Fig. 2: Generation of model of mitochondria-specific single misfolded/aggregated protein-induced proteotoxic stress, in human cells.

A Schematic representation of model stressor proteins, A53T α-synuclein, and PMD (Protein with Misfolded Domain)-fused to GFP, targeted to mitochondria. MTS denotes Mitochondrial Targeting Sequence. B Expression and localization of mitochondria-targeted A53T α-synuclein and PMD proteins in HeLa cells were checked by imaging using confocal microscopy. Mitochondria-targeted mCherry (Mito-mCherry) was used as a mitochondrial marker. Line-scan profile of fluorescence intensity of mitochondria-targeted GFP-tagged proteins (Mito-GFP, Mito-A53T Syn-GFP, and Mito-PMD-GFP) and Mito-mCherry are shown in green and red lines, respectively. C Co-localization analysis of GFP-tagged control/stressor protein with Mito-mCherry by Mander’s overlap shows more than 90% co-localization score of these stressor proteins to mitochondrial marker, exhibiting correct mitochondrial targeting. Values represent means ± SEM (N = 3). D, E Expression of both of these stressor proteins in mitochondria leads to increased mitochondrial fragmentation. However, PMD expression leads to significantly enhanced mitochondrial fragmentation, where the size of mitochondria is reduced to less than one-third of the length of healthy mitochondria. Values in panel E represent mitochondria mean branch lengths in µm ± SEM (N = 3). As data did not follow a normal distribution, a non-parametric Kruskal-Wallis test with Dunn’s multiple comparison test was performed to determine the mean differences in both plots C and E, ****P < 0.0001.