Microtubule-mediated transport of the tumor- suppressor and its mutants

Lorena B. Benseñora, Kari Barlana, Sarah E. Ricea, Richard G. Fehonb, and Vladimir I. Gelfanda,1

aDepartment of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; and bDepartment of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637

Edited by William Saxton, University of California, Santa Cruz, CA, and accepted by the Editorial Board March 11, 2010 (received for review July 3, 2009) The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin Several studies have demonstrated that regu- is a member of the ERM family of that links the lates Merlin function and affects its intracellular localization (18– to the plasma membrane. In , in the NF2 genecause 20). In Drosophila, of a single threonine residue (T616) neurofibromatosis type-2 (NF2), a cancer syndrome characterized by changes its distribution and association with various ligands, such as the development of tumors of the nervous system. Previous reports , another member of the ERM family (20). Both in vivo and have suggested that the subcellular distribution of Merlin is critical to in cultured cells, Merlin together with Expanded, have been shown its function, and that several NF2 mutants that lack tumor-suppressor to regulate cell growth and differentiation by promoting phos- activity present improper localization. Here we used a Drosophila cell phorylation and cytoplasmic retention of the transcriptional coac- culture modelto study the distribution andmechanism ofintracellular tivator Yorkie/YAP1 (12, 21–23). Moreover, recent studies suggest transport of Merlin and its mutants. We found that Drosophila Merlin that through localization at the cortical network, Merlin con- formed cytoplasmic particles that move bidirectionally along micro- trols the internalization and signaling of certain membrane recep- tubules. A single NF2-causing substitution in the FERM tors, including the epidermal (6, 9, 24–27). These domain dramatically inhibited Merlin particle movement. Surpris- results highlight the importance of proper Merlin targeting to this ingly, the presence of this immotile Merlin mutant also inhibited traf- region. Here we explore the mechanism of Merlin intracellular ficking of the WT protein. Analysis of the movement of WT protein trafficking. Our work shows that a DrosophilaMerlin homolog forms using RNAi and pull-downs showed that Merlin particles are associ- particles that move along microtubules through a coordinated ated with and moved by microtubule motors (kinesin-1 and cytoplas- action of two microtubule motors, kinesin-1 and cytoplasmic dynein. CELL BIOLOGY mic dynein), and that binding of motors and movement is regulated This movement is phosphorylation-dependent and is inhibited by a by Merlin phosphorylation. Inhibition of Merlin transport by expres- tumor-causing mutation in the FERM domain of the protein. sion of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coac- Results tivator Yorkie. These results demonstrate the requirement of micro- Subcellular Distribution of Merlin-GFP Particles in S2 Cells. Previous tubule-dependent transport for Merlin function. studies in Drosophila embryos have demonstrated that Merlin is localized at the apical plasma membrane and in cytoplasmic neurofibromatosis | intracellular transport | dynein | kinesin | microtubules puncta (12, 13). To study the mechanisms of Merlin intracellular transport, we used Drosophila S2 cells, which have a consistent erlin is a tumor-suppressor protein of the ERM family morphology and spread well on substrates coated with Con- Mencoded by the NF2 that controls cell growth and canavalin A (Con A). We first analyzed the localization of contact-dependent inhibition of proliferation. Mutations in the endogenous Merlin by immunofluorescence using an antibody NF2 gene are the underlying cause of neurofibromatosis type 2 against Drosophila Merlin. Our results showed that endogenous (NF2), a familial cancer syndrome characterized by the develop- Merlin forms clusters that are distributed underneath the plasma ment of sporadic tumors of the nervous system (1–3). To gain membrane as well as in the perinuclear region (Fig.1A). To char- insight into Merlin’s cellular functions, we have studied a Droso- acterize Merlin clusters