| Human Molecular Genetics | Pages |
Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesion and spreading
Introduction
Overexpression of merlin-17 suppresses cell motility in vitro
Merlin mutants and merlin-16 fail to influence cell motility
Merlin does not influence catenin or ERM protein expression
Induction of merlin results in a transient decrease in cell attachment
Overexpression of merlin results in transient abnormalities in cell spreading
Discussion
Materials And Methods
Antibodies and western blotting
Merlin cDNA constructs
Generation of merlin schwannoma cell lines
Thymidine incorporation
Immunocytochemistry
In vitro migration assay using a membrane invasion culture system (MICS)
Cell attachment
Cell spreading
Acknowledgements
References
Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesion and spreading
INTRODUCTION
Neurofibromatosis 2 (NF2) is an inherited autosomal dominant disorder in which affected individuals typically develop bilateral vestibular nerve schwannomas (1). Meningiomas, ependymomas and schwannomas arising from nerves other than the eighth cranial nerve also occur in NF2 patients (2). Positional cloning resulted in the identification of the NF2 tumor suppressor gene on chromosome 22q in 1993 (3,4). The NF2 protein product, merlin, is a 66-69 kDa protein containing 16 exons and 595 amino acids (5). Sequence alignment has demonstrated that merlin is homologous to the band 4.1 family of proteins linking the actin cytoskeleton to cell surface glycoproteins. Within this family, ezrin, radixin and moesin (ERM proteins) share the highest homology with merlin. ERM proteins have been implicated in cellular remodeling and the formation of membrane microvilli and ruffles (6).
Despite the high degree of homology between merlin and the ERM family, it is clear that merlin has additional properties not ascribed to ERM proteins. For example, merlin can suppress the growth of cells in vitro and in vivo whereas radixin fails to have this effect. Specifically, overexpression of wild-type merlin in NIH-3T3 fibroblasts as well as in rat schwannoma cells results in reduced cell proliferation (7,8). In addition, wild-type merlin-expressing schwannoma cell lines demonstrate reduced growth compared with controls when grown as explants in athymic mice (8). This growth-suppressor function of merlin is consistent with its role as a tumor suppressor gene product.
A property that may be shared between ERM proteins and merlin is the close association with the actin cytoskeleton. ERM proteins bind actin and influence cytoskeletal organization. We recently demonstrated that merlin can associate with polymerized actin in vitro by virtue of a unique actin-binding domain in the N-terminus of the protein (9). Immunocytochemical subcellular localization studies have shown that merlin is expressed at the leading edges of both fibroblasts and meningioma cells, and co-localizes with F-actin at the cell membrane in schwannoma cells (10-14). In rat sciatic nerve, merlin is expressed at paranodal incisures, regions rich in integrins and cadherins (15). Furthermore, cultured human schwannoma cells from NF2 patients have altered cytoskeletal organization and growth properties (16). Collectively, these data suggest that merlin may function to modulate growth-promoting or growth-inhibitory signals transmitted by cell-cell or cell-substrate contact.
To test the hypothesis that merlin participates in pathways important for cell-cell or cell-substrate signal transduction, we examined the ability of merlin to alter three properties of cell-substrate signaling. In this report, we describe for the first time that overexpression of wild-type merlin, but not merlin containing NF2 missense patient mutations, reduces cell motility and cell adhesion and alters cell spreading.
RESULTS
We previously demonstrated that wild-type merlin, but not merlin containing exon 16 (NF2.16) or any of three naturally occurring NF2 missense patient mutations (L64P, K413E and L535P), inhibited rat schwannoma cell proliferation in vitro and in vivo (8,17). The inability of merlin containing exon 16 or the L64P NF2 patient mutation to suppress cell proliferation correlated with the inability of the N- and C-terminal domains of these merlin proteins to interact with each other. To determine whether wild-type merlin, but not merlin proteins defective in growth suppression, could alter JS1 rat schwannoma cell motility, we analyzed clones of JS1 cells that either stably or inducibly expressed merlin, and tested them in an in vitro membrane invasion culture system (MICS) (18,19). The migration rate was calculated by counting cells that had migrated through polycarbonate membrane pores as a function of time after seeding. For experiments with stable cell lines, multiple JS1 rat schwannoma cell lines were established that expressed 8- to 10-fold more merlin or mutant merlin proteins compared with JS1 cells transfected with vector alone (Fig.
