2\u003c/sub\u003e-dependent binding. Most striking was the high relative affinity and the significant increase in binding in the presence of PIP\u003csub\u003e2\u003c/sub\u003e that Merlin showed for RalA and RalB. Given the strong binding between Merlin and RalA and RalB, we searched for Ral-GAP, -GEF and effector proteins in our Merlin-proximal dataset. We identified Exo70, Sec8 and the Ral effector Sec5, three components of exocyst complex (Table II, Supplemental Spreadsheet 1). The exocyst is a conserved 8 protein complex that mediates the initial tethering of the vesicle to the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, stimulates SNARE complex assembly\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and functions in both constitutive\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and regulated exocytosis\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Merlin binding to Exo70, Sec8 and to the Ral effectors Sec5 and Exo84 was detectable and showed a slight increase in the presence of PIP\u003csub\u003e2\u003c/sub\u003e. We conclude that RalA and RalB are good candidates for critical signal transduction proteins that interact directly with active, PIP\u003csub\u003e2\u003c/sub\u003e-bound Merlin.\u003c/p\u003e\n\u003ch3\u003eMerlin Ral Interactions\u003c/h3\u003e\n\u003cp\u003eWe took advantage of the NanoLuc-GFP binding assay to perform bioluminescence resonance energy transfer (BRET) to confirm close interaction between Merlin-NanoLuc and GFP RalA or GFP-RalB in complex. There was significant excitation of the GFP fluorescence, peaking at 510 nm in both GFP-RalA and GFP-RalB proteins when in complex with Merlin-NanoLuc, indicating that Merlin forms close complexes with both RalA and RalB (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Given the relatively high affinity that Merlin has for RalA and RalB we tested whether Merlin affects Ral activity. We assayed for both RalA and RalB activity in control and Merlin knockout iHSC-1l cells, at either sub-confluent or confluent cell densities. In sub-confluent cells, both RalA-GTP and RalB-GTP levels was equal between Merlin knockout and control cells, but in confluent cells RalA/B-GTP levels were significantly increased in Merlin knockout cells relative to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Increased RalA activity is consistent with reports in the literature\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Since our proximity biotinylation data identified RalB as proximal to both Merlin isoforms only confluent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e), we chose to focus on RalB going forward.\u003c/p\u003e \u003cp\u003eThe Merlin-RalB interaction was impaired by deletion of Merlin\u0026rsquo;s unique 20 N-terminus (Merlin-DN18, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and lost in an in-frame deletion within the FERM domain (Merlin-D39-121, Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting that RalB binds to the Merlin FERM domain and that the Merlin N-terminus is necessary for this interaction. RalB binding was also impaired to the closed conformation mutant Merlin-AR\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, the open conformation mutant Merlin-DEL\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e was unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These results suggest that RalB preferentially binds to Merlin\u0026rsquo;s open conformation and are consistent with the PIP\u003csub\u003e2\u003c/sub\u003e-dependent nature of this interaction.\u003c/p\u003e \u003cp\u003eRalA and RalB are small G-proteins, which cycle between a GTP-bound active form and a GDP-bound inactive form. To determine if Merlin specifically binds to active or inactive RalB we performed Merlin-binding assays using 2 constitutively active mutants (RalB\u003csup\u003eG23V\u003c/sup\u003e and RalB\u003csup\u003eQ72L\u003c/sup\u003e) and a dominant negative mutant (RalB\u003csup\u003eD28N\u003c/sup\u003e). All 3 RalB proteins bound to Merlin in the presence of PIP\u003csub\u003e2\u003c/sub\u003e, with slight differences in relative binding efficiencies but no correlation to RalB activation status (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results suggest that Merlin binds RalB in the presence of PIP\u003csub\u003e2\u003c/sub\u003e, suppresses RalB activity during contact inhibition of growth, and that in the absence of Merlin this negative regulation is lost. Next, to determine if Merlin binding either facilitates or inhibits RalB interaction with its exocyst effector proteins, we performed binding assays between RalB and Sec5 or Exo84 in the presence or absence of Merlin. These experiments showed a significant, 50%, reduction of RalB binding to Sec5 and Exo84 in the presence of Merlin (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This suggests that Merlin may function to competitively inhibit RalB binding to its exocyst effectors, Sec5 and Exo84.