and their dynamic behavior, we generated an S2 cell line stably expressing a Merlin-GFP fusion protein phila Merlin homolog and its disease-causing mutant. Drosophila GFP Merlin shares the same fundamental domain composition as its (Mer ) under the control of a heat-shock promoter. The GFP mammalian counterpart, and several groups have obtained com- expression level and inducible nature of Mer in the stable cell fi parable results using the mammalian and Drosophila proteins (4). line were con rmed by immunoblotting with both anti-GFP and anti-Merlin antibodies (Fig. S1). Similar to untagged Merlin, Merlin functions by organizing membrane domains that connect GFP signals coming from the extracellular environment to cytoplasmic Mer forms particles in the cytoplasm and near the plasma factors to ultimately regulate cellular proliferation (5–7). Con- membrane (Fig. 1B and Movie S1). To examine whether Merlin sistent with this role, Merlin is concentrated in actin-rich struc- particles are associated with an endocytic compartment, we incu- fl – bated S2 cells plated in Con A with rhodamine-dextran. Merlin tures, such as membrane ruf es in isolated cells and at cell cell – contacts in dense cultures (8–10). Merlin also exhibits a punctate particles did not colocalize with the rhodamine-dextran contain- ing endosomes after a 4-h postincubation (Fig.1 E–G and Movie distribution that has been attributed to localization to intracellular GFP vesicles (7, 11, 12). Merlin has a dynamic distribution in Drosophila S1). Similarly, Mer particles did not colocalize with the endo- somal marker Rab5 (Fig. S2). S2 cells. Studies have shown that the protein is initially localized along the cell cortical region and is then internalized in the form of particles (12, 13). Author contributions: L.B., S.R., R.F., and V.G. designed research; L.B. performed research; Merlin mislocalization is associated with abnormal tissue K.B. contributed new reagents/analytic tools; L.B., R.F., and V.G. analyzed data; and L.B., growth and proliferation (12, 13). Many NF2 mutations of Merlin S.R., and V.G. wrote the paper. are abnormally distributed in the cells and lack tumor-suppressor The authors declare no conflict of interest. activity (14, 15). Moreover, when overexpressed, these mutant This article is a PNAS Direct Submission. W.S. is a guest editor invited by the versions of Merlin exhibit oncogenic properties, act in a dominant- Editorial Board. negative manner, and interfere with the activity of the WT protein 1To whom correspondence should be addressed. E-mail: [email protected]. (13, 16, 17). How the NF2 mutant protein alters the functional This article contains supporting information online at www.pnas.org/cgi/content/full/ properties of WT Merlin remains unclear, however. 0907389107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0907389107 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 2. Movement of MerGFP particles along processes of S2 cells. (A and B) MerGFP particles move bidirectionally in the processes of Cyto-D–treated S2 cells (Movie S2). Frames in B correspond to the boxed area in A.Time0 Fig. 1. Distribution of Merlin and endocytic vesicles in Drosophila S2 cells. indicates time-lapse start point (1 h after heat shock), and particle plus and – (A D) Drosophila S2 cells plated in Con A without or with Cyto-D treatment; minus end movement is shown in the first and second columns respectively wt GFP and expressing either endogenous Mer (A and C) or Mer under a heat- (white arrowheads). (C) Histogram of velocities for MerGFP particles shows fi shock promoter (B and D). (A and B) Spread S2 cells were xed and stained unbiased bidirectional movement. with anti-Merlin antibody. Merlin particles are found in the proximity of (Inset) and in the perinuclear region (arrow). The distribution is wt GFP GFP identical for both Mer (A) and Mer (B). (C and D) Cyto-D Mer S2 cells (K70E)GFP formed particles within the perinuclear region and pro- form processes that are filled with particles. This distribution is identical for both Merwt (C) and MerGFP (D). (E–G) MerGFP cells incubated with rhodamine- cesses of S2 cells (Fig. 3 A and C); however, analysis of time-lapse GFP dextran and analyzed by fluorescence microscopy. (E) DIC image of S2 cells movies of