Overexpression of merlin-17 suppresses cell motility in vitro
A
B, C
Figure 1. Overexpression of wild-type, but not mutant, merlin reduces rat schwannoma cell motility. (A) Representative western blot demonstrating 8- to 10-fold overexpression of NF2 transgenes in selected JS1 rat schwannoma cell lines. Immunodetection was accomplished using the WA30 rabbit polyclonal antibody (33). (B) (top) Reduced JS1 rat schwannoma cell motility was observed in cell lines overexpressing wild-type merlin isoform I, but not merlin isoform II, compared with vector (pcDNA3)-transfected controls. Standard deviations for each representative cell line are shown. *Statistically significant using the Student’s t-test (P < 0.001). This assay directly measures the migration of JS1 cells in the MICS assay and migration is expressed as µm/h. (C) (bottom) JS1 cell lines expressing wild-type, but not mutant, merlin molecules reduced rat schwannoma cell motility as measured in the MICS assay. The migration per hour is normalized to the pcDNA3 vector controls. Standard deviations for each cell line are shown. *Statistically significant using the Student’s t-test (P < 0.001). Data are representative of at least three different experiments.
As a second means of testing merlin’s effects on cell motility, we developed several merlin-inducible RT4-D6P2T rat schwannoma cell lines in which the expression of merlin was positively regulated by the addition of zinc chloride. Extensive analyses failed to demonstrate any effect of short- or long-term treatment of RT4 cells with zinc chloride with respect to cell proliferation, cell survival or cell attachment (data not shown). Using the synthetic metal-inducible promoter described by Peden et al. (20), we generated RT4 cell lines exhibiting tightly regulated merlin expression. All cell lines had endogenous levels of merlin comparable with untransfected RT4 cells in the absence of zinc, but dramatically increased merlin expression within 3 h after the addition of 100 µM zinc chloride to the media. Representative western blots for several of these cell lines are shown in Figure
Figure 2. Induction of merlin in RT4 rat schwannoma cell containing zinc-inducible merlin constructs. The expression of merlin as a function of time in hours after the addition of 100 µM zinc chloride in several representative cell lines is shown. Merlin expression is detected using the WA30 rabbit polyclonal antibody. Wild-type merlin clone 8 is shown in this experiment. No change in merlin expression was seen in the pMT5.neo vector control cell line. Merlin expression is induced following the addition of zinc chloride in both short-term (0-5 h) (A) and long-term (0-48 h) (B) experiments. Tubulin is included as a control for equal protein loading. Similar induction was observed with another missense mutant merlin cell line (K413E; data not shown). Figure 3. Induction of merlin expression results in decreased RT4 rat schwannoma cell motility. (A) (left) The MICS assay was performed using representative pMT5.neo (vector)- and wild-type merlin-expressing cell lines. Changes in the relative migration of these cell lines in the presence or absence of zinc were determined using the MICS assay. Only the wild-type merlin-containing RT4 schwannoma cell line demonstrated reduced migration upon the addition of zinc. Standard deviations are shown for each cell line. *Statistically significant using the Student’s t-test (P < 0.001). Data are representative of three different experiments. (B) (right) Cell proliferation was performed as described in Materials and Methods on RT4-NF2.17 #8 cells grown in the presence or absence of zinc chloride. No differences in thymidine incorporation were observed between RT4-NF2.17 #8 cells expressing merlin (+ zinc) or not expressing merlin (- zinc) during this 4 h time interval. Using these inducible cell lines, we assayed the ability of merlin to reduce cell motility in vitro. Using the MICS approach, RT4 cells containing empty vector (pMT5.neo) or pMT5.neo.merlin were incubated overnight in the presence or absence of 100 µM zinc chloride and seeded for the motility assay. The results of one representative experiment are illustrated in Figure 
Merlin mutants and merlin-16 fail to influence cell motility