\u003c/p\u003e \u003cp\u003eTo determine if endogenous Merlin co-localizes with the Ral GTPases, iHSC-1l cells were co-stained with antibodies that detect Merlin and pan-RalA/B. Merlin was distributed on the ventral surface of the plasma membrane in a distinct punctate pattern and co-localized, in part, to cell junctions that can contain N-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The exocyst marker Exo70 was also localized to the ventral plasma membrane, where it showed a punctate pattern with significant co-localization with endogenous Merlin (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Antibodies detecting RalA/B showed a similar ventral punctate staining pattern, and significant overlap with Merlin staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). To better verify overlap, we acquired super-resolution microscopic images of Merlin and Ral A/B staining. This confirmed significant co-localization between Merlin and RalA/B, co-localized in ventral puncta (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). This subcellular localization data is consistent with Merlin-Ral interaction and a role for Merlin and RalA/B in the process of exocytosis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMerlin Regulates Exocytosis via RalB\u003c/h2\u003e \u003cp\u003eThe ability of Merlin to bind to RalB in a PIP\u003csub\u003e2\u003c/sub\u003e-dependent manner, to competitively inhibit RalB binding to its exocyst effectors Sec5 and Exo84 and the co-localization of endogenous Merlin with RalA/B punctate structures on the ventral plasma membrane strongly suggested that Merlin might regulate exocytosis via RalB at the exocyst. To test this idea, we used a vesicular membrane marker, VAMP2, fused to the pH sensitive GFP mutant, pHluorin, to measure exocytosis\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Using this marker, endocytic events are indicated by transient increases in fluorescence in small areas on the ventral cell surface, which can be visualized in live cells by TIRF microscopy over for 1\u0026ndash;2 min., in images taken at 100 msec. intervals (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We compared exocytosis between control and Merlin knockout iHSC-1l cells and tabulated the number, size, intensity and duration of events. Examples mapping exocytic events in control and Merlin knockout cells are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and C. There was no significant difference in the number, size or intensity of the exocytic events between control and Merlin knockout. However, we identified a significant difference in event duration. This result is apparent in a frequency histogram of these data (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). In control cells\u0026thinsp;~\u0026thinsp;59% of the events lasted between 1000 and 2000 msec., ~\u0026thinsp;33% from 2000 to 3000 msec. and ~\u0026thinsp;8% lasted longer than 3000 msec. In Merlin knockout cells the figures are ~\u0026thinsp;39%, 41% and 20% respectively. The median duration for control and Merlin knockout is 1300 msec. vs 1600 msec with mean values of 1612 vs 1974 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These data show that loss of Merlin affects the kinetics of exocytosis.\u003c/p\u003e \u003cp\u003eTo test if RalB activity is necessary for this effect on the kinetics of exocytosis, we generated control and Merlin knockout cell lines co-expressing VAMP2-pHluorin with mCherry fused to active RalB\u003csup\u003eG23V\u003c/sup\u003e, dominant negative RalB\u003csup\u003eD28N\u003c/sup\u003e or an mCherry control. The control cells expressing mCherry alone had a mean exocytic duration of 1664 msec (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Expression of active RalB\u003csup\u003eG23V\u003c/sup\u003e significantly increased this value to 1874 msec., phenocopying the effect of Merlin loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). In contrast, Merlin knockout cells expressing mCherry alone had a mean exocytic duration of 1928 msec and expression of active RalB\u003csup\u003eG23V\u003c/sup\u003e did not significantly change this value (1979 msec). In contrast, Merlin knockout cells expressing dominant negative RalB\u003csup\u003eD28N\u003c/sup\u003e had a mean exocytic event duration of 1608 msec, reverting to control event duration. We conclude that Merlin regulates exocytic kinetics by inhibiting RalB activity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMerlin Proximal Proteins\u003c/h2\u003e \u003cp\u003eWe identified 542 Merlin-proximal proteins in Schwann cells, including known Merlin proteins and proteins identified in a previous Merlin proximity biotinylation study\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, validating the dataset. Overall, we confirmed and extended earlier data by identifying the cell surface receptors for cell to cell and cell to substrate cell junctions (Table I). A striking new result was the strong representation of Merlin-proximal proteins involved in intracellular vesicular trafficking, an observation that is consistent with data from the literature\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Finally, we identified 165 proteins involved in general signal transduction, the majority of which are kinases, molecular adapters or phosphatidylinositol binding proteins, including 48 proteins associated with the HIPPO pathway and 19 small GTPases, including 10 members of the Rab subfamily. The other small GTPases were, CDC42, NRAS, RRAS2, RALA, RALB, RAN, RAP1A, RAP2A, and RHOA. Notable by its absence was RAC1, the small GTPase that was most often associated with Merlin in the early literature\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The absence of a protein like Rac1 and a known Merlin binding protein like Lats1/2 does cannot exclude these proteins as Merlin interactors because of biases inherent in the proximity biotinylation system\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMerlin Binding Proteins\u003c/h2\u003e \u003cp\u003eOur goal was to identify proteins that interact with Merlin when it is activated by PIP\u003csub\u003e2\u003c/sub\u003e-mediated dimerization\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Direct binding assays allowed us to test for interaction in the presence and absence of PIP\u003csub\u003e2\u003c/sub\u003e, that is, to test for binding to active Merlin\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Since PIP\u003csub\u003e2\u003c/sub\u003e signaling is transient and spatially restricted to membranes, interactions that are increased when PIP\u003csub\u003e2\u003c/sub\u003e is present are