Figure 2
Akt-induced structures are PM invaginations (a) Fluorescence microscopy of two fields showing four characteristic FM4-64-treated YPH499 yeast cells cultured in SR-Gal expressing GFP-Akt1 from pYES2-GFP-Akt1 and p110α from YCpLG-p110α. (b) Staining of the PM with FM4-64 upon inhibition of endocytosis on either control cells expressing kinase-dead (K179M mutant, upper panel) or wild type GFP-Akt1 (lower panel), both from pYES2-GFP-based plasmids, and co-expressing p110α from YCpLG-p110α. Cells were induced for expression as in (a), but incubation with FM4-64 was done at 0 °C in the presence of NaN3 and NaF. (c) Co-localization of Akt with the PS marker GFP-C2(Lact). Triple transformants bearing pRS410-GFP-LactC2, YCpLG-p110α-CAAX (or a kinase-dead K802R version as a negative control for Akt1 activation; upper panel), and pYES3-mCherry-Akt3 were induced and analyzed as in (a). All scale bars indicate 5 μm.