We next tested the hypothesis that merlin inhibition of schwannoma cell motility was directly related to the ability of merlin to function as a negative growth regulator. Previously, we demonstrated that merlin proteins containing exon 16 or truncated at residue 547 were incapable of forming an intracellular complex (8). Merlin containing a missense mutation at residue 64 (L64P) was also incapable of forming an intramolecular complex whereas merlin containing missense mutations at residues K413 and L535 could form these complexes: all missense proteins were defective as growth regulators (17). Whereas wild-type merlin reduced cell motility by 50% compared with vector controls, neither merlin containing exon 16, merlin truncated at residue 547 nor merlin with patient missense mutations at residues Lys413 (K413E) and Leu535 (L535P) had any effect on cell motility (Fig.
Merlin does not influence catenin or ERM protein expression
To investigate the mechanism by which merlin might alter cell motility and migration, we tested the hypothesis that merlin might modulate the expression of proteins involved in mediating cytoskeletal organization and growth control. Previous work on the adenoma polyposis coli (APC) gene product demonstrated that it functions to regulate cell growth by regulating the intracellular pools of the catenin family of proteins (21). However, upon induction of merlin, no changes in [alpha]- or [beta]-catenin protein expression levels were observed (Fig.
Figure 4. Merlin induction does not result in altered expression of catenins or ERM proteins. (A) (top left) The expression of [alpha]- and [beta]-catenin is shown in this representative experiment as a function of time in hours after the addition of 100 µM zinc chloride. No changes in catenin protein expression were noted. (B) (top right) The expression of moesin and ezrin is shown in this representative experiment as a function of time in hours after the addition of 100 µM zinc chloride. No changes in moesin and ezrin protein expression were noted. (C) (bottom) The expression of moesin and ezrin in cell membrane fractions is shown in this representative experiment as a function of time in hours after the addition of 100 µM zinc chloride. No changes in membrane moesin and ezrin protein expression were noted. The moesin antibody recognizes both moesin and ezrin (protein doublet). Another possible mechanism underlying merlin’s effect on cell migration envisages merlin altering either the distribution or balance of ERM protein expression necessary for the formation of microvillar structures or signaling events at the leading edge. Since merlin has been shown to co-localize with ezrin (12), we examined ezrin and moesin expression after merlin induction. We failed to demonstrate any changes in moesin or ezrin expression after merlin induction (Fig. Figure 5. Immunolocation of ezrin following merlin induction. RT4-NF2.17 #8 cells were induced for 0-6 h with 100 µM zinc chloride on glass coverslips prior to fixation and immunocytochemistry using ezrin-specific monoclonal antibodies (BABCO, 1:1200 dilution). Ezrin was localized using a Cy3-conjugated secondary antibody. No changes in merlin subcellular localization were observed at 2 (data not shown), 4 or 6 h after induction compared with no induction (0 h). Photomicrographs were taken at 40× magnification (A). High power photomicrographs (B) demonstrate the light membrane localization of ezrin in addition to the prominent cytoplasmic labeling. Nuclear staining was observed with both ezrin antibodies. Omission of the primary antibody resulted in no staining (data not shown). Another measure of the ability of merlin to modulate cell-substrate interactions is the effect of merlin expression on cell attachment. We tested the hypothesis that merlin expression could impair the ability of schwannoma cells to adhere to fibronectin-coated plates. RT4 cells containing pMT5.neo.merlin (clone 8) were incubated for 3 h in serum-free media at 37°C in the presence or absence of 100 µM zinc chloride and then seeded onto fibronectin-coated 96-well plates. Cell attachment was determined as a function of time after plating. We consistently observed a reduction in cell attachment in RT4 cells overexpressing wild-type merlin that occurs within the first 3 h after plating (Fig. Figure 6. Induction