Yeast Yeast 2007; 24: 731–739. Published online 14 June 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/yea.1508 Research Article On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae Ann Zahle Andersen*, Allan K. Poulsen, Jens Christian Brasen and Lars Folke Olsen University of Southern Denmark, Department of Biochemistry and Molecular Biology, CelCom, Campusvej 55, 5230 Odense M, Denmark *Correspondence to: Ann Zahle Andersen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, CelCom, Campusvej 55, 5230 Odense M, Denmark. E-mail: zahle@bmb.sdu.dk Received: 26 February 2007 Accepted: 27 April 2007 Abstract We employed the fluorescent cyanine dye DiOC 2 (3) to measure membrane poten- tial in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicoti- namide adenine dinucleotide (NADH). We found that the mitochondrial membrane potential was oscillating, and that these oscillations displayed the same frequency and duration as the NADH oscillations. It was confirmed that DiOC 2 (3) localizes itself in the mitochondrial membrane and thus reports qualitative changes solely in mitochon- drial membrane potential. Our studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolariza- tion of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane potential oscillations and delocalized DiOC 2 (3), while glycolysis continued to oscillate unaffected. Copyright  2007 John Wiley & Sons, Ltd. Keywords: oscillations; glycolysis; mitochondria; membrane potential; cyanine dye Introduction Glycolytic oscillations in a suspension of intact cells of Saccharomyces cerevisiae have been known for several decades; for a review on oscillatory behaviour of yeast cells, see Richard (2003). Yeast cells in a high-density suspension are able to syn- chronize glycolysis, most likely by pulsatile secre- tion of acetaldehyde (Richard et al., 1994, 1996b; Dano et al., 1999; Poulsen et al., 2004, 2007), and each individual cell oscillates in phase with the cell population. The oscillations are monitored rou- tinely by fluorescence measurements of the natu- rally abundant fluorophores NADH and NADPH (Richard et al., 1993), where the NAD + /NADH couple participates in redox reactions in gly- colysis. NADH and NADPH cannot be distin- guished by their fluorescence but only NADH is found to oscillate in yeast, while NADPH remains constant (Richard et al., 1993) during glycolytic oscillations. From here on we therefore refer only to NADH. Glycolytic oscillations are evoked under anaero- bic (Poulsen et al., 2004) or semi-anaerobic condi- tions by the addition of cyanide (Betz and Chance, 1965a). Hence, oxidative phosphorylation in mito- chondria is shut down before oscillations appear. However, whether the mitochondria still have a controlling role with respect to glycolytic oscilla- tions has been subject to speculation. According to Aon et al. (1991), depolarization of the mitochondrial membrane annihilated cyanide- induced oscillations. The authors found that it was not possible to induce a train of glycolytic oscilla- tions when yeast cells were preincubated with car- bonyl cyanide p -(trifluoromethoxy)phenylhydra- zone (FCCP), which discharges the electrochemi- cal proton gradient across the mitochondrial mem- brane. They therefore concluded that mitochon- drial function played a role in the triggering and Copyright  2007 John Wiley & Sons, Ltd.