Figure 3.

Some adPEO Twinkle mutants alter the localization of the protein in mtDNA nucleoids. The subcellular localization of the Twinkle variants was determined by immunofluorescence. While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria, the disease variants K319E Twinkle variants localized only partially with nucleoids, a proportion of the protein was diffusely localized in the mitochondrial lumen, while the nucleoids often appeared enlarged (similar results were observed with the R374Q and dup 352–364 mutants; Supplementary Material, Fig. S2). The W474C variant caused aggregation of nucleoids without a more diffuse distribution of the protein, while the A359T mutation decreased the Twinkle localization in nucleoids without altering their size and number extensively. The effects of expression of the various mutants were categorized as mild, moderate and severe as indicated in the Figure. Cells were induced with 3 ng/ml doxycycline for 3 days, immunocytochemistry was performed using specific antibodies against the myc-tagged Twinkle variants (green) and DNA (red). Images were taken with the same settings for exposure, laser intensity, etc. and in addition were processed for brightness and contrast in the same manner. All mutants are shown in Supplementary Material, Fig. S2.