of merlin results in reduced RT4 cell adhesion. RT4-NF2.17 #8 rat schwannoma cells were incubated in the presence or absence of 100 µM zinc chloride overnight and then harvested by trypsinization. Equal numbers of cells were added to fibronectin-coated 96-well plates for 1 or 3 h. After the specified amount of time, the number of adherent cells was determined using Crystal violet staining and a spectrophotometric assay. Decreased adhesion was seen in the RT4 cell line treated with zinc chloride after 1 h but not after 3 h. This transient decrease in cell adhesion was not observed with the pMT5.neo vector control (data not shown). Data are representative of three different experiments. The transient effect of merlin on cell attachment suggested that merlin may modulate the ability of cells to form meaningful contacts with their substrate. In this regard, cultured human schwannoma cells from NF2 patients demonstrate abnormal cell spreading on laminin or fibronectin substrates (16). To determine whether overexpression of merlin similarly impairs rat schwannoma cell spreading, we assayed the effect of merlin overexpression on actin cytoskeletal organization in cells plated on laminin. As shown in Figure Figure 7. Expression of merlin results in dramatic changes in cell spreading. (A) (top left) RT4 cells transfected with pMT5.neo containing wild-type NF2 (clone 8) were seeded onto laminin-coated plates for 2 h in the presence or absence of 100 µM zinc chloride followed by fixation and analysis using fluorescent phalloidin. As can be seen in this representative experiment, the addition of zinc has a profound effect on cell spreading in both independently generated cell lines. These photomicrographs were taken at a 40× magnification. Phase contrast photomicrographs are included to demonstrate the shape of the cells. Prominent nuclear actin labeling consistently was observed. (B) (top right) RT4 cells transfected with pMT5.neo containing wild-type NF2 (clones 8 and 16) were seeded onto laminin-coated plates for 2 h in the presence or absence of 100 µM zinc chloride followed by fixation and analysis using fluorescent phalloidin. As can be seen in this representative experiment, the addition of zinc has a profound effect on cell spreading in both independently generated cell lines. These photomicrographs were taken at a 63× magnification. Similar results were obtained at 30 min and 1 h (see C). (C) (bottom left) RT4 cells transfected with pMT5.neo vector alone, pMT5.neo vector containing wild-type NF2 and pMT5.neo vector containing mutant NF2 (L64P mutation) were seeded onto laminin-coated plates for 30 min in the presence or absence of 100 µM zinc chloride followed by fixation and analysis using fluorescent phalloidin. As can be seen in this representative experiment, the addition of zinc has no effect on cell spreading (vector control) in contrast to a profound effect of wild-type, but not mutant merlin, expression on cell shape and spreading. These photomicrographs were taken at 40× magnification. Data are representative of four different experiments. (D) (bottom right) RT4 cells transfected with pMT5.neo vector alone, pMT5.neo vector containing wild-type NF2 and pMT5.neo vector containing mutant NF2 (L64P mutation) were seeded onto laminin-coated plates for 6 h in the presence or absence of 100 µM zinc chloride followed by fixation and analysis using fluorescent phalloidin. No differences were observed between vector, wild-type merlin and mutant merlin RT4 cell lines at this time point and after 24 h (data not shown). The NF2 tumor suppressor gene is classified as a negative growth regulator based on the observation that individuals with NF2 mutations develop schwannomas and other tumors at an increased frequency. Formal proof for a growth-regulatory role has been provided by studies in which merlin expression was reduced by antisense treatment, increased by overexpression in cell lines and eliminated by transgenic knockout technology. Reduced merlin expression in glioma and schwannoma cell lines was shown to result in a modest increase in cell proliferation (22). Overexpression of merlin in NIH-3T3 fibroblasts or rat schwannoma cell lines with low or undetectable endogenous merlin expression resulted in significantly reduced cell proliferation both in