Fig. 9
Cell Signaling and Adhesion in Drosophila S2 Cells upon PrP Expression (A–E) Expression of mouse PrP (m PrP [A]), zebrafish PrP-1 (zf PrP-1 [B]) and PrP-2 (zf PrP-2 [C]), Xenopus PrP (xen PrP [D]), and chick PrP (ch PrP [E]) EGFP fusion constructs in Drosophila nonadhesive S2 cells results in the induction of cell–cell contact formation and local PrP accumulation at cell contacts (white arrowheads). (F) Control EGFP fusion constructs do not induce this phenomenon (shown for m PrP at a fortuitous PrP-independent cell contact). (G) Cell contact formation and PrP accumulation (white arrowheads) in mixed S2 cell populations separately transfected with mouse EGFP- and DsRed-monomer-PrP constructs (G) exclude cell division artifacts. (H) The same result is obtained when mouse DsRed-monomer-PrP and zebrafish EGFP-PrP-2 constructs are used, suggesting PrP interaction across species. (I) Blocking of mouse PrP–mediated aggregation of S2 cells (red arrows, lower panel) by a polyclonal antibody against mouse PrP (M-20) shows that the formation of cell clusters (white arrowheads, upper left panel) is specifically induced by PrP. The effect was quantified as the number of cell contacts between S2 cells expressing mouse PrP in the absence (upper left) or presence (upper right) of M-20 or a control antibody at the concentrations indicated in the graph (double asterisks [**] indicate statistical significance at p < 0.01, one-way ANOVA test; error bars indicate SEM). (J–O) Strong anti–phospho-Src kinase immunostaining ([J] α-pSrc), as well as accumulation of rat reggie-1-DsRed-monomer ([L] reggie-1) and Alexa-568 Phalloidin ([N] F-actin) colocalize at mo PrP–mediated cell contacts (white arrowheads), but not at fortuitous PrP-independent cell contacts (K, M, and O). Scale bars in (A–H and J–O) indicate 5 μm; scale bar in (I) indicates 20 μm. |