Figure S1.
Super-resolution microscopy analysis of nuclear pore and outer mitochondrial membrane markers. (A) Localization of the nuclear pore components TgNup302 and TgNup503 via super-resolution (SR) microscopy. A T. gondii line with endogenously tagged TgNup503 and TgNup302 (iHA-TgNup503 TgNup302-myc) was immunostained for the respective tags (pink) and costained with DAPI (grey). (B–F) The same line used in A (iHA-TgNup503 TgNup302-myc) was immunostained with a combination of markers, imaged with SR microscopy, and signal colocalization within a parasite vacuole (Pearson’s coefficient) was calculated: (B)—TgNup302 (anti-myc, pink) and anti-TgMys (green) (three biological replicates, 45 vacuoles in total); (C)—TgNup302 (anti-myc pink) and anti-TgTom40 (green) (three biological replicates, 44 vacuoles in total); (D)—TgNup503 (anti-HA, pink) and anti-TgMys (green) (three biological replicates, 60 vacuoles in total); (E)—TgNup503 (anti-HA, pink) and anti-TgTom40 (green) (three biological replicates, 60 vacuoles in total); (F)—TgNup302 (anti-myc pink) and anti-TgCPN60 (green) (three biological replicates, 59 vacuoles in total). Error bars display mean with SEM (G) TgNup302 (anti-myc, pink) and anti-TgTom40 (green) colocalization was assessed in extracellular parasites (iHA-TgNup503 TgNup302-myc line) where naturally lysed parasites were collected and attached to poly-L-lysine coated coverslips followed by immunostaining, SR microscopy, and calculation of colocalization (Pearson’s coefficient) for each mitochondrial morphology: lasso, sperm-like, and collapsed (three biological replicates, 60 parasites in total per mitochondrial morphology). Error bars display mean with SEM and data were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test. (H and I)T. gondii immunostained for mitotracker (green) and costained with the pellicle marker IMC1 (H) or the cytosolic marker CDPK1 (I) (pink). (J) Immunofluorescence detection of TgNup302 via anti-Myc antibody (pink) costained with the DNA dye DAPI (grey), shows that TgNup302 remains nuclear upon TgNup503 depletion (24 h ATc). (K) Representative 3D reconstruction of SR images used for the colocalization measurements in M, costained with anti-TgTom40 (top) and anti-TgMys (bottom) (both green) and TgNup302 (pink). (L) TgNup302 signal volume was measured in untreated and treated (24 h) parasites (iHA-TgNup503 TgNup302-myc) from 3D-reconstruction SR images. Each measurement represents the average TgNup302 volume per parasite within a vacuole (three biological replicates, 75 measurements total). Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test (NS). (M) TgTom40 and TgMys signal volumes were measured in untreated and treated (24 h) parasites (iHA-TgNup503 TgNup302-myc) from 3D-reconstruction SR images (left graph for each marker). Each measurement represents the average volume per parasite within a vacuole (three biological replicates, 71–75 measurements total). TgTom40 and TgMys colocalization with TgNup302 (myc) was calculated (Pearson’s coefficient, three biological replicates with 75 measured vacuoles in total for each mitochondrial marker, right graph for each marker). Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test. (N) Western blots showing levels of TgNup302 (myc), TgMys, and TgTom40 upon TgNup503 downregulation (24 h ATc) with TgCDPK1 as a loading control. The signal was quantified using secondary fluorescent antibodies. Two or three biological replicates were performed per protein. Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test (NS). Source data are available for this figure: SourceData FS1.

Super-resolution microscopy analysis of nuclear pore and outer mitochondrial membrane markers. (A) Localization of the nuclear pore components TgNup302 and TgNup503 via super-resolution (SR) microscopy. A T. gondii line with endogenously tagged TgNup503 and TgNup302 (iHA-TgNup503 TgNup302-myc) was immunostained for the respective tags (pink) and costained with DAPI (grey). (B–F) The same line used in A (iHA-TgNup503 TgNup302-myc) was immunostained with a combination of markers, imaged with SR microscopy, and signal colocalization within a parasite vacuole (Pearson’s coefficient) was calculated: (B)—TgNup302 (anti-myc, pink) and anti-TgMys (green) (three biological replicates, 45 vacuoles in total); (C)—TgNup302 (anti-myc pink) and anti-TgTom40 (green) (three biological replicates, 44 vacuoles in total); (D)—TgNup503 (anti-HA, pink) and anti-TgMys (green) (three biological replicates, 60 vacuoles in total); (E)—TgNup503 (anti-HA, pink) and anti-TgTom40 (green) (three biological replicates, 60 vacuoles in total); (F)—TgNup302 (anti-myc pink) and anti-TgCPN60 (green) (three biological replicates, 59 vacuoles in total). Error bars display mean with SEM (G) TgNup302 (anti-myc, pink) and anti-TgTom40 (green) colocalization was assessed in extracellular parasites (iHA-TgNup503 TgNup302-myc line) where naturally lysed parasites were collected and attached to poly-L-lysine coated coverslips followed by immunostaining, SR microscopy, and calculation of colocalization (Pearson’s coefficient) for each mitochondrial morphology: lasso, sperm-like, and collapsed (three biological replicates, 60 parasites in total per mitochondrial morphology). Error bars display mean with SEM and data were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test. (H and I)T. gondii immunostained for mitotracker (green) and costained with the pellicle marker IMC1 (H) or the cytosolic marker CDPK1 (I) (pink). (J) Immunofluorescence detection of TgNup302 via anti-Myc antibody (pink) costained with the DNA dye DAPI (grey), shows that TgNup302 remains nuclear upon TgNup503 depletion (24 h ATc). (K) Representative 3D reconstruction of SR images used for the colocalization measurements in M, costained with anti-TgTom40 (top) and anti-TgMys (bottom) (both green) and TgNup302 (pink). (L) TgNup302 signal volume was measured in untreated and treated (24 h) parasites (iHA-TgNup503 TgNup302-myc) from 3D-reconstruction SR images. Each measurement represents the average TgNup302 volume per parasite within a vacuole (three biological replicates, 75 measurements total). Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test (NS). (M) TgTom40 and TgMys signal volumes were measured in untreated and treated (24 h) parasites (iHA-TgNup503 TgNup302-myc) from 3D-reconstruction SR images (left graph for each marker). Each measurement represents the average volume per parasite within a vacuole (three biological replicates, 71–75 measurements total). TgTom40 and TgMys colocalization with TgNup302 (myc) was calculated (Pearson’s coefficient, three biological replicates with 75 measured vacuoles in total for each mitochondrial marker, right graph for each marker). Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test. (N) Western blots showing levels of TgNup302 (myc), TgMys, and TgTom40 upon TgNup503 downregulation (24 h ATc) with TgCDPK1 as a loading control. The signal was quantified using secondary fluorescent antibodies. Two or three biological replicates were performed per protein. Error bars display mean with SEM. Data were analyzed with two-sided unpaired T test (NS). Source data are available for this figure: SourceData FS1.

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