FIGURE 5.

Akt recruitment to lysosomes is accompanied by Golgi disassembly and nuclear condensation. (A) HeLa Kyoto cells were co-transfected with a trans-Golgi fluorescent marker (TGN38-FRB-CFP, magenta) and either WT-YFP or Akt1-C344S-YFP (yellow). Dissociation of the typical Golgi structure into vesicles can be seen in cells displaying Akt puncta. DAPI was used to reveal cell nuclei (blue); scale bar, 5 μm. (B) Akt puncta (green) do not colocalize with Golgi (magenta). (C) Puncta formation in Akt1-C344S cells is accompanied by cell blebbing, nuclear condensation and fluorescence loss (yellow numbers indicate the percentage of yellow fluorescence remaining in the cell at each time point, compared to t = 0). HeLa Kyoto cells constitutively expressing H2B-mCherry (blue) were transfected with a plasmid coding for Akt1-C344S-YFP (yellow). Live cells were imaged every 20 min. Every time point (minutes, white numbers), we acquired a z-stack with 3 z-steps of 1 μm in 2 different wavelengths (RFP, exposure: 0.2 s and YFP, exposure: 0.5 s). Bar, 10 μm. (D) Akt1-C344S cells displaying Akt puncta show reduced nuclear area compared to WT Akt1-YFP expressing cells (p < 0.01 according to a Student’s t-test).