SUBCELLULAR-LOCALIZATION AND EXPRESSION PATTERN OF THE NEUROFIBROMATOSIS TYPE-2 PROTEIN MERLIN SCHWANNOMIN/

To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the ex pression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibo dy raised against the C-terminus of merlin. Three of the six merlin is oforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been rep orted earlier and can be explained by alternative splicing. In additio n, we detected higher molecular weight bands of about 110 kDa, 100 kDa and 84 kDa. Although the merlin bands of 100 kDa and 110 kDa may repr esent homo- or heterodimers, oligomerization due to formation of disul fide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kD a were quantitatively extractable in Lubrol WX, indicating a localizat ion in or close to the plasma membrane. The 45 kDa band, however, was not soluble in Lubrol WX compatible with a localization of this NF2 is oform in the endoplasmic reticulum. Applying confocal laser scanning m icroscopy, merlin was shown to be located in four subcellular compartm ents: (i) perinuclear in a compartment resembling endoplasmic reticulu m, (ii) in ruffling membranes and at the leading edges, (iii) in filop odia, and (iv) at cell/substrate adhesion points. Codistribution of me rlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesio n junctions as shown by phalloidin-rhodamine staining. Double immunofl uorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the ac tin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the re gulation of cell growth by interaction with cytoskeleton-associated pr oteins.