Scrib is not localized in focal adhesions in CV-1 and MDCKII cells, and is dispensable for targeting LPP to these structures
We have shown before that LPP is localized in cell-cell contacts [11] and also for human Scrib, it was shown that it is localized in these structures [17] (also shown in Fig. 3C and 5, upper right panel). Since LPP is not only localized in cell-cell contacts but also in focal adhesions [11,13], we investigated whether also Scrib had the ability to localize at these structures. For this purpose, we used two different cell lines: the epithelial cell line MDCKII and the fibroblast cell line CV-1. However, in contrast to LPP, Scrib could not be detected in focal adhesions as shown by staining CV-1 cells with Scrib-472 antibodies (Fig. 4, upper left panel). Identical results were obtained in MDCKII cells (results not shown). Focal adhesions were indeed present, as these structures could be stained using vinculin antibodies used as a marker for focal adhesions (CV-1 cells: Fig. 4, upper right panel; MDCKII cells: results not shown). If Scrib had been present in focal adhesions, we would have detected it there, because, as shown in Fig. 3A, Scrib is highly expressed in CV-1 as well as in MDCKII cells, and as shown in Fig. 3C, Scrib-472 antibodies are able to detect Scrib in its native conformation in fixed cells. Moreover, a hScrib-GFP protein expressed in CV-1 or MDCKII cells was never detected in focal adhesions (results not shown) but was localized in cell-cell contacts (MDCKII cells: Fig. 5, lower left panel). The nature of the nuclear staining observed in CV1-cells stained with the Scrib-472 antibody (Fig. 4, upper left panel) is aspecific, as it is also obtained with the corresponding pre-immuneserum. In addition, nuclear staining was never obtained when an hScrib-GFP protein was transiently overexpressed in these cells (results not shown). Nuclear staining was also not detected in MDCKII cells as shown in Fig. 3C and 5, upper right panel. These results indicate that, in contrast to LPP, which is localized both in focal adhesions and in cell-cell contacts in CV-1 and MDCKII cells, Scrib is only localized in cell-cell contacts but not in focal adhesions in these cells.
Figure 4  Scrib is not localized in focal adhesions in CV-1 cells, and is dispensable for targeting LPP to these structures. Upper panels: CV-1 cells, grown on glass coverslips, were double labelled with Scrib-472 antibodies (left panel) and anti-vinculin antibodies (right panel) used as a marker for focal adhesions. Lower panels: CV-1 cells were transiently transfected with wild-type human LPP (left panel), or LPP with a mutated carboxy-terminus (T610A) (right panel), as GFP-fusions. GFP-fluorescence was visualized by epifluorescence microscopy.
Figure 5  Scrib and LPP are localized in cell-cell contacts but are dispensable for targeting each other to these structures. Upper panels: MDCKII cells, grown on glass coverslips, were double labelled with anti-LPP antibodies (left panel) and anti-Scrib antibodies (right panel). Lower panels: MDCKII stable cell lines, expressing GFP-fusion proteins containing wild-type human LPP (upper left panel), LPP with a mutated carboxy-terminus (T610A) (upper right panel), human wild-type Scrib (lower left panel), or Scrib with a deletion of all its PDZ domains (lower right panel), were grown on glass coverslips (Scrib) or on Transwell-Clear polyester membranes (LPP). GFP-fluorescence was visualized by epifluorescence microscopy (Scrib) or by confocal microscopy (LPP).   As deduced from these results, we hypothesized that Scrib was not involved in targeting LPP to focal adhesions. Indeed, evidence for this hypothesis was obtained by transfecting CV-1 cells with a construct expressing GFP-LPPWT containing full length wild-type LPP, or GFP-LPPT610A, which is identical to GFP-LPPWT except for a point mutation to alanine introduced at threonine610, which abolishes binding to Scrib. No difference in focal adhesion localization could be detected between wild-type and mutated GFP-LPP fusion proteins (Fig. 4, lower panels).