likely to represent a small but critical proportion of Merlin molecules within cells. Because the assay is quantitative and, although we did not formally measure K\u003csub\u003ed\u003c/sub\u003e, we were able to compare the relative affinity between Merlin and its binding proteins and to reveal a hierarchy of Merlin binding affinities. Anigomotin showed the highest affinity for Merlin. Binding was independent of PIP\u003csub\u003e2\u003c/sub\u003e, suggesting a distinct function for the Merlin-angiomotin complex. The HIPPO pathway proteins Lats1, ASPP2 and YAP1 showed strong Merlin binding, albeit with lower affinity than angiomotin; of these proteins only YAP1 showed significantly increased binding in the presence of PIP\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe small GTPases RalA and RalB had a higher relative affinity for Merlin, roughly 7-fold over the GFP control, which increased dramatically to more than 50-fold over control in the presence of PIP\u003csub\u003e2\u003c/sub\u003e. This level of binding is significantly higher than any other tested proximal proteins and on par with that of angiomotin. RalA and RalB are proximal to both isoform 1 and isoform 2 in confluent cells, with RalB being one of the seven proteins that are proximal to both isoforms exclusively in confluent cells. Endogenous Merlin colocalizes with RalA/B on the ventral surface of cells, in a punctate pattern similar to that of the exocyst component, Exo70, and consistent with a subcellular localization where exocytosis occurs. RalA/B activity is significantly increased in confluent Merlin knockout cells, but not in sub-confluent cells. Increased RalA activity upon Merlin loss was described more than 20 years ago\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, although the mechanism described in that paper, Merlin binding to and inhibiting the Ral activator, RalGDS, is not supported by our data since RalGDS is not among the Merlin proximal proteins we identified. However, we cannot rule this out as a mechanism by which RalA/B is activated in confluent cells upon Merlin loss. Merlin competitively inhibited RalB binding to the exocyst effectors Sec5 and Exo84. Since both RalA and RalB bind to Sec5 and Exo84 it is likely that Merlin competitively inhibits RalA binding to the exocyst as well. Loss of Merlin caused a RalB dependent change in exocytosis kinetics that is phenocopied by active RalB in wild type cells. Taken together these data identify RalA and RalB as a key binding partner for active Merlin and show that Merlin regulates exocytosis in a RalB dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRalA and RalB\u003c/h2\u003e \u003cp\u003eBoth RalA and RalB play critical roles a diverse set of cellular functions including proliferation, cell survival, differentiation motility and cellular polarization\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Mechanistically, many of these biological effects are dependent upon RalA/B regulation of endocytosis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and exocytosis\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Ral regulation of endocytosis is achieved via the Ral effector RalBP which binds to the endocytic machinery via AP-2 to mediate EGF receptor mediated endocytosis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. RalBP was not among the Merlin proximal proteins we identified. Rather, the three exocyst components, Exo70, Sec8 and the Ral effector protein Sec5 were identified as Merlin proximal. These exocyst components plus the other Ral effector, Exo84, are all low affinity Merlin binding proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that the Merlin-Ral interaction may affect exocytosis rather than endocytosis, although the close coordination between endocytosis and exocytosis suggests that we cannot exclude a role for Merlin-Ral interaction in endocytosis as well as exocytosis.\u003c/p\u003e \u003cp\u003eThe exocyst is responsible for tethering the exocytic vesicle to the inner face of the plasma membrane. It is a critical point of regulation via its interactions with vesicular and plasma membrane proteins, phosphoinositides, kinases, and GTPases\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, including cdc42, RhoA\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, RalA and RalB\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The exocyst is a Ral effector\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and active, GTP bound forms of both RalA and RalB can bind to Sec5 and Exo84\u003csup\u003e52\u003c/sup\u003e and mediate the assembly of mature exocyst complexes from 4-protein precursor subcomplexes I and II\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. We note that other proteins involved in exocytic vesicle fusion were identified as Merlin-proximal, including Rab11b and its effector WDR44, the v-SNAREs SNAP-29 and YKT6 and the syntaxin binding proteins STXBP1 and STXBP2, key regulators of the SNARE complex that is essential for exocytic vesicle fusion to the plasma membrane (Table II, Supplemental Spreadsheet 1). Indeed, SNAP-29 is one of the 7 proteins that, like RalB, is proximal to both Merlin isoforms only at confluence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExocytosis in Cancer Biology\u003c/h2\u003e \u003cp\u003eFusion of exocytic vesicles with the plasma membrane is the mechanism by which integral membrane proteins and membrane lipids are incorporated into the plasma membrane and intra-vesicular contents are released into extracellular space\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The RalB-dependent changes in exocytosis kinetics seen upon Merlin loss may reflect distinct subtypes of exocytic events that can be visualized by the VAMP2-pHluorin reporter. Exocytic events with a