Vps34 dynamics during omegasome formation. (A) Clone 201 cells were transfected with mRFP-Vps34, and a stable population expressing both proteins was selected (last three lanes). Note that exogenous mRFP-Vps34 was comparable to endogenous Vps34 and stable during amino acid starvation. (B) Cells as in A were starved for 60 min, fixed, and stained for Lamp-2. Note that DFCP1 punctate structures are frequently in proximity to Vps34 membranes (arrows on the bottom left) and that the majority but not all of Lamp-2 vesicles (∼80%) colocalize with Vps34, whereas all of the Vps34 vesicles colocalize with Lamp-2 (arrows on the bottom right indicate examples of Lamp-2 vesicles devoid of Vps34). (C) Selected frames from live imaging of mRFP-Vps34 in cells also expressing CFP-ER. Note that the Vps34 vesicle is in constant proximity to the ER and frequently appears to use the ER strands to move long distances (arrows on the bottom). The times refer to the period after amino acid starvation, but similar types of movement are evident without starvation. (D and E) Selected frames from live imaging of mRFP-Vps34, GFP-DFCP1, and CFP-ER during amino-acid starvation. (D) An omegasome being formed in constant and close proximity to a Vps34 particle. Bar, 1 μm. For selected frames of this video, as indicated (10–12 and 35–37), E shows the relationship of Vps34 to DFCP1 (top), Vps34 to ER (middle), and all three (bottom). Also see Video 7 . (F) An additional example from live imaging experiments showing an omegasome forming in close proximity to a Vps34 particle.