vitro and in vivo (7,8). The ability of merlin to modulate cell growth was only observed with merlin molecules capable of intramolecular associations between the N- and C-termini, and not with merlin proteins that fail to form such intramolecular complexes or that contain missense or nonsense NF2 patient mutations (8,17). Interestingly, mice heterozygous for a targeted disruption of the Nf2 gene develop highly malignant tumors (23). Recent work from our laboratory has demonstrated that merlin is most potent as a negative growth regulator in RT4 schwannoma cells grown to confluence, suggesting that merlin tumor suppressor function may be maximal in the context of cell-cell contact (17). To study the possibility that merlin provides its negative growth regulator signal via pathways requiring cell-cell or cell-substrate contact, we examined the effect of merlin overexpression on cell motility, cell adhesion and cell spreading. In this report, we demonstrate that wild-type merlin, but not merlin molecules defective as negative growth regulators, inhibits cell motility and cell adhesion as well as altering cell spreading. Each of these processes is heavily dependent on actin cytoskeleton-mediated events. Several findings support the possibility that merlin, like other ERM family members, modulates signaling through the actin cytoskeleton. First, merlin expression is regulated by contact inhibition (24). Increased merlin expression was observed in fibroblasts arrested by contact inhibition or serum starvation but not by [gamma]-irradiation, nocodazole or hydroxyurea. In addition, loss of adhesion is associated with changes in merlin serine phos-phorylation, suggesting that merlin may function in the context of contact inhibition (25). Second, immunocytochemistry studies have demonstrated that merlin is expressed at the plasma membrane both endogenously and in cells overexpressing merlin transgenes (13-15). In these studies, merlin is enriched in microvilli and ruffling edges of cells in vitro and at paranodal incisures of the rat sciatic nerve in vivo (15). Both of these regions are rich in cell adhesion molecules and signaling proteins important for transducing cell-cell and cell-substrate signals. Third, merlin has been shown to interact with both actin and [beta]-2 spectrin (fodrin) (26). Fodrin belongs to a family of proteins that bind actin and several other cytoskeletal proteins. The association between merlin and fodrin may serve to bring merlin to the actin cytoskeleton where it can participate most effectively in cell growth-regulatory pathways. Recently, we have demonstrated a putative merlin actin-binding site distinct from the actin-binding domain at the C-terminus of ERM proteins, located in the N-terminal region of merlin (residues 280-340) (9). This region may overlap with the N-terminal residues required for the formation of merlin intramolecular complexes, suggesting the intriguing possibility that merlin’s association with the actin cytoskeleton may regulate merlin growth suppressor function. Similar associations have been proposed for other ERM proteins in which binding to actin is negatively regulated by ERM protein folding. ERM proteins are expressed in microvilli, ruffling membranes and cleavage furrows (27-29). Antisense oligonucleotide down-regulation of ezrin or radixin expression disrupts the initial step of cell-cell and cell-substrate contact and adhesion to result in defective spreading. Microvilli structures were unaffected by reduced ezrin or radixin expression. In contrast, microvillar structures were profoundly affected by antisense oligonucleotide down-regulation of moesin expression without any impairment of cell contact or spreading. Antisense oligonucleotide down-regulation of ezrin, radixin and moesin expression simultaneously resulted in detachment from the substrate. Similar experiments using oligonucleotides targeted against merlin resulted in cell detachment concomitant with modestly increased cell proliferation (22). The mechanism(s) underlying merlin’s ability to regulate cell growth may relate to actin-associated signal transduction events. Moesin, ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to Rho and Rac activation (30). Rac/RhoA activation results in phosphatidylinositol (PIP)-5[prime] kinase