longer duration are characteristic of \u0026ldquo;full fusion\u0026rdquo; exocytosis in which the vesicle membrane completely integrates with the plasma membrane, along with new membrane components and integral membrane proteins including cell junctional proteins and growth factor receptors\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Short duration exocytic events include \u0026ldquo;kiss-and-run\u0026rdquo; exocytosis, in which the vesicle opens and closes transiently, leading to the release of soluble intra-vesicular contents into the extracellular space, without incorporation of the vesicular membrane and associated integral membrane proteins into the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The shift from shorter to longer duration events upon loss of Merlin is consistent with a shift from the \u0026ldquo;kiss and run\u0026rdquo; to the \u0026ldquo;full fusion\u0026rdquo; exocytic events that require the exocyst. Loss of exocyst function impairs \u0026ldquo;full fusion\u0026rdquo; and increases the proportion of \u0026ldquo;kiss and run\u0026rdquo; events\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Therefore, the changes in exocyst kinetics that we observed may reflect a shift to more \u0026ldquo;full fusion\u0026rdquo; events mediated by the exocyst in response to the increased RalB activity seen in Merlin KO cells at confluence or by the active RalB\u003csup\u003eG23V\u003c/sup\u003e mutant in control cells.\u003c/p\u003e \u003cp\u003eRal-regulation of exocytosis is crucial for cell growth, cell-cell communication, and establishment of cell polarity\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. During development of highly polarized Schwann cells, Ral GTPases promote radial axonal sorting in peripheral nerves through the exocyst complex\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. NF2 mutant schwannoma are epithelioid cells characterized by dysregulated polarity, caused by loss of Merlin function\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. A key mechanism by which cell polarity is established and maintained is asymmetric membrane trafficking controlled by the exocyst\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and Ral activity is implicated in this process\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. We hypothesize that upon contact-inhibition, cells are not actively growing and do not require plasma membrane expansion, full fusion exocytosis is down regulated by active-PIP\u003csub\u003e2\u003c/sub\u003e-bound Merlin dimers that bind to RalA/B and to prevent association with the exocyst and inhibit full fusion exocytosis. In NF2-schwannomatosis the absence of Merlin the failure to inhibit the RalA/B interaction with the exocyst causes increased \u0026ldquo;full fusion\u0026rdquo; exocytosis, leading to changes in the cell surface proteome that affects cell polarity in tumor cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic Implications\u003c/h2\u003e \u003cp\u003eThe identification of RalA and RalB as binding partners for active, PIP\u003csub\u003e2\u003c/sub\u003e-bound Merlin define a mechanism underlying previously described trafficking and cell polarity defects in \u003cem\u003eNF2\u003c/em\u003e null cells. The coordinated nature of exocytosis and endocytosis and the correlation of increased exocytic duration with \u0026ldquo;full fusion\u0026rdquo; exocytosis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e raises the possibility that Merlin loss may cause differential expression of key proteins on the cell surface. The cell surface is the critical interface between tumor cells and the extracellular environment and is the point of interaction with pharmacological agents. Increased trafficking of differentially expressed cell surface proteins may also make these cells vulnerable to endocytic nanoparticle-based drug delivery strategies\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Constitutive RalB activity has been shown to be critical for survival in other human tumor cells\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. This effect is mediated by Sec5-dependent activation of the IkB kinase family member TBK1\u003csup\u003e64\u003c/sup\u003e, raising the possibility that pharmacological inhibition of the Ral itself, or of its downstream effector pathways, may also induce death in schwannoma cells. The observation that loss of Merlin leads to increased RalA/B activity in confluent Schwann cells also identifies them as potential therapeutic target in NF2 schwannoma. As Ral-specific inhibitors for \u003cem\u003ein vivo\u003c/em\u003e use become available, it will be of great interest to test their effects in this tumor type. That Ral small GTPases represent a new signal transduction pathway that may be exploited, either alone or in combination with agents targeting the HIPPO pathway, to treat NF2-related schwannomatosis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eRecombinant DNA\u003c/h2\u003e\n \u003cp\u003eAll recombinant DNA subcloning was performed using NEBuilder\u0026reg; HiFi DNA Assembly Cloning Kit (New England Biolabs, Ipswich, MA, Cat# E5520s). DNA fragments were PCR amplified using Q5 High-Fidelity 2X Master Mix (New England Biolabs, Ipswich, MA, Cat# M0492S) and purified Monarch\u0026reg; DNA Cleanup Columns (New England Biolabs, Ipswich, MA, Cat# T1034L). Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, Iowa). The products of recombinant DNA manipulations were confirmed by sequencing (CCHMC Genomics Sequencing Facility, Cincinnati, OH). The pLNh-APEX vector was derived from plasmids lentiCRISPR, APEX2-GBP and pLenti-CMV-GFP using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Cat# E5520s). The cDNAs for Merlin isoform1 and isoform 2 were cloned in pLNh-APEX in frame N-terminal to APEX2. pLenti-CMVie-XX-mCh was constructed by cloning the mCherry ORF from pcDNA3-FlipGFP(Casp3 cleavage seq) T2A mCherry into pLenti CMVie-IRES-BlastR. RalB was subcloned into the vector pLenti-CMVie-XX-mCh-BlastR in frame, C-terminal to mCherry to generate the lentiviral expression vector pLnCMV-Ch-RalB. The RalB mutants G23V, Q72L and D28N were constructed by PCR mediated site directed mutagenesis using primer containing the appropriate mutant. The vector pLentiCMV-VAMP2-pHluorin-Hygro was constructed by subcloning the VAMP2-pHluorin ORF in place of GFP in pLenti-CMV-GFP. The plasmids expressing GFP fusions for a-Catenin, d-catenin, PPP1R12A, RhoA, RALA and RALB cDNA ORF Clones cDNA ORF Clone, were purchased from Sino Biological US, Inc. (Wayne, PA, Cat#: HG-12388-ACG, HG-16488-ACG, HG-13990-ACG, HG12110-ANG, HG52476-ANG, HG15052-ANG). The plasmids pEGFP-C3-Exo70, pEGFP-C3-Sec8, pEGFP-C3-Exo84 and pEGFP-C3-Sec5 were a gift from Channing Der. (Addgene plasmids # 53761, # 53758, #53762, #53756. pClneoMyc human ERBIN was a gift from Yutaka Hata (Addgene plasmid # 40214. Full-length MLCK-GFP was a gift from Anne Bresnick (Addgene plasmid # 46316. pcDNA3-EGFP-Cdc42(wt) was a gift from Klaus Hahn (Addgene plasmid # 12599). Cadherin-EGFP was a gift from Valeri Vasioukhin (Addgene plasmid # 18870. Integrin b1-GFP was a gift from Martin Humphries (Addgene plasmid # 69804). EGFP-Rab7A was a gift from Qing Zhong (Addgene plasmid # 28047). Human b-catenin GFP was a gift from Alpha Yap (Addgene plasmid # 71367). pEGFP-Akt1(WT) and pEGFP-Akt2 were a gift from Thomas Leonard \u0026amp; Ivan Yudushkin (Addgene plasmids # 86637, #86593. pLVX-EF1a-EGFP-RAB11B-IRES-Puromycin was a gift from David Andrews (Addgene plasmid # 134860). pLenti-PGK-RAP1A-WT-neo was a gift from Roland Friedel (Addgene plasmid # 156173). pCMV-Vamp2-pHluorin was a gift from Brad Zuchero (Addgene plasmid # 190151). The plasmids pEGFP-C3-Exo70, pEGFP-C3-Sec8, pEGFP-C3-Exo84 and pEGFP-C3-Sec5 were a gift from Channing Der. (Addgene plasmids # 53761, # 53758, #53762, #53756). pLenti CMVie-IRES-BlastR was a gift from Ghassan Mouneimne (Addgene plasmid # 119863). pcDNA3-FlipGFP(Casp3 cleavage seq) T2A mCherry was a gift from Xiaokun Shu (Addgene plasmid # 124428. The plasmid lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961. APEX2-GBP was a gift from Rob Parton (Addgene plasmid # 67651). pLenti-CMV-GFP Hygro (656-4) was a gift from Eric Campeau \u0026amp; Paul Kaufman (Addgene plasmid # 17446. IKK1-EYFP was a gift from Johannes A. Schmid (Addgene plasmid # 111206). The plasmids mEmerald-Ezrin-N-14, mEmerald-Moesin-N-14, mEmerald-PTK2-C-10, mEmerald-ILK-N-17 were a gift from Michael Davidson (Addgene plasmid #54090, #54185, #54240, #54127). IKK1-EYFP was a gift from Johannes A. Schmid (Addgene plasmid # 111206). R777-E187 Hs.PPP1CA was a gift from Dominic Esposito (Addgene plasmid # 70471). Lenti-CAG-GFP-hKRAS4B-Zeocin was a gift from Dr. Liang Hu, (Cincinnati, Children\u0026rsquo;s Hospital). The lentiviral packaging plasmids psPAX2 and pMD2.G were a gift from Didier Trono (Addgene plasmids #12260, #12259)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eCell Culture\u003c/h2\u003e\n \u003cp\u003eThe immortalized human Schwann cell line, iHSC1-l, was gift from Dr. M. R. Wallace\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. HEK-293T cells were purchased from ATCC (CRL-2316). Cells were grown in DMEM supplemented with 10% FBS and Pen/Strep at 37\u0026ordm;C 5% CO\u003csub\u003e2\u003c/sub\u003e. 293T cells were transfected with 25000 MW, linear polyethylemimine (PEI, Polysciences, Warrington, PA. (cat# 23966-1). For lentiviral production, HEK-293T cells were co-transfected with the lentiviral expression plasmid and the packaging plasmids psPAX2 and pMD2.G using PEI (1.62 pmol:1.32 pmol:1.32 pmol ratio). After 24 hours, the media was changed and cells were grown in a minimal volume of fresh media for 48 hours. Conditioned media was collected, passed through a 0.22\u0026micro; filter, aliquoted and stored at -80\u0026ordm;C. iHSC-1l cells were infected with a titration of lentiviral conditioned media in the presence of 8 \u0026micro;g/ml polybrene (MilliporeSigma, St. Louis, MO). After 48 hours, the cells were washed 3x with sterile PBS and grown in selection media, with either 2 \u0026micro;g/ml puromycin, 100 \u0026micro;g/ml hygromycin, or 5 \u0026micro;g/ml blasticidin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eImmunofluorescence\u003c/h2\u003e\n \u003cp\u003eCells were plated in 8-well chamber slides (Cat# 80806-90, Ibidi, Fitchburg, WI). Cells were fixed in 4% paraformaldehyde, permeabilized, blocked and probed with primary antibodies, washed in PBS probed with Alexa\u003csup\u003e488\u003c/sup\u003e or Alexa\u003csup\u003e555\u003c/sup\u003e conjugated secondary antibodies (Cat# A32731, A32727, ThermoFisher Waltham, MA) and counterstained with DAPI and Alexa-647 conjugated-phalloidin (Cat# 8940S, Cell Signaling Technology, Danvers, MA). Images were acquired on a Nikon C2plus Confocal microscope using either a 20x, 0.75 NA, Nikon PlanApo l or a 60x 1.4 NA, Apo lS Oil objective. Super-resolution microscopy was performed in the CCHMC Bio-Imaging and Analysis Facility (RRID:SCR_022628). Images were acquired on a Nikon Ti2, AX confocal equipped with an NSPARC ISM spatial array confocal detector in SR mode using a 100x, 1.45 NA, PLAN APO \u0026lambda;D, Oil objective.\u003c/p\u003e\n \u003cp\u003eThe antibodies used were: Merlin (Cat# 6995, Cell Signaling Technology, Davners, MA), RalA/B (Cat# sc-374582, Santa Cruz Biotechnology, Dallas, TX), Active Ral (Cat# 26913, NewEast Biosciences, Glenmoore, PA), RalA (Cat# 21034, NewEast Biosciences, Glenmoore, PA), RalB (Cat# 21033, NewEast Biosciences, Glenmoore, PA), YAP1 (Cat# PA1-46189, ThermoFisher, Waltham, MA), N-Cadherin (Cat# 14215, Cell Signaling Technology, Davners, MA), Exo70 (Cat# MABT186, Millipore Sigma, St Louis, MA), Exo70 (Cat# sc-365825, Santa Cruz Biotechnology, Dallas, TX), APEX (Cat# CAC09177, Biomatik, Kitchener, ON, Canada), Sec5 (Cat# sc-393230, Santa Cruz Biotechnology, Dallas, TX).