Fig. 4: TMBIM5 and MICU1 are found in the same macromolecular complex and their deficiency results in similar changes in mitochondrial shape and inner boundary membrane.

a Blue native gel electrophoresis of TMBIM5, MCU and MICU1 in human HAP1 (left) and HEK293 cells with (wild type, WT) and without TMBIM5 (knockout, KO) expression. Approximate molecular weight is indicated. b Co-immunoprecipitation of TMBIM5 with HA-tagged MICU1 (arrow). Left panel shows successful pull-down of tagged MICU1 and the right panel coimmunoprecipitation of endogenous TMBIM5. Molecular weight is indicated. c/c’ Morphometric analysis of TMRM-labeled mitochondria in WT, TMBIM5 (T5) KO, and MICU1 (M1) KO HEK cells imaged with structured illumination microscopy. C’ shows typical pictures, size is indicated. d/d’ Evaluation of mitochondrial membrane potential (ΔΨm) gradients of WT, T5KO, and M1KO HEK cells using structured illumination microscopy. d’ illustrates the findings. Vertical lines in D indicate the TMRM concentration threshold for half-maximal IBM association index changes. Lower thresholds reflect more even membrane potential distribution between cristae and inner boundary membrane; higher thresholds indicate greater potential differences between these compartments. Data in (c) are presented as box and whisker plots with the box representing the interquartile range, spanning from the 25th to the 75th percentile. Whiskers extend from the minimum to the maximum data points. Each data point represents a single cell accumulated from 4 independent experiments. Data in (d) show the mean ± standard errors of mean. Statistical significance in c was determined using the Kruskal-Wallis test followed by Dunn’s multiple comparisons test, p values are indicated.