Mer(K70E) revealed a dramatic difference between plated in Con A. (F) Distribution of MerGFP particles. (G) Rhodamine-dextran– the behavior of the WT and mutant protein (Fig. 3 A′–C′). Unlike labeled endosomes. See Movie S1. normal Merlin particles, the clusters formed by Mer(K70E)GFP protein were completely stationary (Fig. 3C′ and Movie S3). We then tested whether overexpression of WT Merlin could res- MerGFP Moves Bidirectionally Along Microtubules. S2 cells can be induced to form long, thin processes when treated with cytocha- cue bidirectional transport of the disease-causing mutant. For this lasin-D (Cyto-D) and plated on Con A (Fig.1 C and D) (28). Cyto- D–induced processes contain parallel arrays of microtubules of uniform polarity with their plus-end directed toward the tips and are an ideal model for assessing microtubule-based transport. Both endogenous Merlin and MerGFP particles were found in the processes and the perinuclear region of Cyto-D–treated S2 cells (Fig.1 C and D). MerGFP particles move bidirectionally in these pro- cesses (Fig. 2 A and B and Movie S2). Approximately 20% of par- ticles undergo fast and repeated bidirectional movements charac- teristic of microtubule motor–driven transport (Fig. 2C). MerGFP particles exhibited an unbiased bidirectional motion and displayed similar distributions of velocities for plus-end and minus-end movement, with an average plus-end velocity of 0.50 ± 0.22 μm/s and an average minus-end velocity of 0.49 ± 0.21 μm/s (Fig. 2C). Together, these results indicate that MerGFP particles engaged in bidirectional transport are likely to be moved by the microtubule- based transport system.

Disease-Causing Mutants of Merlin Are Defective in Transport. Sev- eral groups have used disease-causing mutants of the NF2/Merlin tumor suppressor to elucidate the molecular mechanisms of Merlin function. Certain point mutations in the NF2 gene are known to produce a full-length, stable protein that is functionally inactive. Fig. 3. An NF2 mutant of Merlin, Mer(K70E)GFP, is defective in intracellular Consequently, we examined the intracellular transport of a disease- transport. (A and B) Cyto-D–treated S2 cells stably expressing MerGFP (A)or causing mutant of Merlin known to lack tumor-suppressor activity MermCherry (B)(Movie S4). (A′ and B′) Kymographs of the boxed areas and found that it is distributed abnormally compared with WT showing particles moving bidirectionally along processes. (C) Expression of protein. The Merlin mutant K70E contains a replacementof a single the NF2-mutant Mer(K70E)GFP in S2 cells. (C′) Kymographs of the boxed areas showing absence of movement (Movie S3). (D–F) Coexpression of MermCherry conserved Lys to Glu at position 70 within the FERM domain, which GFP ′ is equivalent to residue K79 in NF2 (Fig. S3) (14). To study (D) and Mer(K70E) (E) in S2 cells. (F and F ) Colocalization of the mutant and the WT protein. (D′ and E′) Kymographs of the boxed areas. (G) Pull- the intracellular transport of this mutant in Drosophila S2 cells, we down using GBP. Input, crude cell extract from WT cells (untransfected) and generated a stable cell line expressing a GFP-tagged mutant, Mer cells expressing MerGFP or Mer(K70E)GFP after heat shock. GBP, pull-down GFP (K70E) , under a heat-shock promoter and observed its dis- from WT extracts (untransfected) or extracts from S2 cells expressing MerGFP tribution and behavior in live cells. Similar to the WT protein, Mer or Mer(K70E)GFP. Blots were probed with antibodies against Merlin.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0907389107 Benseñor et al. Downloaded by guest on September 30, 2021 purpose, we coexpressed mCherry-tagged Merlin (MermCherry) under Merlin Transport Machinery Includes Kinesin-1 and Cytoplasmic Dynein. a constitutively active promoter in Mer(K70E)GFP cells. Before heat To gain insight into the mechanism of Merlin transport along mi- shock, Mer(K70E)GFP was not expressed, and MermCherry formed crotubules, we used a combined approach including RNA interfer- particles that move bidirectionally along processes, as was seen in its ence (RNAi), fluorescence microscopy, and biochemical analysis. GFP counterpart (Fig. 3 B and B′ and Movie S4). After induction of Merlin has been shown to coimmunoprecipitate with