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eProximity Biotinylation\u003c/h2\u003e\n \u003cp\u003eThree cell lines were generated by infecting iHSC-1l cells with lentivirus expressing the fusion proteins Merlin isoform 1-APEX2, Merlin isoform 2-APEX2 or control APEX2. Hygromycin resistant cell populations were then selected and pooled. APEX activity was measured in lysates using ECL Chemiluminescent HRP substrate (Cat# WBKLS0050, Millipore Sigma, St Louis, MO). Proximity biotinylation was performed in triplicate in 15 cm dishes. For sub-confluent samples cells were plated at 5000 cells per cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, confluent samples were plated at 15,000 cells per cm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Cells were incubated for 48 hours then processed for biotinylation. Proximity biotinylation was performed as described in the literature\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Briefly, media was changed to fresh media supplemented with 500 \u0026micro;M biotin-tyramide (Cat#. SML2135-50MG, MilliporeSigma, St. Lousi, MO) and incubated for 30 min @ 37\u0026ordm;C. To initiate biotinylation, fresh 100 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS was added to 1 mM final concentration, cells were incubated at 37\u0026ordm;C for 1 minute. The reaction was then quenched in final concentration of 5 mM Trolox (Cat# 238813-5g, MilliporeSigma, St Louis, MO), 10 mM sodium ascorbate and 10 mM sodium azide. Cells were then rinsed with ice cold PBS and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (HALT, Pierce) and Benzonase (Sigma-Aldrich). Biotinylated proteins were isolated from cleared lysates by incubating with Streptavidin-Mag agarose beads (Cat# GE28-9857-99, MilliporeSigma, St Louis, MO) overnight at 4\u0026ordm;C with agitation. Beads were captured in a magnetic stand, washed 3x with RIPA, 3x with 2M urea in TBS pH 7.4, 3x with TBS pH 7.4 and 5% aliquots were resuspended in a fluorescent protein staining loading buffer (Instant Bands, Cat# PFS001P, Thomas Scientific, Swedesboro, NJ) run on 4\u0026ndash;20% gradient SDS\u0026ndash;polyacrylamide gel electrophoresis (PAGE) (Bio-Rad). Fluorescent total proteins were visualized in an Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Gels were then blotted and probed with streptavidin-IRDye680 and antibodies to Merlin. The remaining beads were washed 10x with 50 mM ammonium bicarbonate then sent to the Taplin Biological Mass Spectrometry Facility at Harvard Medical School and processed for matrix-assisted laser desorption/ionization mass spectroscopy. Mass spec data consisting of peptide number mapping to specific proteins were normalized to the total number peptides per sample, expressed per 10,000 peptides. Data was displayed as a proximity network using Cytoscape\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Gene ontogeny and functional enrichment was performed within Cytoscape using the String database \u003csup\u003e312\u003c/sup\u003e. Hierarchical cluster analysis was performed using the web application Morpheus (Broad Institute, MIT, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://software.broadinstitute.org/morpheus\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistics\u003c/h2\u003e\n \u003cp\u003eCollated mass spec data was analyzed for statistically significant increases over the APEX only control by multiple unpaired T-test and a 5% false discovery rate using the two-stage step-up method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e with Graphpad Prism 10. For binding assays, unpaired T-test were used to test significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eMerlin Binding Assays\u003c/h2\u003e\n \u003cp\u003eDirect Merlin binding assays were performed, with modifications, as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Assays were performed in triplicate, by incubating a probe consisting of purified Merlin-NanoLuc fusion protein with magnetic beads coated with GFP-fused bait proteins, isolated from transiently transfected 293T cells, in the presence or absence of 200 \u0026micro;M PI(4,5)P\u003csub\u003e2\u003c/sub\u003e diC8 (Cat# P-4058-2, Echelon Biosciences, Salt Lake City, UT).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eMerlin NanoLuc Probe Purification\u003c/h2\u003e\n \u003cp\u003eTwelve 15 cm dishes with 1.3 x 10\u003csup\u003e7\u003c/sup\u003e 293T cells per plate were transfected with plasmid expressing Merlin fused N-terminal to NanoLuc with a dual Streptag affinity tag at its C-terminus (pMerNLst2) using 1 mg/ml PEI, at a 5:1 \u0026micro;l PEI to \u0026micro;g DNA ratio (250 \u0026micro;l PEI to 50 \u0026micro;g plasmid, per plate). Cells were incubated for 48 hours. Cell lysates were harvested by washing with ice cold PBS, then lysing in a high salt lysis buffer (20 mM TrisCl pH 8.0, 500 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5% NP-40 supplemented with 1x HALT protease and phosphatase inhibitor, Cat# 78430, ThermoFisher, Waltham, MA) and 1:5000 Benzonase (Cat# E1014-5KU, Millipore Sigma, St Louis, MA). Lysates were cleared by centrifugation at 21,000 xg for 10 min. at 4\u0026ordm;C. Merlin-NanoLucST2 protein was affinity purified by gravity flow over a 0.2 ml bed volume Strep-TactinXT 4Flow high-capacity resin column (Cat# 2-5030-002, ThermoFisher, Waltham, MA). Columns were washed with 15 column volumes (CV) High Salt Wash Buffer (500 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.1% Triton