Folia Microbiol. 52 (4), 381–390 (2007) http://www.biomed.cas.cz/mbu/folia/ Alterations in Mitochondrial Morphology of Schizosaccharomyces pombe Induced by Cell-Death Promoting Agents V. PEVALA, J. KOLAROV, P. POLČIC* Department of Biochemistry, Faculty of Science, Comenius University, 842 15 Bratislava, Slovakia e-mail polcic@fns.uniba.sk Received 5 December 2006 Revised version 23 April 2007 ABSTRACT. The effect of the yeast cell-death inducing agents, Bax and acetic acid, on mitochondrial structure of Schizosaccharomyces pombe was studied. Comparison of mitochondrial structures in cells grown on different substrates and visualized with different probes revealed variations in their morphology. Cells grown on respiratory C sources as well as in the presence of antimycin A exhibited punctuated mitochondria when visualized with mitochondrially targeted green fluorescent protein, while they still appeared as tubular structures when stained with DiOC 6 (3). Both expression of Bax and acetic acid treatment induced fragment- ation and aggregation of mitochondrial network, which could be prevented by coexpression of Bcl-XL. Aberrant mitochondrial morphology generated by either Bax or acetic acid was not accompanied with the loss of mitochondrial genome (mtDNA), indicating that alterations of mitochondrial morphology following death stimuli follow different mechanisms than those involved in mitochondrial inheritance mutants. Abbreviations DAPI 4´,6-diamidino-2-phenylindole MT mitochondrial(ly) DiOC 6 (3) 3-hexyl-2-[3˝-(3´-hexyl-2´(3´H)benzoxazolylidene)- mtGFP MT-targeted green fluorescent protein 1˝-propenyl]benzoxazolium iodide (see formula) NCS nonfermentable carbon source(s) FCS fermentable carbon source(s) MT morphology is dictated by the equilibrium between fusion and fission of mitochondria, two processes that occur in unicellular as well as in multicellular organisms (Okamoto and Shaw 2005). There is increasing evidence that the MT dynamic behavior is critical for many cellular functions including MT inheritance (Rapaport et al. 1998; Nunnari et al. 1997; Jones and Fangman 1992), energy transduction (Sku- lachev 2001), cellular aging (Ono et al. 2001), and apoptosis (Karbowski et al. 2002; Frank et al. 2001). In yeast, the MT fusion and fission are under the control of a set of nuclear genes, the mutations in which affect both the MT inheritance and morphology (Jensen 2005; Bleazard et al. 1999; Guan et al. 1993; Jones and Fangman, 1992). When these genes, including DNM1, MGM1, MDM10, MDM12, MMM1, MMM2, are deleted in Saccharomyces cerevisiae, MT dynamics is inpaired and mitochondria lose their DNA (mtDNA) but the mutant cells are viable on a FCS. The corresponding mutations in Schizosaccharomyces pombe are lethal because the cells cannot survive when they lose their mtDNA (Pelloquin et al. 1998, 1999). MT dynamics also differs between S. cerevisiae and S. pombe with respect to the cytoskeletal elements in- volved, which are microtubules in S. pombe and mammals (Yaffe et al. 1996) rather than actin and inter- mediate filaments in S. cerevisiae (Boldogh et al. 2004; Boldogh and Pon 2001). The mechanisms that influence MT morphogenesis in mammals may thus be closer to those in S. pombe than in S. cerevisiae. Yeast MT fission proteins have been implicated in apoptosis-like cell death, supporting a primordial origin of programmed cell death involving mitochondria (Fannjiang et al. 2004). Apoptosis-like cell death in yeast can be induced by various agents (reviewed in Madeo et al. 2004) including expression of mammalian pro-apoptotic protein Bax (Zha et al. 1996) and treatment with acetic acid (Ludovico et al. 2001). Given the link between MT dynamics and programmed cell death, we investigated the impact of both death stimuli on the MT morphology in S. pombe. We next investigated how the MT morphology and mtDNA are maintained upon cell death induction. In order to examine MT morphology by fluorescence microscopy and to find the optimal approach to visualization of mitochondria we first compared the MT phenotypes of cells grown on different carbon sources using two different florescent probes, MT-targeted GFP and DiOC 6 (3). The former probe consists of protein (GFP) imported to MT matrix by MT protein *Corresponding author.