activity which regulates PIP2 synthesis. PIP2 regulates actin polymerization and stabilizes interactions between moesin and CD44 (31) in such a manner as to induce unfolding of ERM proteins. LPA treatment results in RhoA-dependent relocalization of radixin, ezrin and moesin into apical membranes and actin protrusions (25). In contrast, merlin did not translocate to apical protrusions in response to LPA treatment, but was less membrane associated in the presence of an activated RhoA molecule. It is not known whether PIP2 regulates merlin growth regulation or actin binding relevant to merlin intra- or inter-molecular complex formation. In this report, we demonstrate that merlin negatively regulates several cellular properties important for cell-cell and cell-substrate interactions. Merlin specifically impairs cell motility by modulating events at the leading or ruffling edge of the membrane. Similarly, merlin impairs cell attachment and spreading. These effects were shown to be merlin-specific and depend on the presence of a functional merlin molecule. In fibroblasts, induction of DNA synthesis is inhibited when anchorage is prevented (32). Merlin may function analogously by modulating the signal imparted by cell adhesion to provide a non-permissive message for cell proliferation. Failure to regulate this adhesion signal may result in increased cell proliferation. Likewise, it is possible that merlin positively contributes to the contact inhibition growth arrest signal, such that absent merlin expression results in impaired transmission of a growth arrest signal. The transient effect of merlin on these cell interaction functions most likely reflects the brief window in which these signals are transmitted and the cellular context in which these signals are delivered. Future studies will be required to determine whether merlin modulates cell surface signaling relevant to contact inhibition growth arrest and to dissect the intracellular signaling mechanisms operative in transducing this signal. The WA30 merlin antibody is a rabbit polyclonal antiserum generated against merlin peptide sequences as previously reported (15,33). Proteins were separated by SDS-PAGE and transferred onto Immobilon-P filters (Millipore) for western blotting as previously described (15). WA30 antibodies were used at a dilution of 1:2000-1:4000. The rabbit FLAG polyclonal and myc monoclonal (clone 9E10) antibodies were purchased from Santa Cruz Biotechnology and used as recommended by the manufacturer. Tubulin monoclonal antibodies (Sigma; clone DM-1A), rabbit [alpha]- and [beta]-catenin polyclonal antibodies (Sigma), monoclonal ezrin antibodies (BABCO; clone 4A5) and monoclonal moesin antibodies (Transduction Laboratories; clone 38) were used according to the manufacturer’s recommendations. Development was accomplished using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL; Amersham). Cell membrane preparations were performed as previously described (15,17). Merlin cDNA constructs were generated by PCR using the full-length NF2 cDNA as template (JJR.1 generously provided by Dr James Gusella, MGH). Merlin containing exon 16 was generated by RT-PCR as previously described. Patient mutations were generated using oligonucleotides as previously reported (17). The mutant NF2 fragments were cloned back into the full-length NF2 cDNA for expression studies. All mutants were sequenced using the Sequenase reagent to verify the introduction of the specific NF2 mutation. Epitope-tagged merlin constructs were generated by PCR using the NF2 cDNA as template. Each tagged construct was sequenced (Sequenase) and tested using the appropriate FLAG or myc antibody. All merlin cDNAs generated proteins of the predicted molecular weights. Inducible merlin constructs were generated by cloning the full-length NF2 cDNA into pMT5.neo as previously reported (20). RT4 rat schwannoma cell lines were established as previously described (8,17). Briefly, transfections of RT4 cells were performed using DOTAP lipofection reagent (Gibco BRL) according to the manufacturer’s recommendations. Stable cell lines were selected in 500 µg/ml G418 (geneticin) and expanded for western blot analysis using WA30 merlin antibodies. At least three cell lines with equivalent overexpression of merlin (8- to 10-fold overexpression