X-100, 0.1% Tween-20\u0026thinsp;+\u0026thinsp;HALT), then 15 CV Normal Salt Wash Buffer (150 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.1% Triton X-100, 0.1% Tween-20\u0026thinsp;+\u0026thinsp;HALT) followed by 15 CV of TBS (150 mM NaCl, 20 mM Tris-Cl pH 8.0). Merlin-NanoLucST2 was eluted stepwise with 0.3 ml of Elution Buffer (100 mM Tris-Cl pH 8.0, 150 mM NaCl, 50 mM Biotin\u0026thinsp;+\u0026thinsp;HALT) per step for a total of 8 fractions. Purification was monitored by NanoLuc luciferase assays on all fractions (Cat# N1110, Promega, Madison WI), measured using white round bottom 96-well plates in a FlexStation 3 plate reader (Molecular Devices, San Jose, CA). Positive fractions were pooled, passed through a 0.22 \u0026micro; filter then loaded onto a NCG Quest plus FPLC for gel filtration over a 10x300 Enrich SEC 650 size exclusion column (Bio-Rad, Hercules, CA) equilbtated in TBS (20 mM Tris-Cl pH 7.4, 150 mM NaCl). Collected fractions were assayed for NanoLuc activity. High activity fractions corresponding to the closed conformation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e eluting at 13\u0026ndash;14 ml were pooled. Concentration was determined by A\u003csup\u003e280\u003c/sup\u003e in a Nanodrop 2000c (ThermoFisher, Waltham, MA). Purity was determined by SDS-PAGE using Instant Bands loading dye imaged on the Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Preps were adjusted 0.5 mg/ml BSA and 0.1% Tween-20 and stored at 4\u0026ordm;C for no more than two weeks.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eGFP-Bait Protein Purification\u003c/h2\u003e\n \u003cp\u003eGFP-bait expressing plasmids were transfected into 5 x 10\u003csup\u003e6\u003c/sup\u003e HEK 293T cells in a 10 cm dish and incubated for 48 hours. Cells were rinsed with cold PBS, harvested in Lysis Buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 2 mM, MgCl\u003csub\u003e2,\u003c/sub\u003e 0.5 mM EDTA, 0.5% NP-40\u0026thinsp;+\u0026thinsp;1x HALT and 1:5000 Bensonase), cleared by centrifugation at 21,000 xg for 10 min. at 4\u0026ordm;C. Lystates were incubated with 30 \u0026micro;l washed GFP-Trap_MA magnetic agarose beads (Cat# GTMA020, Bulldog Bio Inc. Portsmouth, NH) overnight at 4\u0026ordm;C with agitation. The beads were captured, washed three times with High Salt Wash Buffer (20 mM Tris-Cl pH 7.4, 0.5 M NaCl, 0.1% Triton X-100, 0.1% Tween 20), followed by three washes with Low Salt Wash Buffer (20 mM Tris-Cl pH 7.4, 50 mM NaCl, 10% Glycerol, 0.2% NP-40) then three washes with Normal Salt Wash Buffer (20 mM Tris-Cl pH 7.4,150 mM NaCl, 0.1% Triton X-100, 0.1% Tween 20) and finally resuspended in Blocking Buffer (20 mM Tris-Cl pH, 7.4, 150 mM NaCl, 2.5 mg/ml BSA, 0.05% Tween-20). Final bead fluorescence was measured on a FlexStation 3 plate reader (Molecular Devices, San Jose, CA).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003eDirect Binding Assays\u003c/h2\u003e\n \u003cp\u003eEach preparation GFP-bait-bound was divided into six wells of a white round bottom 96- well plates. The beads were captured on a 96-well magnetic stand (EpiMag HT (96-Well) Magnetic Separator, Cat# Q10002-1, EpiGenTek, Farmingdale, NY), resuspended in a 30-\u0026micro;l Blocking Buffer and incubated for 1 hour at room temperature with agitation. The beads were recovered magnetically then resuspended in 100 nM Merlin-NLuc protein in Blocking Buffer with or without 200 \u0026micro;M PI(4,5)P\u003csub\u003e2\u003c/sub\u003e diC8 and incubated at room temperature for 1 hour with agitation. Beads were then captured magnetically, washed four times with TBST, resuspended in 25 \u0026micro;l TBST and NanoLuc luciferase activity was measured using NanoGlo Luciferase Substrate Buffer (Promega, Madison, WI). NanoLuc activity, GFP fluorescence and BRET spectra were measured on a FlexStation 3 (Molecular Devices, San Jose, CA). For display purposes the plates were also imaged on that Azure C-600 imaging system (Azure Biosystems, Dublin, CA). Data was expressed a % NanoLuc activity bound to the beads relative to the input Merlin-NanoLuc probe, normalized to the GFP-only negative control.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003eRal Activity and Exocytosis Assays\u003c/h2\u003e\n \u003cp\u003eRal activity was assayed in sub-confluent and confluent Control and Merlin knockout iHSC-1l cells using the RalB activity assay (Cat# 81501, NewEast Biosciences, Glenmoore, PA) by immunoprecipitation with antibodies to active Ral and then probes with antibodies specific for RalA and RalB, (Cat # 26913, 21034, 21033, NewEast Biosciences, Glenmoore, PA). For exocytosis assays, Control and Merlin knockout iHSC-1\u0026lambda; cells were infected with lentivirus expressing VAMP2-pHluorin and selected with 100 \u0026micro;g/ml hygromycin. Subsequently, both resulting cell lines were infected with lentivirus expressing mCherry-RalB\u003csup\u003ewt\u003c/sup\u003e, mCherry-RalB\u003csup\u003eG23V\u003c/sup\u003e, mCherry-RalB\u003csup\u003eD28N\u003c/sup\u003e and control mCherry and expressing clones were selected with 5 \u0026micro;g/ml blasticidin. Exocytosis was measured in live VAMP2-pHluorin expressing cells in the CCHMC Bio-Imaging and Analysis Facility (RRID:SCR_022628). Imaging was performed on a TIRF equipped Nikon TiE microscope equipped with a Tokai Hit stage top incubation system and an Andor DU-897 C-6347 EMCCD camera using a Nikon Apo TIRF 100x Oil DIC, 1.49 NA. Time lapse images of live VAMP2-pHluorin expressing cells were acquired using TIRF illumination for 2 min. at 100 msec intervals. Exocytosis was analyzed using the ExoJ plugin\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e on FIJI/Image J software run on the CCHMC High-Performance Computing cluster(RRID:SCR_022622).