JCB: ARTICLE Lysosomal biogenesis and function is critical for necrotic cell death in Caenorhabditis elegans Marta Artal-Sanz, Chrysanthi Samara, Popi Syntichaki, and Nektarios Tavernarakis Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Heraklion 71110, Crete, Greece Y G O L O I B L L E C F O L A N R U O J E H T N ecrotic cell death is deﬁned by distinctive mor- phological characteristics that are displayed by dying cells (Walker, N.I., B.V. Harmon, G.C. Gobe, and J.F. Kerr. 1988. Methods Achiev. Exp. Pathol. 13:18–54). The cellular events that transpire during ne- crosis to generate these necrotic traits are poorly under- stood. Recent studies in the nematode Caenorhabditis elegans show that cytoplasmic acidiﬁcation develops during necrosis and is required for cell death (Syntichaki, P., C. Samara, and N. Tavernarakis. 2005. Curr. Biol. 15:1249–1254). However, the origin of cytoplasmic acid- iﬁcation remains elusive. We show that the alkalization of endosomal and lysosomal compartments ameliorates Introduction Previous studies implicate necrotic cell death in devastating hu- man pathologies such as stroke and neurodegenerative diseases (Walker et al., 1988; Martin, 2001). In Caenorhabditis elegans, specific mutations in several genes that encode ion channel subunits and regulators trigger the degeneration of specific sets of neurons (for review see Syntichaki and Tavernarakis, 2003). Dying neurons exhibit macroscopic and ultrastructural cha- racteristics that are reminiscent of the excitotoxic neuronal death that occurs during stroke in mammals (Hall et al., 1997; Lee et al., 1999; Nicotera et al., 1999). Thus, vertebrates and C. elegans share a death mechanism that involves the hyperacti- vation of ion channels. These observations are consistent with the hypothesis that a threshold of ion influx is needed to initiate the degenerative process. Perturbation of cellular ionic homeostasis contributes deci- sively to necrotic neuronal death (Syntichaki and Tavernarakis, 2003). In addition to ion homeostasis, intracellular pH has emerged as an important modulator of necrosis in C. elegans. Cytoplasmic acidification develops during necrosis, whereas the vacuolar H+-ATPase, which is a pump that acidifies lyso- somes, is required downstream of cytoplasmic calcium over- load to promote necrotic cell death (Syntichaki et al., 2005). Correspondence to Nektarios Tavernarakis: tavernarakis@imbb.forth.gr © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 173, No. 2, April 24, 2006 231–239 http://www.jcb.org/cgi/doi/10.1083/jcb.200511103 necrotic cell death triggered by diverse stimuli. In addi- tion, mutations in genes that result in altered lysosomal bio- genesis and function markedly affect neuronal necrosis. We used a genetically encoded ﬂuorescent marker to follow lysosome fate during neurodegeneration in vivo. Strikingly, we found that lysosomes fuse and localize ex- clusively around a swollen nucleus. In the advanced stages of cell death, the nucleus condenses and migrates toward the periphery of the cell, whereas green ﬂuorescent protein–labeled lysosomal membranes fade, indicating lysosomal rupture. Our ﬁndings demonstrate a prominent role for lysosomes in cellular destruction during necrotic cell death, which is likely conserved in metazoans. Interestingly, similar acidosis accompanies necrotic cell death after stroke in mammals (Sapolsky et al., 1996; Nicotera et al., 1999). Moreover, the examination of postmortem human brains associates neuronal pH alterations with several pathological and neurodegenerative states (Li et al., 2004). Investigations in both nematodes and mammals converge to implicate specific calpain and aspartyl proteases (cathepsins) in the execution of necrotic cell death (Syntichaki et al., 2002; Yoshida et al., 2002). Calpain proteases are normally dependent on calcium for activation, whereas aspartyl proteases require a highly acidic environment for full activity and are primarily confined to lysosomes and other acidic endosomal compartments (Ishidoh and Kominami, 2002; Goll et al., 2003). Studies in primates indicate that dam- age to the lysosomal membrane is inflicted enzymatically by activated calpains. Calpains localize to lysosomal membranes after the onset of ischemic episodes, with subsequent spillage of cathepsins to the cytoplasm (Yamashima et al., 2003). This observation led to the formulation of the “calpain–cathepsin hypothesis,” whereby the calcium-mediated activation of cal- pains results in the rupture of lysosomes and leakage of killer cathepsins that eventually dismantle the cell (Yamashima et al., 1998; Yamashima, 2000, 2004). Although these observations collectively indicate that lysosomes participate actively in the process of cell death, their contribution is poorly understood. JCB 231