Fig. 7
From: Modulation of synaptic plasticity, motor unit physiology, and TDP-43 pathology by CHCHD10
Mutant CHCHD10 promotes mitochondrial TDP-43 aggregation and increases the size of TDP-43 aggregates in vitro. a Schematic of hTDP-43 mitochondria import and turnover assay for (b–e). HEK293T cells were transfected with Flag-CHCHD10WT or Flag-CHCHD10S59L and subjected to isolated of mitochondria. Human recombinant TDP-43 (hTDP-43) protein was imported into isolated mitochondria by incubation for 1 h at 33 °C, spun & washed, and chased for 0–12 h at 33 °C, after which mitochondria were lysed with RIPA to separate RIPA-soluble and RIPA-insoluble fractions. b Representative blots showing equal protein amounts of RIPA-soluble and RIPA-insoluble mitochondria immunoblotted for hTDP-43 and CHCHD10. c Quantification of recombinant hTDP-43 remaining in mitochondria over 12 h from RIPA-soluble fractions from Fig. (2b) (2-way ANOVA, group F(1, 3) = 9.69, P = 0.05: posthoc Sidak, *p < 0.05, **p < 0.01, n = 4/group). d Equal amounts of sonicated RIPA-insoluble fractions subjected to filter trap assay for hTDP-43. e Quantification of filter-trapped hTDP-43 aggregates from Fig. 2d classified by 1-4 h and 8-12 h time points (2-way ANOVA, group F(1, 8) = 12.07, P = 0.0084: posthoc Sidak, *p < 0.05, **p < 0.01, n = 4/group). f Recombinant CHCHD10WT or CHCHD10S59L protein incubated alone with vigorous shaking at 37 °C for 8 h and subjected to filter trap assay for CHCHD10. g Recombinant TDP-43 protein incubated alone or co-incubated with CHCHD10WT or CHCHD10.S59L protein with vigorous shaking at 37 °C for 8 h and subjected to filter trap assay for TDP-43. h–i Representative AFM images of recombinant proteins from Fig. 2f and g. Maximal particle height indicated by color-coded side bars. Arrows indicate relatively high-height protein particles per side bar scale. j Quantification of protein particle area among particles > 10 nm in height (1-way ANOVA, F(2, 182) = 51.36, P < 0.0001: posthoc Tukey, *p < 0.05, ##p < 0.0001, n = 57-67 particles/group). k Representative 3-dimensional reconstruction of AFM images showing particle contour, height, and area