Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms

Abstract

Mitochondrial [Ca²⁺] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca²⁺ overload. This regulation depends on Ca²⁺ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca²⁺ uniporter (MCU). Here, we have studied the mitochondrial Ca²⁺ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca²⁺ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca²⁺] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca²⁺ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca²⁺ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca²⁺ pathway.

Keywords: C. elegans; MCU; calcium dynamics; knockout; mitochondria; mitochondrial calcium uniporter.

Figure 1

Mitochondrial [Ca²⁺] dynamics in the AQ3055 and mYCmcu-1 strains. Panels a and b show typical records of fluorescence ratio obtained in the AQ3055 control strain (panel (a)) and the mYCmcu-1 mutant strain, clone #1 (panel (b)). When indicated, carbachol (Cchol) was added to the chamber to reach a final concentration of 10 mM. The rectangles in the traces correspond to the region that has been amplified in panels c and d. Panels c and d show the traces corresponding to the two individual F535 and F480 fluorescences (in arbitrary units), and the F535/F480 ratio, both for the AQ3055 strain (panel (c)) and for the mYCmcu-1 strain (panel (d)). Panel (e) shows the mean peak width and mean peak height obtained in each strain by averaging data obtained from 8 AQ3055 worms (2015 peaks) and 6 mYCmcu-1 worms (1183 peaks). ***, p < 0.001, ANOVA test.

Figure 2

Electropharyngeograms (EPGs) obtained in N2 and mcu-1 mutants. Panels (a,b) show typical EPGs obtained either in control N2 worms or in mcu-1 mutants, respectively. Panel (c–f) shows the mean values of several parameters obtained from 3-min EPG records of 52 control N2 worms and 77 mcu-1 mutants. The meaning of the parameters is shown in panels (a,b) waves E (depolarization) and R (repolarization), pump duration and interpump interval (IPI). ***, p < 0.001; *, p < 0.05, ANOVA test.

Figure 3

Effect of carbachol on pharynx [Ca²⁺]_M in both the control AQ3055 strain and the mutant mcu-1 strain. Panel (a) shows the mean values of fluorescence ratio obtained from 8 experiments performed in control AQ3055 worms and 10 experiments performed in mcu-1 mutants. Panel (b) shows the statistical data on the percentage increase in the ratio induced by carbachol, and panel (c) shows the basal ratio in both strains. Comparisons made with ANOVA test. ***, p < 0.001.