Fig. 2: Drosophila Tmbim5 knockout impairs wing posture, mobility, and lifespan with phenotypes resembling mammalian models: altered cristae, mitochondrial swelling, and reduced Ca²⁺ buffering capacity.

a Tmbim5 expression assessed by quantitative PCR in wild type (WT, w¹¹¹⁸), heterozygous (KO/+), homozygous (KO/KO), and hemizygous (KO/Y) knockout flies demonstrating loss of expression. Each data point contains five 4-7-day-old flies. b Abnormal wing posture in 6-week-old male KO/Y flies. Each data point contains the mean percentage of flies with normal wings in a sample of 25 flies. c Climbing ability of WT and KO/Y flies on the indicated days post-eclosion with an age-dependent decline in KO/Y. Each data point contains the mean speed of 25 flies. d Reduced lifespan of KO/Y flies, sample size >100 flies/group. Mild ubiquitous expression of Tmbim5 using daughterless (da)-gal4 but not mitoGFP in KO/Y flies confirmed that the lifespan defect was specifically due to Tmbim5 loss. e Ubiquitously reduced ATP levels in 7-day-old male Tmbim5 knockout flies (KO/Y). Values were normalized to protein content. f Exemplary transmission electron microscopy images showing a disrupted cristae architecture of flight muscle mitochondria in 7-day-old KO/Y flies. Red arrows and green shading indicate mitochondria, asterisks mark indirect flight muscle. Size is indicated. g Larger delta in absorbance following osmotic shrinking with polyethylene glycol (PEG, added where indicated) in 7-day-old Tmbim5 KO mitochondria indicating swelling. Values were normalized to the last values after PEG-addition. Baseline absorbance and the ratio of absorbance before and after shrinking are quantified on the right. Data are shown as mean ± SEM, n = 9. h Reduced mitochondrial calcium buffering capacity in 7-day-old KO/Y flies shown by quantifying Ca²⁺ uptake of isolated mitochondria in a bath with Calcium Green 5 N challenged by sequential addition of 5 µM CaCl₂ pulses. Fluorescence was normalized to the initial value (F₀). Data are shown as mean ± SEM, n = 5-9. Data are represented as box and whisker plots with the box representing the interquartile range, spanning from the 25th to the 75th percentile. A horizontal line within the box indicates the median. Whiskers extend from the minimum to the maximum data points. Each data point in A represented the mean value of 5 flies, in (b, c) 25 flies, in (e) 2 flies, in (g and h) 100 flies. Statistical significance was calculated by Šídák’s multiple comparisons test in A, the Mann Whitney test in B, unpaired t tests in (c, e, g). p-values are indicated.