kinesin in cul- NF2 mutant expression by heat shock, MermCherry Mer(K70E)GFP tured HeLa cells (31), which suggests its association with microtubule colocalized with Mer(K70E)GFP (Fig. 3 D–F). Remarkably, the motors. Direct functional evidence of the involvement of kinesin-1 or particles containing both MermCherry and Mer(K70E)GFP were dynein in Merlin transport is lacking, however. Therefore, to identify completely immotile (Fig. 3 D′ and E′ and Movie S5). This result components of the microtubule transport machinery for Merlin could be explained if Mer(K70E)GFP associated in a heterocomplex particles, MerGFP cells were treated with dsRNA for various motors, with WT Merlin. We performed a pull-down of Mer(K70E)GFP using and the specificity and efficiency of RNAi knockdown was examined immobilized GFP binder protein (GBP) (29). Blotting with anti- by immunoblot analysis (Fig. S4). When the heavy chain of kinesin-1 Merlin antibody revealed that endogenous Merlin forms a complex (KHC) was knocked down, the motility of MerGFP particles was with Mer(K70E)GFP (Fig. 3G). Note that anti-Merlin antibody rec- dramatically reduced (Fig. 5 A and B). Tracking and kymographic ognizes both slow-migrating and fast-migrating bands of endogenous analysis of particle movement clearly showed that KHC-RNAi Merlin (hyperphosphorylated and hypophosphorylated forms, treatment reduced the number of movements by >10-fold (Fig. 5 A respectively). Interestingly, K70E, the disease-causing mutant of and B). As a control for motor specificity, we treated S2 cells with Merlin, was bound preferentially to the hyperphosphorylated form of RNAi against another member of the kinesin superfamily, Klp68D (a the endogeneous protein (Fig. 3G, last lane). subunit of Drosophila kinesin-2). As shown in Fig. 5A,depletionof We then tested whether expression of the NF2 mutant caused kinesin-2 had no effect on MerGFP movement. GFP a global inhibition of transport along microtubules, including The bidirectional nature of Mer transport implies that a minus- cargoes unrelated to Merlin. We selected mitochondria move- end–directed microtubule motor is also involved in the observed ment for this analysis because mitochondria, like Merlin particles retrograde movement. Indeed, depletion of dynein heavy chain (see below), are moved by conventional kinesin (30). Time-lapse (DHC) inhibited the bidirectional movement of particles by >4-fold, movies of mitochondria labeled with MitoTracker Red show that as demonstrated by both the number of moving particles (Fig. 5A) expression of Mer(K70E)GFP did not affect movement of mito- and kymographic analysis of time-lapse movies (Fig. 5B). Similar to chondria (Movie S6). This finding demonstrates that the inability KHC knockdown, depletion of DHC inhibited both plus-end and of Mer(K70E)GFP to move in the cell does not result from global minus-end transport. This bidirectional transport inhibition cannot CELL BIOLOGY suppression of motor-driven motility, but is likely explained by be explained by the simultaneous knockdown of DHC and KHC, the intrinsic inability of mutant Merlin to interact with micro- because the expression of one of these motors was not affected when tubule transport system. the other motor was knocked down (Fig. S4). Based on these results, we conclude that both kinesin-1 and cytoplasmic dynein are required Expression of the NF2 Mutant Mer(K70E)GFP Relocalizes Yorkie to the for transport of the Merlin particles along microtubules. Nucleus. Merlin regulates cell growth by signaling through the Hippo pathway to inhibit the function of the transcriptional coactivator Yorkie (Yki) (22). We used Yki translocation into the nucleus of S2 cells as readout to test whether Merlin’s function requires a motile protein (Fig. 4A). In control cells, expression of YkimCherry alone or in coexpression with MerGFP resulted in ∼10% of mCherry fluorescence in the nucleus. Knock-down of Merlin using specific RNAi probes or expression of immotile Mer (K70E)GFP resulted in an almost 3-fold increase in nuclear fluo- rescence (Fig. 4 A and B and Fig. S4). We hypothesized that immobilization of Merlin by Mer(K70E)GFP is the cause of Yki redistribution, and thus directly tested the physiological relevance of Merlin transport in another series of experiments.