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowlegements\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Department of Defense, Congressionally Directed Medical Research Program Neurofibromatosis Research Program Awards NF120118, NF160078 and NF190083 and the University of Cincinnati Brain Tumor Center Jejurikar Fellowship Program to RFH. \u0026nbsp;We thank Dr. Shyra Tedesco and Dr. Brad Ozanne for critically reading the manuscript. \u0026nbsp; We would like to acknowledge the support of the CCHMC Bio-Imaging and Analysis Facility (RRID:SCR_022628) and the Information Services for Research (IS4R) (RRID:SCR_022622) shared facilities at Cincinnati Children\u0026rsquo;s Hospital Medical Center.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLloyd, S. 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Exocytotic fusion pore under stress. \u003cem\u003eCell Stress\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 218-226 (2020). https://doi.org/10.15698/cst2020.09.230\u003c/li\u003e\n \u003cli\u003eRizzoli, S. O. \u0026amp; Jahn, R. Kiss-and-run, collapse and \u0026apos;readily retrievable\u0026apos; vesicles. \u003cem\u003eTraffic\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 1137-1144 (2007). https://doi.org/10.1111/j.1600-0854.2007.00614.x\u003c/li\u003e\n \u003cli\u003eAn, S. J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e An active tethering mechanism controls the fate of vesicles. \u003cem\u003eNature communications\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 5434 (2021). https://doi.org/10.1038/s41467-021-25465-y\u003c/li\u003e\n \u003cli\u003eHsu, S. C., TerBush, D., Abraham, M. \u0026amp; Guo, W. The exocyst complex in polarized exocytosis. \u003cem\u003eInternational review of cytology\u003c/em\u003e \u003cstrong\u003e233\u003c/strong\u003e, 243-265 (2004). https://doi.org/10.1016/S0074-7696(04)33006-8\u003c/li\u003e\n \u003cli\u003eOmmer, A.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Ral GTPases in Schwann cells promote radial axonal sorting in the peripheral nervous system. \u003cem\u003eThe Journal of cell biology\u003c/em\u003e \u003cstrong\u003e218\u003c/strong\u003e, 2350-2369 (2019). https://doi.org/10.1083/jcb.201811150\u003c/li\u003e\n \u003cli\u003eGladden, A. B., Hebert, A. M., Schneeberger, E. E. \u0026amp; McClatchey, A. I. The NF2 tumor suppressor, Merlin, regulates epidermal development through the establishment of a junctional polarity complex. \u003cem\u003eDevelopmental cell\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 727-739 (2010). https://doi.org/10.1016/j.devcel.2010.10.008\u003c/li\u003e\n \u003cli\u003eLalli, G. 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Characterization of RalB-Sec5-TBK1 function in human oncogenesis. \u003cem\u003eMethods Enzymol\u003c/em\u003e \u003cstrong\u003e438\u003c/strong\u003e, 321-329 (2008). https://doi.org/10.1016/S0076-6879(07)38022-1\u003c/li\u003e\n \u003cli\u003eShannon, P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Cytoscape: a software environment for integrated models of biomolecular interaction networks. \u003cem\u003eGenome Res\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2498-2504 (2003). https://doi.org/10.1101/gr.1239303\u003c/li\u003e\n \u003cli\u003eBenjamini, Y., Krieger, A. M. \u0026amp; Yekutieli, D. Adaptive linear step-up procedures that control the false. \u003cem\u003eBiometrika\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 491-507 (2006).\u003c/li\u003e\n \u003cli\u003eLiu, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e \u003cem\u003eExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection and analysis of exocytosis\u003c/em\u003e (Cold Spring Harbor Laboratory, 2022).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables I and II are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6890466/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6890466/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLoss of \u003cem\u003eNF2\u003c/em\u003e tumor suppressor activity causes NF2-related schwannomatosis. Proximity biotinylation identified proteins proximal to Merlin isoform 1 and isoform 2 at confluence, when Merlin is active, but not in sub-confluent, growing cells. These data confirmed Merlin involvement in cell-cell and cell-substrate junctions, identified new signal transduction pathways, and highlighted a role for Merlin in intracellular transport. Direct binding assays identified the small GTPases RalA and RalB as high affinity PIP\u003csub\u003e2\u003c/sub\u003e-dependent Merlin binding proteins that co-localized with RalA/B on the plasma membrane. Merlin loss resulted in aberrant activation of RalA and RalB at high cell density. Merlin competitively inhibited RalB binding to its exocyst effectors Sec5 and Exo84 and regulated the kinetics of exocytosis in a RalB dependent manner. Thus, RalB is a novel binding partner for active Merlin, and the RalA/B pathway is a possible therapeutic target to treat NF2-related schwannoma.\u003c/p\u003e","manuscriptTitle":"Active Merlin Binds RalB to Regulate Exocytosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 06:50:44","doi":"10.21203/rs.3.rs-6890466/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1b6fff17-30f4-42a5-a4f9-20ee25a84920","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51359336,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":51359337,"name":"Biological sciences/Cell biology/Membrane trafficking/Exocytosis"},{"id":51359338,"name":"Biological sciences/Cancer/Tumour-suppressor proteins"}],"tags":[],"updatedAt":"2025-08-01T14:35:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 06:50:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6890466","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6890466","identity":"rs-6890466","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}