Microscopic analysis of peroxisome dynamics during sucrose starvation. (A–D) BY-2 cells were subjected to sucrose starvation for 2 days in the absence (A,B; –3-MA) or presence of 3-MA (C,D; +3-MA). Note peroxisome clustering around the cell nucleus in the absence of 3-MA (at this magnification seen as green clots in the cell center). (E–H) Monodansylcadaverine (MDC)-positive organelles (E,F) and LysoTracker red (LTR)-positive organelles (G,H), both indicative of autolysosomes (Al), appeared in the cells and were detected by staining after sucrose starvation (32 h) in the absence of 3-MA. (I–L) To achieve autolysosome accumulation, BY-2 cells were subjected to sucrose starvation alone for 8 h and in the presence of E-64 for an additional 24 h. The arrow points to a subcellular aggregate of putative autolysosomes (I,J) around the cell nucleus and peroxisomes (K) with image overlay (L = J+K). (M–P) After 2 days of sucrose starvation and in the presence of ConcA autophagic bodies (Ab, in M) and EYFP fluorescent structures (N) were detected in the central vacuole (M,N). (O,P) Application of 3-MA inhibited ConcA-dependent autophagic body formation (O) and the appearance of EYFP fluorescent structures in the central vacuole (P). Images were taken with Nomarski optic (A,C,E,G,I,M,O) and epifluorescence (B,D,F,H,J,K,N,P). Scale bar: 20 μm.