The neurofibromatosis type 2 gene product, merlin, reverses the F-actin cytoskeletal defects in primary human Schwannoma cells

Abstract

Schwannoma tumors, which occur sporadically and in patients with neurofibromatosis, account for 8% of intracranial tumors and can only be treated by surgical removal. Most schwannomas have biallelic mutations in the NF2 tumor suppressor gene. We previously showed that schwannoma-derived Schwann cells exhibit membrane ruffling and aberrant cell spreading when plated onto laminin, indicative of fundamental F-actin cytoskeletal defects. Here we expand these observations to a large group of sporadic and NF2-related tumors and extend them to schwannomatosis-derived tumors. Mutation at NF2 correlated with F-actin abnormalities, but the extent of morphological change did not correlate with the type of NF2 mutation. We used a recently described molecular strategy, TAT-mediated protein transfer, to acutely introduce the NF2 protein, merlin, into primary human schwannoma cells in an attempt to reverse the cytoskeletal phenotype. Abnormal ruffling and cell spreading by cells with identified NF2 mutations were rapidly reversed by introduction of TAT-merlin. The effect is specific to TAT-merlin isoform 1, the growth-suppressive isoform of merlin. TAT-merlin isoform 2, a TAT-merlin mutant (L64P), and merlin lacking TAT were ineffective in reversing the cytoskeletal phenotype. Results show that merlin isoform 1 is sufficient to restore normal actin organization in NF2-deficient human tumor cells, demonstrating a key role for merlin in the NF2 phenotype. These results lay the foundation for epigenetic complementation studies in NF2 mouse models and possibly for experiments to evaluate the utility of merlin transduction into patients as protein therapy.

FIG. 1.

Abnormal F-actin organization in schwannoma and schwannomatosis cells. Cells were plated onto laminin-coated coverslips, serum was added, and cells were fixed and stained with BODIPY-phalloidin to visualize F-actin (A to D) and then analyzed by confocal microscopy. Membrane ruffles are present on cells from a sporadic schwannoma (A) and a schwannoma from a schwannomatosis patient (B) but not normal human Schwann cells (C) or neurofibromatosis type 1 neurofibroma-derived Schwann cells (D). Bar, 10 μm.

FIG. 2.

Purification and analysis of TAT-merlin. (A) Schematic of TAT-merlin constructs. (B) Coomassie blue-stained gel showing bacterial extract expressing TAT-merlin (lane 1) and purified TAT-merlin (6 μg) (lane 2). Lane 3 shows a Western blot of lane 2 stained with antimerlin antibody. (C) Normal human Schwann cells were untreated (lane 1), treated with TAT-merlin isoform 1 for 5 h (lane 2), or treated for 5 h followed by 16 h without TAT-merlin (lane 3). Western blots of cell lysates were probed with anti-Myc. Control schwannoma cells (D) and schwannoma cells incubated with 100 μg of TAT-merlin isoform 1 per ml for 5 h (E) were fixed and then stained with anti-Myc, which localizes TAT-merlin isoform 1 to the plasma membrane (E). (E) Particulate likely represents clumps of TAT-merlin that did not enter cells.

FIG. 3.

TAT-merlin isoform 1 specifically reverses schwannoma cell membrane ruffling. (A) Schwannoma cells were exposed to the designated concentrations of purified TAT-merlin. Cells were then replated onto laminin-coated coverslips and exposed to serum. Cells were stained, and the percentage of S100β-positive cells with ruffling membranes was quantitated. (B to G) Alexa-phalloidin-labeled cells are shown. In each case, schwannoma cells were treated with 60 μg of designated protein per ml. (B) No protein was added. Schwannoma cells were exposed to TAT-merlin 1 (C), merlin 1 (no TAT) (D), TAT-merlin isoform 2 (E), TAT-β-galactosidase (F), and TAT-L64P merlin isoform 1 (G). Bar, 10 μm.

FIG. 4.

TAT-merlin isoform 1 specifically reverses the F-actin cytoskeletal defects in schwannoma cells: quantitation. (A and B) TAT-merlin isoform 1 was added to cells from the tumor type designated under the x axis. Cells were replated onto laminin in the presence of serum. The percentage of cells with membrane ruffles was measured as for Fig. 1 and 3. (A) Left of the vertical line, TAT-merlin isoform 1 (I) but not TAT-merlin isoform 1 with a mutation (L64P) reverses schwannoma cell membrane ruffling. The effects of TAT-merlin isoform 1 were reversed when cells were treated with TAT-merlin 1 for 5 h and then exposed overnight to medium without TAT-merlin isoform 1 (1w/o). To the right of the vertical line, membrane ruffling was inhibited by TAT-merlin isoform 1 (I) but not TAT-merlin isoform 2 (II) or TAT-β-galactosidase (β-gal). (C) From each tumor type designated under the x axis, cells were exposed to 60 μg of TAT-merlin isoform 1 and then washed, replated, and stained as for panel A, and their spread areas were measured.