Figure 4. Lysosomal morphology and distribution during neurodegeneration. (A) Confocal images of wild-type touch receptor neurons expressing a pmec-17 LMP-1::GFP transgene. LMP-1::GFP expression, dif- ferential interference contrast (DIC), and merged im- ages are shown. Healthy neurons show a scattered and punctate pattern of lysosomal distribution. (i) Wild- type PVM touch receptor neuron. (ii) Wild-type ALM (left and right) touch receptor neurons. (B) Confocal images of PLM touch receptor neurons of mec-4(d) animals expressing a pmec-17LMP-1::GFP transgene. During the early to middle (mid) stages of degene- ration, lysosomes enlarge and appear to coalesce around a swollen nucleus (i–iv). Later on, the nucleus migrates to the periphery of the cell and condenses (iv–vi). At the late stage, no lysosome structure is evident and the vacuolated cell becomes diffusely ﬂuo- rescent (vii and viii). (C) Acridine orange staining of a middle to late degenerating PLM touch receptor neuron. Acridine orange, DIC, and merged images are shown. Bars, 5 μm. degeneration in mammals (Yamashima et al., 2003). Diffusion of the highly localized GFP staining during the late stages of neurodegeneration indicates lysosome rupture. To further con- firm the loss of lysosomal integrity, we stained mec-4(d) ani- mals with acridine orange, which is an acidophilic dye that distinctively stains lysosomes. Acridine orange accumulates diffusely in the cytoplasm of neurons during the late stages of necrosis, indicating extensive cytoplasmic acidification (Fig. 4 C). This observation, coupled with the loss of specific LMP-1::GFP localization, suggests that lysosome rupture and the spillage of acidic lysosomal contents mediate cytoplasm acidification during necrosis. Figure 5. DAPI staining of mec-4(d) animals expressing pmec-17 LMP-1::GFP. (A) ALM touch receptor neuron. (B and C) PLM touch receptor neurons. Arrows point to DAPI-positive nuclei of the dying neurons. Bars, 5 μm. LYSOSOMAL FATE IN NECROTIC CELL DEATH • ARTAL-SANZ ET AL. 235