Fig. 9. Localization of GFP-fused chimeric proteins in transfected COS7 cells. Protein domains are presented as in Fig. 3. All GFP fusion protein comprising ABDs (A-F) were colocalized with actin stress fibers (A,C,D,F, big arrows) and actin meshwork throughout the cell as well as with the cortical actin in lamellae (C,F, arrowheads). The GFP-ABD-S was enriched at the tips of stress fibers (B,arrow) and diminished at the plasmalemma (B), whereas the GFP-ABDsr1-2 (C) associated stronger with the submembrane actin. Nascent focal complexes did not attract GFP-ABD-S (small arrowheads). The amino acids AYKN at the C-terminus of the GFP-ABD-S protein were encoded by the additional exon 8a found by PCR analysis. GFP-ABDsr1-2 protein (C,F) harbors two spectrin repeats in addition to the ABD. GFP-sr15-21, corresponding to the middle part of NUANCE from spectrin repeat 15 to 20, seems to target intracellular membranes and vesicular structures colocalizing with ß-COP-positive structures (G,J, arrow) but not to the NE. By contrast, the GFP-Cterm1 protein harboring the TMD together with the two preceding spectrin repeats is recruited to the NE and adjacent ER (H). Note the lack of nuclear rim staining in the transfected cell (K, arrowhead). The GFP-Cterm2tm fusion protein is associated with vesicles but not with the NE (I,L, arrowheads). Cells transfected with the GFP-Cterm2tm construct were fixed with methanol (see details in the text). The transfected COS7 cells were double-labeled with TRITC-phalloidin (D), vinculin (E) and NUANCE (F,K,L) and ß-COP (J) mAbs. Bar, 20 µm.

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