Fig. 5. Bidirectional movement of Merlin clusters depends on kinesin-1, dynein motors, and adaptor proteins KLC and dynactin. (A) Frequencies of plus-end (white bars) and minus-end (black bars) movements of MerGFP particles. Bars represent the percentage of vectors of length >0.35 μm. Fig. 4. Expression of Merlin mutant affects Yorkie distribution. (A) From Depletion of either KHC or DHC was sufficient to inhibit bidirectional left to right, expression of YkimCherry alone, coexpression with MerGFP, Merlin movement of Merlin particles. (B) Kymographs of MerGFP particles moving in RNAi, coexpression with Mer(K70E)GFP, and KHC RNAi. (B) Quantification of the processes. RNAi targets are listed above the corresponding panels. Error Yorkie fluorescence in the nucleus. Error bar indicates SD (ANOVA). P = 0.01. bar indicates SD (ANOVA). P = 0.005.

Benseñor et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 We then tested whether kinesin light chain (KLC) and dynactin, tubule motors (Fig. 6B). These data indicate that Merlin clusters are known adapters for kinesin-1 and cytoplasmic dynein, respectively associated with kinesin-1 and dynein molecular motors, and these (32, 33) are required for motor-driven transport of Merlin particles. motors are responsible for movement of the tumor-suppressor For this test, we depleted S2 cells of KLC or p150 glued, the key subunit protein along microtubules. of the dynactin complex, using specific RNAi probes, and analyzed MerGFP particle movement. The anti-KLC and anti-p150 antibodies Phosphorylation Regulates Association of Merlin With Molecular Mo- used to examine the depletion efficiency decreased dramatically tors. Previous studies demonstrated that in Drosophila the sub- after RNAi treatment (Fig. S4). Knockdown of KLC and p150 glued cellular localization of Merlin is regulated by phosphorylation. A inhibited the movement of MerGFP particles (Fig. 5A and B). In single mutation in a threonine residue—a putative phosphorylation conclusion, our functional data indicate that Merlin intracellular site, Thr616—was shown to be responsible for changes in the pro- transport is driven by kinesin-1 and cytoplasmic dynein, and that tein distribution presumably affecting intramolecular interactions these motors use KLC and dynactin, respectively, as their adapters. between FERM and C-terminal domains (20). Thus, we tested To test the functional significance of the microtubule-dependent whether phosphorylation of Thr616 serves as a regulatory mecha- transport of Merlin, we tested the effect of kinesin-1 depletion on nism for Merlin transport. We used stable S2 cell lines expressing a mCherry the distribution of Yki between the nucleus and the cyto- nonphosphorylatable mutant of Merlin, Mer(T616A)GFP,ora plasm. Remarkably, inhibition of kinesin-driven transport of Merlin phosphomimetic mutant, Mer(T616D)GFP, under a heat-shock particles caused a 3-fold increase in the nuclear accumulation of promoter (Fig. S3). These mutants have been previously shown to Yorkie. This result is similar to the effect of either depletion of behave like the dephosphorylated and phosphorylated forms of endogenous Merlin or WT Merlin immobilization by expressing the Merlin, respectively (20). To analyze the distribution of MerGFP and Mer(K70E) mutant (Fig. 4 A and B and Fig. S4). mutant Merlin particles in 3D, we used confocal microscopy and built Z-stacks (Fig. S5 A–F). In control MerGFP cells, at 4 h post- Merlin Binds Kinesin-1 and Cytoplasmic Dynein. When endogenous GFP induction, particles were found at the cell cortex, in processes, and in Merlin or Mer were pulled-down with either anti-Merlin anti- ′′ bodies or GBP, respectively, both KHC and DHC were detected by the perinuclear region of S2 cells (Fig. S5 A, A ,andD). Expression of Mer(T616A)GFP results in the formation of rather large particles, immunoblotting (Fig. 6 A and B). As a control for endogenous GFP Merlin immunoprecipitation, we substituted the Merlin