Schmucker B, Ballhausen W G, Kressel M
Institute of Human Genetics, Friedrich Alexander University of Erlangen-Nürnberg, Germany.
Eur J Cell Biol. 1997 Jan;72(1):46-53.
To elucidate the physiological function of the neurofibromatosis type 2 (NF2) tumor suppressor protein merlin/schwannomin, we studied the expression pattern and subcellular localization in human fibroblasts by Western blot analyses and immunofluorescence using a polyclonal antibody raised against the C-terminus of merlin. Three of the six merlin isoforms identified in this study (75 kDa, 58 kDa, 45 kDa) have been reported earlier and can be explained by alternative splicing. In addition, 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 represent homo- or heterodimers, oligomerization due to formation of disulfide bonds was excluded. Furthermore, the isoforms of 84 kDa and 58 kDa were quantitatively extractable in Lubrol WX, indicating a localization 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 isoform in the endoplasmic reticulum. Applying confocal laser scanning microscopy, merlin was shown to be located in four subcellular compartments: (i) perinuclear in a compartment resembling endoplasmic reticulum, (ii) in ruffling membranes and at the leading edges, (iii) in filopodia, and (iv) at cell/substrate adhesion points. Codistribution of merlin and F-actin filaments was found in filopodia, ruffling membranes and at the insertion points of stress fibers at cell/substrate adhesion junctions as shown by phalloidin-rhodamine staining. Double immunofluorescence analyses of merlin and moesin revealed a colocalization in filopodia and ruffling membranes. The localization of merlin in the actin-rich cortical cytoskeleton corresponds to the ezrin-radixin-moesin family of proteins suggesting the NF2 protein to contribute to the regulation of cell growth by interaction with cytoskeleton-associated proteins.
为阐明2型神经纤维瘤病（NF2）肿瘤抑制蛋白默林/施万诺明的生理功能，我们通过蛋白质免疫印迹分析以及使用针对默林C端的多克隆抗体进行免疫荧光实验，研究了其在人成纤维细胞中的表达模式和亚细胞定位。本研究中鉴定出的六种默林同工型中的三种（75 kDa、58 kDa、45 kDa）此前已有报道，可通过可变剪接来解释。此外，我们还检测到了分子量约为110 kDa、100 kDa和84 kDa的更高分子量条带。尽管100 kDa和110 kDa的默林条带可能代表同二聚体或异二聚体，但排除了因二硫键形成导致的寡聚化。此外，84 kDa和58 kDa的同工型可在Lubrol WX中定量提取，表明其定位于质膜或靠近质膜的位置。然而，45 kDa条带不溶于Lubrol WX，这与该NF2同工型定位于内质网一致。应用共聚焦激光扫描显微镜观察发现，默林定位于四个亚细胞区室：（i）核周一个类似于内质网的区室；（ii）褶皱膜和前缘；（iii）丝状伪足；（iv）细胞/底物黏附点。如罗丹明鬼笔环肽染色所示，在丝状伪足、褶皱膜以及应力纤维在细胞/底物黏附连接处的插入点发现默林与F-肌动蛋白丝共分布。默林与埃兹蛋白的双重免疫荧光分析显示，在丝状伪足和褶皱膜中共定位。默林在富含肌动蛋白的皮质细胞骨架中的定位与埃兹蛋白 - 根蛋白 - 埃莫辛蛋白家族相对应，提示NF2蛋白通过与细胞骨架相关蛋白相互作用参与细胞生长调控。
