Pax6 intercellular transfer is blocked by extracellular anti-Pax6 antibodies. (a) Sequence alignment of Pax6 and En2 homeodomains. Consensus amino acids are indicated in green letters between the two sequences; alpha helices are shaded. Amino acids involved in secretion and internalization are underlined (in red and blue, respectively). (b-d) COS-7 cells were transfected with a Pax6 expressing plasmid and co-cultured with HeLa cells marked by a GFP-H2b fusion. Pax6 transfer was monitored in every optical field by quantifying the Pax6 signal (overall signal shown in (b)) only in GFP-expressing HeLa cells (shown in (c)), giving (in (d)) the transfer signal as described in Materials and methods. Scale bar = 60 μm. (e,f) Subcellular localization of single-chains aP6 (e) or spaP6 (f) transiently expressed in COS-7 cells. Schematic representations of the antibodies are presented: V-k, kappa chain variable region; l, linker; V-H, heavy chain variable region; hm, his-myc tag. Whereas aP6 is detected throughout the cytoplasm and in the nucleus, spaP6 is restricted to vesicular structures and the Golgi apparatus (asterix). Scale bar = 10 μm. (g,h) Inhibition of Pax6 intercellular transfer. (g) COS-7 cells expressing Pax6 were cultured together with HeLa cells in medium supplemented with anti-myc hybridoma (Hamyc) or anti-Pax6 hybridoma (HaP6). (h) COS-7 cells co-expressing Pax6 and secreted single-chain antibodies against either Engrailed (spaEn) or Pax6 (spaP6) were cultured together with HeLa cells. In (g,h), Pax6 transfer to HeLa cells (analyzed as described in Materials and methods, in three independent experiments) was significantly reduced by hybridoma addition (p < 0.015) or single-chain co-expression (p < 0.005).