Figure 5.

PIP₂ conducts membrane delivery to Atg16L1 precursors. (A) HeLa cells transiently expressing CFP-FRB and either mRFP-FKBP or mRFP-FKBP-5ase for 20 h were treated as indicated with 2.5 µM rapamycin and incubated for 30 min with CTX subunit B conjugated to Alexa Fluor 488 (CTX-488). Cells were fixed and subjected to confocal microscopy. Representative confocal pictures are shown. (B) The data represent the means ± SD of Atg16L1-CTX colocalization (Pearson’s coefficient) from A. n > 30 cells for each condition. Arrows indicate Atg16L1-CTX colocalization, whereas the arrowhead indicates the absence of colocalization between Atg16L1 and CTX. (C) HeLa cells transfected with two rounds of control, Arf6, or PIP5K siRNA for 5 d were transfected during the last 20 h with Atg16L1-GFP. Cells were incubated for 30 min with CTX subunit B conjugated to Alexa Fluor 555 (CTX), fixed, and subjected to confocal microscopy analysis. The data represent the means ± SD of Atg16L1-CTX colocalization (Pearson’s coefficient). n > 30 cells for each condition. Higher magnifications of the colocalizations are shown in the insets. kd, knockdown. Bars, 5 µm.