Figure 4.
TgNup503 depletion results in a specific mitochondrial morphology defect. (A) Representative SR microscopy images of mitochondria (stained with TgTom40 antibody, green) of wild type (normal) mitochondrial morphology and mitochondrial morphological defects with more compact, “stringy” shape (atypical). Left image of each morphology shows the mitochondria of four parasites and the right images show eight. (B) Quantification of mitochondrial morphology (using confocal microscopy images, where the mitochondria were immunostained with anti-TgTom40) in iHA-TgNup503 TgNup302-myc line upon TgNup503 depletion (+ATc) for 16 and 24 h (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test. (C) Quantification of mitochondrial morphology in parental line treated with ATc for 16 and 24 h as control (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test (NS). (D) Quantification of organelle morphology (apicoplast—anti-TgCPN60; IMC—anti-TgIMC1; microneme—anti-TgMic5; and rhoptries—anti-TgRop7) in iHA-TgNup503 TgNup302-myc line upon TgNup503 depletion (+ATc) for 16 and 24 h (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test (all NS). (E) Representative fluorescent microscopy images used for the quantification in F. Parasite lines iHA-TgNup503 TgNyp302-myc (top) and parental (bottom) were treated with ATc for 16 or 24 h and co-stained with anti-TgIMC1 (green) and the DNA dye DAPI (pink). (F) Quantification of nuclear area (marked by DAPI) relative to the total area of the parasite (marked by IMC1) in iHA-TgNup503 TgNup302-myc and parental lines (three biological replicates per time point, total of 150 parasites). Error bars display mean with SEM and data were analyzed with two-tailed unpaired T test. (G) iHA-TgNup503 TgNup302-myc and parental line were treated with ATc for 24 h and stained with TgENO2 to identify parasites where nuclear cytosolic shuttling is still functional and stained with mitotracker to visualize mitochondria and analyze their morphology. The graph shows the quantification of mitochondrial morphologies (two biological replicates, a total of 40 parasites, statistical analysis not carried out).

TgNup503 depletion results in a specific mitochondrial morphology defect. (A) Representative SR microscopy images of mitochondria (stained with TgTom40 antibody, green) of wild type (normal) mitochondrial morphology and mitochondrial morphological defects with more compact, “stringy” shape (atypical). Left image of each morphology shows the mitochondria of four parasites and the right images show eight. (B) Quantification of mitochondrial morphology (using confocal microscopy images, where the mitochondria were immunostained with anti-TgTom40) in iHA-TgNup503 TgNup302-myc line upon TgNup503 depletion (+ATc) for 16 and 24 h (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test. (C) Quantification of mitochondrial morphology in parental line treated with ATc for 16 and 24 h as control (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test (NS). (D) Quantification of organelle morphology (apicoplast—anti-TgCPN60; IMC—anti-TgIMC1; microneme—anti-TgMic5; and rhoptries—anti-TgRop7) in iHA-TgNup503 TgNup302-myc line upon TgNup503 depletion (+ATc) for 16 and 24 h (three biological replicates per time point, total of 150 parasites). Data were analyzed with two-sided Fisher’s exact test (all NS). (E) Representative fluorescent microscopy images used for the quantification in F. Parasite lines iHA-TgNup503 TgNyp302-myc (top) and parental (bottom) were treated with ATc for 16 or 24 h and co-stained with anti-TgIMC1 (green) and the DNA dye DAPI (pink). (F) Quantification of nuclear area (marked by DAPI) relative to the total area of the parasite (marked by IMC1) in iHA-TgNup503 TgNup302-myc and parental lines (three biological replicates per time point, total of 150 parasites). Error bars display mean with SEM and data were analyzed with two-tailed unpaired T test. (G) iHA-TgNup503 TgNup302-myc and parental line were treated with ATc for 24 h and stained with TgENO2 to identify parasites where nuclear cytosolic shuttling is still functional and stained with mitotracker to visualize mitochondria and analyze their morphology. The graph shows the quantification of mitochondrial morphologies (two biological replicates, a total of 40 parasites, statistical analysis not carried out).

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