ULK1 and 2 localize to the isolation membrane (phagophore) under starvation conditions. (A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min. They were then cultured in fresh complete medium for an additional 30 min (starvation→complete). (C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660–conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1. (E–H) Wild-type (E and G) and Atg5^−/− (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.

The kinase-dead ULK mutants inhibit autophagy. (A) In vitro kinase assay of endogenous ULK1. MEFs were cultured in complete or starvation medium for 60 min. ULK1 kinase activity was determined as described in Materials and methods. Relative kinase activity is shown. Data are the mean ± SE of five independent experiments. (B and C) NIH3T3 cells were transiently transfected with HA-ULK1, HA-ULK2, or their kinase-dead mutants and subjected to immunofluoresence microscopy using monoclonal anti-HA and polyclonal anti-Atg16L1 antibodies for primary staining and Alexa Fluor 488–conjugated goat anti–mouse IgG and Alexa Fluor 568–conjugated goat anti–rabbit IgG antibodies for secondary antibodies. Transfected cells are indicated by arrows. Bars, 20 μm. (D) NIH3T3 cells stably expressing GFP-ULK1^K46N and GFP-ULK2^K39T were cultured in starvation medium for 120 min. The cells were subjected to immunofluorescence microscopy using anti-Atg16L1 antibody. Bar, 20 μm. (E) NIH 3T3 cells were transfected with the retroviral vectors encoding HA-ULK1^K46N or with the corresponding empty retrovirus (mock). They were cultured in complete or starvation medium for 120 min. Cell lysates were then analyzed by immunoblot analysis with the indicated antibodies.

ULK1 interacts with FIP200. (A) HEK293T cells were cotransfected with FLAG-FIP200 and HA-ULKs. Cell lysates were subjected to immunoprecipitation (IP) using antibodies against FLAG. The resulting precipitates were examined by immunoblot (IB) analysis with the indicated antibodies. The asterisk indicates nonspecific band. (B) Lysates of MEFs were immunoprecipitated with anti-ULK1 or anti-FIP200 antibody or preimmune rabbit serum, and the resulting precipitates were subjected to immunoblot analysis with antibodies against ULK1 and FIP200. (C) Schematic representation of ULK1 mutants used in D. (D) HEK293T cells were cotransfected with FLAG-FIP200 and various ULK1 mutants and analyzed as in A using anti-HA and anti-FLAG antibodies. (E) NIH3T3 cells stably expressing GFP-ULK1 (left) and GFP-ULK1^ΔC (right) were cultured in starvation medium for 120 min. Bar, 20 μm.

FIP200 localizes to phagophore after starvation treatment. (A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed. (B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568–conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1. (C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568–conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.

FIP200 is required for autophagy. (A) FIP200^+/+, FIP200^−/−, Atg5^+/+, and Atg5^−/− MEFs were cultured in complete or starvation medium for 60 min. In the recovery experiments, starved MEFs were cultured in fresh complete medium for an additional 60 min (replenishment). The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) Wild-type and FIP200^−/− MEFs were cultured in the complete or starvation medium for indicated time with or without 100 nM bafilomycin A₁. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (C) Wild-type and FIP200^−/− MEFs were transfected with retroviral vectors encoding GFP-Atg5 or GFP-LC3. Resulting cells were cultured in the starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (D–G) Wild-type (D and E) and FIP200^−/− (F and G) MEFs were cultured in complete (D and F) or starvation (E and G) medium for 120 min and then fixed and subjected to EM analysis. Autophagosome-like structures (open arrowheads), and autolysosomes (closed arrowheads) are indicated. Bar, 1 μm. (H) The ratio of total area of autophagosomes (AP) and autolysosomes (AL) to total cytoplasmic area in D–G was determined by morphometric analysis.

FAK is not required for autophagy. (A) FAK^+/− and FAK^−/− MEFs were cultured in the complete or starvation medium for the indicated times with or without 100 nM bafilomycin A₁. The cell lysates were subjected to immunoblot analysis with the indicated antibodies. (B) FAK^+/− and FAK^−/− MEFs stably expressing GFP-LC3 were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. (C) Wild-type MEFs stably expressing GFP-FAK were cultured in complete or starvation medium for 120 min. The cells were fixed and subjected to immunofluorescence microscopy using anti-Atg16 antibody. Black squares indicate the enlarged areas shown in insets. Bars, 20 μm.

FIP200 functions downstream of mTOR in autophagosome formation. (A) Wild-type and FIP200^−/− MEFs were treated with 100 ng/ml rapamycin (rapa) or vehicle (DMSO) for 120 min in the presence or absence of 100 nM bafilomycin A₁. The cell lysates were subjected to immunoblot analysis with anti-LC3 antibody. (B) Wild-type and FIP200^−/− MEFs stably expressing GFP-Atg5 or GFP-LC3 were cultured in the presence of 100 ng/ml rapamycin for 120 min. The formation of GFP-Atg5 (top) and GFP-LC3 (bottom) puncta was examined by fluorescence microscopy. Bar, 20 μm. (C) Wild-type and FIP200^−/− MEFs were treated with 10 mM lithium chloride for 24 h or 100 μM C₂-ceramide for 2 h.

FIP200 is required for membrane targeting and proper function of ULK. (A) FIP200^+/− and FIP200^−/− MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (B) FIP200^+/+ and FIP200^−/− MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control). (C) Phosphatase sensitivity of ULK1. FIP200^+/+ and FIP200^−/− MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM Na₂VO₄, 50 mM KF, 15 mM Na₄P₂O₇, and 1 mM EGTA).