antibody but unlike Mer , Z-stacks show that these particles were prefer- with preimmune IgG (Fig. 6A), and to test GBP specificity, we used entially accumulated in the perinuclear region (Fig. 7D and Fig. S5 ′′ GFP extracts from untransfected S2 cells that do not express MerGFP B, B , E,andG). Conversely, a Mer(T616D) phosphomimetic (Fig. 6B). Neither KHC nor DHC could be detected in any of these mutant formed smaller particles primarily at the cell cortex (Fig. S5 ′′ controls, demonstrating that the interactions of both endogenous C, C , F,andG). The dramatic effect of a single amino acid sub- Merlin and MerGFP with KHC and DHC are specific. Thus, the stitution at Thr616 on the subcellular localization of Merlin suggests biochemical data support the functional evidence that Merlin indeed forms a complex with KHC and DHC. The foregoing results led us to hypothesize that the observed lack of transport of Mer(K70E)GFP could be due to a defect in the association with a motor complex. Therefore, we examined whether the Mer(K70E)GFP mutant was able to associate with KHC and DHC. Pull-down of cell extracts from Mer(K70E)GFP using GBP shows that the disease-causing protein did not interact with micro-

Fig. 7. Thr-616 phosphorylation regulates Merlin bidirectional transport and motor association. (A and A′) S2 cells expressing MerGFP particles move bidirectionally in processes (Inset) as shown by kymograph analysis (A′). (B and B′) Expression of Mer(T616A)GFP, a nonphosphorylatable mutant, pro- duces particles that move bidirectionally (Inset and B′), but mostly accumu- late at the perinuclear region (Movie S7). (C and C′) Expression of Mer (T616D)GFP, a phosphomimetic mutant, generates small particles that lack bidirectional movement (C′ and Movie S8) and are located at the cortex (Inset). (D) Comparison of particle distributions in MerGFP and its phospho- Fig. 6. Merlin forms a complex with kinesin-1 and dynein motors. (A) mutants. The bar graph displays the percentage of particles found in the Immunoprecipitation of endogenous Merlin from WT S2 cell extracts with processes. (E) Immunoprecipitation of cell extracts with anti-GFP antibody. anti-Merlin antibody. Input, crude cell extract; IgG, control IgG preimmune Input, crude cell extract; MerGFP, IP from extracts of cells expressing Merlin- serum; anti-Mer, anti-Merlin antibody. (B) Pull-down using GBP. Input, crude GFP after heat shock; induction, IP from extracts from MerGFP cells before cell extract from WT cells (untransfected) and cells expressing MerGFP or Mer heat shock (control); T616DGFP, IP from extracts from cells expressing Mer (K70E)GFP after heat shock; GBP, pull-down from WT extracts (untransfected) (T616D)GFP; T616AGFP, IP from extracts from cells expressing Mer(T616A)GFP. or extracts from S2 cells expressing MerGFP or Mer(K70E)GFP. Blots were Blots were done with anti-dynein and anti–kinesin-1 antibodies. The bottom probed with antibodies against kinesin (KHC) or dynein (DHC). part of the gel was stained with Coomassie blue for loading control (load).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0907389107 Benseñor et al. Downloaded by guest on September 30, 2021 that phosphorylation of Thr616 plays an important regulatory role defective in transport act as dominant negative forms of Merlin and in protein transport. could lead to loss offunction oftheWTprotein (38).Inaddition, our To further address the effect of phosphorylation on Merlin results correlate with the fact that the K70E mutation occurs in particle movement and subcellular distribution, we analyzed time- 3 within the FERM domain known to generate an inactive mutant lapse images of Merlin particles for each individual mutant (Fig. 7 protein with dominant-negative properties (15, 39). A–C). In agreement with the confocal microscopy data, Mer Phosphorylation modulates Merlin subcellular localization and (T616A)GFP particles were rarely found in processes and mostly growth suppression, as