Point mutation in the NF2 gene of HEI-193 human schwannoma cells results in the expression of a merlin isoform with attenuated growth suppressive activity.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by the formation of bilateral schwannomas of the eighth cranial nerve. Although the protein product of the NF2 gene (merlin) is a classical tumor suppressor, the mechanism by which merlin suppresses cell proliferation is not fully understood. The availability of isolated tumor cells would facilitate a better understanding of the molecular function of merlin, but primary schwannoma cells obtained from patients grow slowly and do not yield adequate numbers for biochemical analysis. In this study, we have examined the NF2 mutation in HEI-193 cells, an immortalized cell line derived from the schwannoma of an NF2 patient. Previous work showed that the NF2 mutation in HEI-193 cells causes a splicing defect in the NF2 transcript. We have confirmed this result and further identified the resultant protein product as an isoform of merlin previously designated as isoform 3. The level of isoform 3 proteins in HEI-193 cells is comparable to the levels of merlin isoforms 1 and 2 in normal human Schwann cells and several other immortalized cell lines. In contrast to many mutant forms of merlin, isoform 3 is as resistant to proteasomal degradation as isoforms 1 and 2 and can interact with each of these isoforms in vivo. Cell proliferation assays showed that, in NF2(-/-) mouse embryonic fibroblasts, exogenously expressed merlin isoform 3 does exhibit growth suppressive activity although it is significantly lower than that of identically expressed merlin isoform 1. These results indicate that, although HEI-193 cells have undetectable levels of merlin isoforms 1 and 2, they are, in fact, not a merlin-null model because they express the moderately active growth suppressive merlin isoform 3.

Cell motility assays on tissue culture dishes via non-invasive confinement and release of cells.

In vitro cell migration assays are useful for screening bioactive agents that regulate angiogenesis, tumor metastasis, would healing, and immune responses by effecting changes in the rate of cell migration. Here we have developed a noninvasive in vitro migration assay that operates through release of confluent groups of cells initially confined within patterns of cell-resistant polyelectrolyte. Cell-resistant patterns of polyelectrolyte, separating groups of confluent cells, are rendered cell adhesive by adsorption of a second, cell adhesive polyelectrolyte of opposite charge; thereby, resulting in migration of cells into the separating regions. By dynamically controlling cell-surface interactions through self-assembly of cell-adhesive and cell resistant polyelectrolytes, this method eliminates the need to mechanically wound cells, as is done in current cell migration assays. The utility of this technique in identifying molecules and mechanisms that regulate cell migration is demonstrated by its application as an assay for the effects of platelet derived growth factors, cytoskeleton disrupting agents, and Merlin overexpression, on the migration of NIH 3T3 fibroblasts.

Methods to study protein-protein interactions.

Protein-protein interactions are the underpinnings of a vast number of cellular processes. In recent years, the convergence of biochemistry, cellular, and molecular biology has made available a number of powerful techniques for studying such interactions. These techniques vary in their sensitivity, efficiency, and rapidity, but judicial deployment of a combination of them has proved to be effective and reliable. Here, we highlight a version of the yeast two-hybrid assay originally pioneered by Fields and Song (1989) and subsequently enhancements by other investigators. We also briefly describe a number of new fluorescent imaging-based biophysical techniques for studying protein-protein interactions FRET, FCS, and BiFC. Together, these constitute an impressive collection of tools for studying interactions among proteins.