American Society of Hematology

Figure 2.

$Figure 2. Stage-specific expression and localization of K13 in relation to ER-PI3P, PfBiP, and PfEMP1. (A) Custom antibodies to K13 (supplemental Methods) detect an 83-kDa band in Pf3D7-infected red cells (IRs) but not uninfected red cells (URs) in Western blots (molecular weights in kDa) and localize K13 (green) by IFA in trophozoite and schizont stages (counterstained for host band 3, red), as imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope using DeltaVision Deconvolution microscopy.25 (B-C) Cryo-IEM of PfNF54K13WT dually probed for PI3P (6 nm gold) and ER marker BiP (15 nm gold) (B) or K13 (15 nm gold) (C). Black arrow indicates PI3P in lumen of ER tubule; yellow arrows indicate cytoplasmic PI3P; double yellow arrows indicate PI3P vesicular clusters outside of ER tubules on the cytoplasmic face; red arrow indicates low level of PI3P vesicles devoid of K13. Scale bar, 100 nm. (D) Membrane association of K13. Lysates of Pf3D7 were treated as indicated, separated by centrifugation (15 000 g for 30 min) into membrane pellet and soluble supernatant fractions and probed in Western blots for parasite and host (human) markers. Adding 6 M urea (a strong chaotropic agent) for 30 min at 23°C to parasite cell lysates failed to release K13 from the pellet (of parasite cell lysates), although the cytosolic parasite protein PfFKBP was quantitatively detected in the soluble fraction, suggesting that K13 was membrane associated. Sodium carbonate 100 mM, pH 11.5, for 30 min on ice released K13 from the pellet, suggesting that it was peripherally (but not integrally) associated with membranes (and consistently, K13 was also released by 1% Tx-100 for 30 min at room temperature or 1% SDS for 30 min at room temperature). Human band 3 was only released by 1% Triton or 1% SDS, confirming that it was integrally membrane associated. Molecular weight standards (in kDa) are as shown. (E) Stereological analyses of K13-gold particle distribution by cryo-IEM. Vesicles close to the ER appear to bud from ER tubules. Vesicles distal to the ER may be derived from other organellar membranes and cannot be ascribed solely to the ER. (F) IFA single optical sections localizing K13 in segmenter, merozoite, and ring stages. Scale bars are as shown. (G) Anti-ATS antibodies recognize a band >250 kDa in Western blots (as was expected for PfEMP1) in IRs but not URs. Molecular weight standards (in kDa) are as shown. (H) IFA showing single optical sections colocalizing K13 and PfEMP1 (labeled by anti-ATS) in Pf3D7 segmenter and merozoites. Pearson’s correlation coefficients are as indicated. Experimental replicates, n = 3. Scale bars are as indicated. Parasite nucleus (blue) is stained with Hoechst 33242. HuBand3, Human band 3; P, pellet; PC, Pearson’s correlation coefficient; S, supernatant.$

Stage-specific expression and localization of K13 in relation to ER-PI3P, PfBiP, and PfEMP1. (A) Custom antibodies to K13 (supplemental Methods) detect an 83-kDa band in Pf3D7-infected red cells (IRs) but not uninfected red cells (URs) in Western blots (molecular weights in kDa) and localize K13 (green) by IFA in trophozoite and schizont stages (counterstained for host band 3, red), as imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope using DeltaVision Deconvolution microscopy.²⁵ (B-C) Cryo-IEM of PfNF54K13WT dually probed for PI3P (6 nm gold) and ER marker BiP (15 nm gold) (B) or K13 (15 nm gold) (C). Black arrow indicates PI3P in lumen of ER tubule; yellow arrows indicate cytoplasmic PI3P; double yellow arrows indicate PI3P vesicular clusters outside of ER tubules on the cytoplasmic face; red arrow indicates low level of PI3P vesicles devoid of K13. Scale bar, 100 nm. (D) Membrane association of K13. Lysates of Pf3D7 were treated as indicated, separated by centrifugation (15 000 g for 30 min) into membrane pellet and soluble supernatant fractions and probed in Western blots for parasite and host (human) markers. Adding 6 M urea (a strong chaotropic agent) for 30 min at 23°C to parasite cell lysates failed to release K13 from the pellet (of parasite cell lysates), although the cytosolic parasite protein PfFKBP was quantitatively detected in the soluble fraction, suggesting that K13 was membrane associated. Sodium carbonate 100 mM, pH 11.5, for 30 min on ice released K13 from the pellet, suggesting that it was peripherally (but not integrally) associated with membranes (and consistently, K13 was also released by 1% Tx-100 for 30 min at room temperature or 1% SDS for 30 min at room temperature). Human band 3 was only released by 1% Triton or 1% SDS, confirming that it was integrally membrane associated. Molecular weight standards (in kDa) are as shown. (E) Stereological analyses of K13-gold particle distribution by cryo-IEM. Vesicles close to the ER appear to bud from ER tubules. Vesicles distal to the ER may be derived from other organellar membranes and cannot be ascribed solely to the ER. (F) IFA single optical sections localizing K13 in segmenter, merozoite, and ring stages. Scale bars are as shown. (G) Anti-ATS antibodies recognize a band >250 kDa in Western blots (as was expected for PfEMP1) in IRs but not URs. Molecular weight standards (in kDa) are as shown. (H) IFA showing single optical sections colocalizing K13 and PfEMP1 (labeled by anti-ATS) in Pf3D7 segmenter and merozoites. Pearson’s correlation coefficients are as indicated. Experimental replicates, n = 3. Scale bars are as indicated. Parasite nucleus (blue) is stained with Hoechst 33242. HuBand3, Human band 3; P, pellet; PC, Pearson’s correlation coefficient; S, supernatant.

This Feature Is Available To Subscribers Only