Mislocalization of mitochondria and compromised renal function and oxidative stress resistance in Drosophila SesB mutants

Abstract

Mitochondria accumulate at sites of intense metabolic activity within cells, but the adaptive value of this placement is not clear. In Drosophila, sesB encodes the ubiquitous isoform of adenine nucleotide translocase (ANT, the mitochondrial inner membrane ATP/ADP exchanger); null alleles are lethal, whereas hypomorphic alleles display sensitivity to a range of stressors. In the adult renal tubule, which is densely packed with mitochondria and hence enriched for sesB, both hypomorphic alleles and RNA interference knockdowns cause the mitochondria to lose their highly polarized distribution in the tissue and to become rounded. Basal cytoplasmic and mitochondrial calcium levels are both increased, and neuropeptide calcium response compromised, with concomitant defects in fluid secretion. The remaining mitochondria in sesB mutants are overactive and maintain depleted cellular ATP levels while generating higher levels of hydrogen peroxide than normal. When sesB expression is knocked down in just tubule principal cells, the survival of the whole organism upon oxidative stress is reduced, implying a limiting role for the tubule in homeostatic response to stressors. The physiological impacts of defective ANT expression are thus widespread and diverse.

Fig. 1.

Mitochondrial distribution in tubules from wild-type and sesB mutants. For orientation, arrows indicate apical mitochondria (yellow), basolateral mitochondria (white), principal cell nuclei (blue), and the tubule lumen (red) in A. A: immunocytochemistry using anti-sesB rabbit polyclonal antibody and anti-rabbit IgG-FITC conjugate reveal mitochondrial sesB protein localization in the main, fluid-transporting segment of the Malpighian tubule. B–F: expression of mitochondrial GFP reporter targeted to principal cells in adult tubules using the c42-GAL4 driver. B: control (c42mtGFP); C: SesB knockdown (c42mtGFP > sesB RNAi); D: SesB¹ mutants (c42mtGFP > sesB¹). In A–D, tubule diameter is taken as 30 μm. E–G: enlargement of basolateral plane, showing rod-like and branching normal mitochondria from c42mtGFP (E), compared with small globular mitochondria from c42mtGFP > sesB RNAi (F), and c42mtGFP > sesB¹ tubules (G). In E–G, scale bar represents 1 μm.

Fig. 2.

SesB knockdowns increase basal calcium levels and blocks neuropeptide stimulation. A, B: expression of sesB RNA interference (RNAi) was targeted to principal cells in adult tubules expressing either the cytosolic (A) or mitochondrial (B) targeted calcium reporter, apoaequorin. Tubules were stimulated with the neuropeptide Capa-1 (10⁻⁷ M, arrowed), and the typical calcium response shown in black. Data are expressed as mean [Ca²⁺] (nM) ± SE; n > 10, P < 0.05, compared with wild-type tubules. C: bar graphs represent cytosolic and mitochondrial basal [Ca²⁺] from tubule principal cells overexpressing targeted sesB RNAi, average [Ca²⁺] (nM); n = 6, where ***P < 0.0001, Student's t-test. D: expression of sesB RNAi was targeted to stellate cells in adult tubules expressing the cytosolic calcium reporter. Tubules were stimulated with the neuropeptide Drosophila leucokinin (Drosokinin, 10⁻⁷ M, arrowed). Overexpression of sesB RNAi resulted in a significantly decreased Drosokinin calcium response.

Fig. 3.

Cellular ATP is modulated by sesB gene expression. Pools of 20 tubules were assayed for ATP content. Data are shown as means ± SE (n = 6). Significant difference *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.

Impact of sesB mutations on tubule function. A: fluid transport by Drosophila renal tubule is significantly decreased in hypomorphic sesB¹ allele when the tubule is maximally stimulated by application of the neuropeptides Capa-1(10⁻⁷ M) and Drosokinin (10⁻⁷ M). *P < 0.05. B: tubules, in which UAS-sesB RNAi was driven by hs-GAL4, were stimulated at 30 min by the addition of Capa-1 and subsequently at 60 min with Drosokinin (arrows) cannot sustain high rates of stimulated fluid transport compared with wild type.

Fig. 5.

Mitochondria are overactive in sesB mutants. A, B: imaging of live tubules using mitochondrial potential-sensitive dyes. Activated mitochondria, in which the inner membrane potential is hyperpolarized, accumulate red fluorescence with JC-1 (48). A: mitochondrial activity in tubules from hs-GAL4 flies. B: note the increase in the red fluorescence compared with A in basal surface of tubule principal cells from hs-GAL4 > UAS-sesB RNAi flies.

Fig. 6.

Hydrogen peroxide (H₂O₂) production in tubules and in isolated mitochondria. A: detection of H₂O₂ in Malpighian tubules. Tubules from c42mtGFP > UAS-sesB RNAi and hs-GAL4 > UAS-sesB RNAi flies (heat-shocked) show increased H₂O₂ production compared with all tested genotypes. Data are shown as mean μM H₂O₂ ± SE (n = 6), where *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. B: detection of H₂O₂ production in mitochondria. Mitochondria from heat-shocked hs-GAL4 > UAS-sesB RNAi flies show increased H₂O₂ production compared with parental lines. Data are shown as mean μM H₂O₂ ± SE (n = 3), where ***P < 0.001, Student's t-test.

Fig. 7.

Expression of sesBs exclusively in tubule principal cells modulates survival of the whole fly under oxidative stress. A, B: survival of flies after ingestion of H₂O₂. A: survival of flies in which sesB-1 and sesB-2 were targeted to tubule principal cells. B: survival of flies in which sesB RNAi was targeted to tubule principal cells and from male viable hypomorphic allele of sesB¹. Outcrossed parental lines are shown in gray (UAS-sesB-1, UAS-sesB-2, and UAS-sesB RNAi) or black (UO-GAL4 > WhCS) and were generated to control for genetic background of the sesB transgenics stocks. Data are expressed as numbers of surviving flies up to 150 h ± SE, n > 30 flies per vial, repeated 3 times for each genotype.