(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green). (B–D) GFP–Atg8, overexpressed using the Dmef2–Gal4 driver, labels autophagosomes. Dmef2–Gal4, UAS–GFP–Atg8 animals were fed on high-nutrient food (B), starved on low-nutrient food for 6 h (C), or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (D). GFP–Atg8-labeled vesicles appeared only in the starved animals (C–D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP–Atg8-labeled vesicles. (E–G) Dmef2–Gal4, UAS–GFP–Atg8/UAS–HRP–Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP–Atg8 and HRP–Lamp1-labeled vesicles. (F) Colocalization of GFP–Atg8 and HRP–Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP–Atg8 and HRP–Lamp-labeled vesicles, but they failed to colocalize. (H) Quantification of the number of GFP–Atg8, HRP–Lamp1, or GFP–Atg8+HRP–Lamp1 vesicles in starved or starved +CQ muscles. (I–M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2–Gal4, UAS–GFP–Atg8/UAS–Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP–Atg8-labeled autophagosomes (compare I–J to C–D). (K–L) Dmef2–Gal4, UAS–GFP–Atg8, Atg1Δ3d larvae failed to form GFP–Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2–Gal4, UAS–GFP–Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS–Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP–Atg8 vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals. (N–O) EM of muscles from Dmef2–Gal4, UAS–whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N). (P) CQ treatment increased the larval crawling time of Dmef2–Gal4, UAS–whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2–Gal4, UAS–whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).