well as Merlin function. Our data show that accumulated in the perinuclear region (Fig. 7 B and D); however, a phosphorylation of Merlin in Thr616 eliminates its association with few particles found in processes showed fast bidirectional move- the motor complex and leaves Merlin unable to move from the cell ments (Fig. 7B′ and Movie S7). These movements were com- membrane. Conversely, Merlin T616A forms particles that associate parable to the movements of particles in control MerGFP cells with microtubule motors; however, these particles accumulate pri- characterized by kymographic analysis (Fig.7 A, A′, B, and B′). In marily at the perinuclear region. These data correlate with previously contrast, Mer(T616D)GFP phosphomutant formed smaller and reported findings in Drosophila showing that T616D phosphomi- immotile particles distributed beneath the cell boundaries shown metic mutant remains membrane-associated and is internalized at a in Fig. 7C (Inset) and Fig. S5 C, C′′, F, and G. These observations much slower rate, whereas the nonphosphorylatable T616A is rap- suggest that phosphorylation of Merlin in Thr616 might affect idly internalized (20). Phosphorylation has been shown to regulate Merlin’s ability to assemble into a motile complex (Fig. 7C′ and intramolecular head-to-tail interactions of Merlin. It is possible then Movie S8). Consistent with these findings, immunoprecipitation that either phosphorylation of Merlin or NF2 mutations that disrupt studies showed that Mer(T616A)GFP interacted with both kinesin-1 the folding of Merlin not only affect protein retention at a particular and dynein, whereas Mer(T616D)GFP did not (Fig. 7E). Taken location within the cell, but also disrupt Merlin’s ability to reach its together, our functional and biochemical data suggest that the location by interfering with its microtubule-dependent transport. association of Merlin with a microtubule transport system is Human Merlin is also regulated by phosphorylation of both dependent on the phosphorylation state of Thr616, and that and threonine residues at the C terminus of the protein. In particular, phosphorylation of this residue is critical to regulation of targeting the phosphorylation state of the regulatory serine residue (Ser518) in and distribution of Merlin to specific cell regions. the human protein is known to affect intracellular distribution of the protein and its association with other proteins (14, 18, 19). This serine Discussion residue is not conserved in Drosophila, although phosphorylation of Merlin is a tumor-suppressor protein found both at the plasma Thr616, which is conserved in ERM proteins and has well-described CELL BIOLOGY membrane and in clusters in the cell. The subcellular distribution regulatory functions, retains the same regulatory characteristics. of the Merlin protein is critical for its tumor-suppression activity. Based on our data, it is possible that the corresponding Thr616 res- Merlin has been shown to inhibit mitogenic signaling by binding to idue in Drosophila Merlin acts as the regulatory phosphorylation site the E3 CRL4DCAF1 in the nucleus (34). How the controlling the intracellular transport of Merlin (Fig. S6A). We pre- protein translocates between different cellular compartments and dict that, similar to Thr616 in Drosophila, phosphorylation of Ser518 reaches the nucleus remains unknown, however. Here we provide inhumanMerlinregulatesitsassociationwithmicrotubulemotors. evidence that Merlin forms clusters that move along microtubules, Collectively, our findings reveal the mechanism of intracellular and that microtubule-dependent transport of Merlin is an essential transport of Merlin particles by molecular motors, and show that part of the regulatory mechanism of the tumor suppressor. Merlin trafficking is essential for its function. Our results call Our data demonstrate that Merlin is bidirectionally transported into question whether Merlin’s intracellular transport also reg- in the