relative to RT4 cells) were selected for analysis. Likewise, pMT5.neo.merlin clones were selected and tested for induction of merlin expression in the presence of zinc chloride. We experimentally determined that 100 µM zinc chloride was the optimal dose for induction. Significant merlin expression was detected in all cell lines by 3 h after the addition of zinc chloride. There was no observed toxicity in cells maintained for weeks in zinc chloride as judged by cell proliferation and viability as assessed by Trypan blue exclusion. Inducible merlin cell lines were chosen with merlin expression equivalent to endogenous RT4 levels in the absence of zinc. Thymidine incorporation was performed by pulsing RT4-NF2.17 #8 cells with 1 µCi of [3H]thymidine (Amersham) per ml for 4 h in 24-well plates (six wells per culture or condition). Cells were induced for 4-6 h with 100 µM zinc chloride and then seeded at 10 000 cells per well prior to [3H]thymidine addition. Labeled cells were washed in phosphate-buffered saline (PBS) and solubilized in 200 µl of 0.2 M NaOH. Counts were then determined in a scintillation counter. RT4-NF2.17 #8 cells were seeded onto glass coverslips and induced for 2, 4 or 6 h by the addition of 100 µM zinc chloride. Cells were then washed in 1× PBS and fixed for 20 min with 4% paraformaldehyde prior to processing for immunocytochemistry. All antibody incubations were performed in PBS containing 10% normal goat serum for 1 h at 37°C. Primary ezrin antibodies (BABCO, 1:1200 dilution and Transduction Laboratories, 1:250) were employed in combination with Cy3-conjugated anti-mouse or rabbit secondary antibodies (Jackson Immunochemicals). Coverslips were mounted onto glass slides and images were acquired on a Nikon Optiphot fluorescent microscope. The MICS was used to assess the migratory ability of the JS-1 and RT4 transfectants (18,19). A 10 µm pore size polycarbonate membrane (Osmonics, Livermore, CA) soaked in 0.1% gelatin was placed between the upper and lower plates of the MICS chamber with 1 × 105 cells and incubated for 4 h at 37°C. Those cells that had migrated through the pores to the lower wells were harvested with 2 mM EDTA in PBS, stained with a LeukoStat staining kit (Fisher Scientific) and counted visually by 6-8 random high powered fields to calculate the migration rate. The migration rate for the stable merlin cell lines was determined by normalizing the pcDNA3 vector JS1 clones value to 100% and comparing its relative migration values with the experimental merlin-expressing cell lines. The migration rate using the merlin-inducible clones was determined by normalizing the RT4 clones (untreated) value to 100% and comparing its relative migration values with the ZnCl2-treated samples. Cell attachment was measured by seeding 10 000 merlin-inducible RT4 cells that had been pre-incubated for at least 4-6 h in serum-free media in the presence or absence of zinc into 96-well plates pre-coated with 10 µg/ml fibronectin (Gibco Life Sciences). Six wells were used for each condition. After 1 or 3 h, the plates were gently washed in 1× PBS and the number of adherent cells determined by incubation with 0.5% Crystal violet for 30 min followed by extraction in 1% SDS overnight and spectrophotometric analysis at 540 nm. Glass coverslips were coated with 10 µg/ml of laminin (Sigma) in PBS overnight at 4°C. Coverslips were then aspirated and placed in 24-well dishes. Inducible merlin RT4 cells, cultured in Dulbecco’s modified Eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) were treated with ZnCl2 for 4-6 h, then removed from dishes by trypsinization. Cells were washed twice in PBS, then resuspended in DMEM + 10% FBS and plated onto the coverslips at ~20 000 cells/well. After 30 min, 1, 2, 3 or 6 h, cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized in PBS containing 0.2% Triton X-100, then stained with BODIPY-conjugated phalloidin (0.2 U in 50 µl; Molecular Probes) for 30 min. Coverslips were then washed in PBS, mounted in one drop of Fluoromount G (EM Sciences), and examined on a Zeiss Axiophot microscope. We appreciate the technical assistance of Drs Alice Gardner and Yujing Zhang during the execution of these experiments. We thank Helen Morrison for insights and suggestions. This work is supported by NIH grant NS35848.