cytoplasm along microtubules guided by the microtubule ulates its function as a growth regulator. We speculate that this motors kinesin-1 and cytoplasmic dynein. Movement of Merlin- indeed is the case, based on our findings on the translocation of containing particles also requires the cofactors KLC and dynactin the transcriptional coactivator Yorkie. (Fig. S6A). These results agree with previous studies showing that Finally, point mutations of critical NF2 residues occur naturally Merlin associates with a 400-KDa complex containing KHC (31). In in patients. Therefore, we believe that our results form the foun- addition, we show that Merlin transport affects its function. We dation for further experiments aimed at defining how mutations of found a marked increase in nuclear accumulation of the transcrip- Merlin affect Merlin’s association with other proteins that are tional coactivator Yorkie, a downstream effector of Merlin, under important for its transport and intracellular signaling. conditions in which Merlin bidirectional transport is inhibited by KHC knockdown or coexpression with an NF2 mutant (Fig. 4). Materials and Methods Our results showing that K70E colocalizes with WT Merlin in Cell Imaging, Particle Tracking, and Image Analysis. Images were captured every immotile particles agree with previous studies demonstrating that 1 s for a period of 1 min. Particle movement was analyzed with Diatrack version the subcellular distribution of the NF2-mutants of Merlin with 3.01 (Semasopht) using vector analysis. Vectors are defined as displacement FERM domain abnormalities was primarily perinuclear, without between two consecutive frames. A threshold of 0.25 μm was used, and vectors accumulation at the membrane (35, 36). These results clearly with a length below this threshold were excluded from the velocity calculations. indicate a defect in protein trafficking with the potential to affect The number of vectors was normalized by the total number of particles. At least 10 independent experiments were performed for each condition, and 5–10 cells membrane-cytoskeleton signaling and block growth-inhibiting from each experiment were chosen randomly for analysis. Kymographic analy- responses, leading to tumor development. Our observations on the ses of time-lapse movies were obtained using ImageJ software (National Insti- effect of coexpression of the NF2 mutant protein with WT Merlin tutes of Health). To quantify particles in the cell body and processes (Fig. 7D), resulted in an ideal model to study the physiological effect of fluorescent images were subjected to processing with the ImageJ Auto-Local Merlin intracellular transport. The nuclear accumulation of Threshold plug-in (Bernsen algorithm), and the distribution of threshold par- Yorkie is consistent with a loss of function in these experiments. ticles in the cell body and processes (outlined using phase-contrast images) was Yorkie is the substrate of the regulated quantified. The same filter was used to analyze the particle size distributions. by Merlin and Expanded (12, 21–23). Therefore, restriction of Detailed information on the equipment, reagents and conditions, endo- motor-driven transport of an otherwise normal WT Merlin protein some and mitochondria labeling, RNAi, transfection, pull-down assays, fl by an NF2 mutant protein results in the loss of function. immuno uorescence, DNA constructs, and antibodies used in this study is provided in SI Materials and Methods. A possible mechanism that can explain the targeting of WT Merlin to particles carrying the mutant Merlin protein is the het- ACKNOWLEDGMENTS. L.B. is the recipient of an American Heart Association erodimerization of WT Merlin with Merlin mutant proteins (Fig. Postdoctoral Fellowship (Midwest Affiliate). This work was funded by the S6B) (14, 37). Our findings indicate that NF2 mutants of Merlin National Institutes of Health (Grant GM52111 to V.G.).

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