Induction of merlin results in a transient decrease in cell attachment
Overexpression of merlin results in transient abnormalities in cell spreading
DISCUSSION
MATERIALS AND METHODS
Antibodies and western blotting
Merlin cDNA constructs
Generation of merlin schwannoma cell lines
Thymidine incorporation
Immunocytochemistry
In vitro migration assay using a membrane invasion culture system (MICS)
Cell attachment
Cell spreading
ACKNOWLEDGEMENTS
REFERENCES
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D. H. Gutmann, A. C. Hirbe, and C. A. Haipek Functional analysis of neurofibromatosis 2 (NF2) missense mutations Hum. Mol. Genet., July 1, 2001; 10(14): 1519 - 1529. [Abstract] [Full Text] [PDF]
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H. Morrison, L. S. Sherman, J. Legg, F. Banine, C. Isacke, C. A. Haipek, D. H. Gutmann, H. Ponta, and P. Herrlich The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44 Genes & Dev., April 15, 2001; 15(8): 968 - 980. [Abstract] [Full Text]
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D. H. Gutmann The neurofibromatoses: when less is more Hum. Mol. Genet., April 1, 2001; 10(7): 747 - 755. [Abstract] [Full Text] [PDF]
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D. H. Gutmann, C. A. Haipek, S. P. Burke, C.-X. Sun, D. R. Scoles, and S. M. Pulst The NF2 interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), associates with merlin in the 'open' conformation and suppresses cell growth and motility Hum. Mol. Genet., April 1, 2001; 10(8): 825 - 834. [Abstract] [Full Text] [PDF]
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 D G. R Evans, M Sainio, and M. E Baser Neurofibromatosis type 2 J. Med. Genet., December 1, 2000; 37(12): 897 - 904. [Abstract] [Full Text]
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D. R. Scoles, D. P. Huynh, M. S. Chen, S. P. Burke, D. H. Gutmann, and S.-M. Pulst The neurofibromatosis 2 tumor suppressor protein interacts with hepatocyte growth factor-regulated tyrosine kinase substrate Hum. Mol. Genet., July 1, 2000; 9(11): 1567 - 1574. [Abstract] [Full Text] [PDF]
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M. Giovannini, E. Robanus-Maandag, M. van der Valk, M. Niwa-Kawakita, V. Abramowski, L. Goutebroze, J. M. Woodruff, A. Berns, and G. Thomas Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2 Genes & Dev., July 1, 2000; 14(13): 1617 - 1630. [Abstract] [Full Text]
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D. H. Gutmann, J. Donahoe, A. Perry, N. Lemke, K. Gorse, K. Kittiniyom, S. A. Rempel, J. A. Gutierrez, and I. F. Newsham Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas Hum. Mol. Genet., June 12, 2000; 9(10): 1495 - 1500. [Abstract] [Full Text] [PDF]
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 B. McCartney, R. Kulikauskas, D. LaJeunesse, and R. Fehon The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation Development, January 3, 2000; 127(6): 1315 - 1324. [Abstract] [PDF]
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G.-H. Xiao, A. Beeser, J. Chernoff, and J. R. Testa p21-activated Kinase Links Rac/Cdc42 Signaling to Merlin J. Biol. Chem., January 4, 2002; 277(2): 883 - 886. [Abstract] [Full Text] [PDF]
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M. Jannatipour, P. Dion, S. Khan, H. Jindal, X. Fan, J. Laganiere, A. H. Chishti, and G. A. Rouleau Schwannomin Isoform-1 Interacts with Syntenin via PDZ Domains J. Biol. Chem., August 24, 2001; 276(35): 33093 - 33100. [Abstract] [Full Text] [PDF]
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