Volume 20, Issue 1, Pages 60-71 (July 2016) Essential Role of the PfRh5/PfRipr/CyRPA Complex during Plasmodium falciparum Invasion of Erythrocytes  Jennifer C. Volz, Alan Yap, Xavier Sisquella, Jenn K. Thompson, Nicholas T.Y. Lim, Lachlan W. Whitehead, Lin Chen, Marko Lampe, Wai-Hong Tham, Danny Wilson, Thomas Nebl, Danushka Marapana, Tony Triglia, Wilson Wong, Kelly L. Rogers, Alan F. Cowman  Cell Host & Microbe  Volume 20, Issue 1, Pages 60-71 (July 2016) DOI: 10.1016/j.chom.2016.06.004 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 DiCre Recombinase-Mediated Gene Excision of pfripr and cyrpa (A) Southern blots showing excision of pfripr and cyrpa in RiprloxCre and CyRPAloxCre. (+) and (−) rapamycin (rap) over 48 (1), 96 (2), 144 (3), and 192 (4) hr. Top panel, RiprloxCre, “non-excised” band 7.1 kb, “excised” band 3.8 kb. Lower panel, CyRPAloxCre, “excised” band 10.1 kb, “non-excised” band 2.7 kb. (B) Densitometry of pfripr and cyrpa excision in RiprloxCre and CyRPAloxCre. (C) Conditional expression of PfRipr and CyRPA in RiprloxCre and CyRPAloxCre. RiprloxCre and CyRPAloxCre parasites (+) and (−) rapamycin (rap) over 48 (1), 96 (2), 144 (3), and 192 (4) hr. Lysates probed with anti-HSP70 as loading controls (lower panel). Asterisks: full-length PfRipr and processed bands. (D) Densitometric quantification of PfRipr and CyRPA expression in RiprloxCre1 and CyRPAloxCre in rapamycin over 48 (1), 96 (2), 144 (3), and 192 (4) hr. (E) RiprloxCre schizonts (−) or (+) rapamycin localized with anti-HA (green) and anti-RON4 (red). Scale bar, 5 μm. (F) CyRPAloxCre schizonts (−) or (+) rapamycin localized with anti-CyRPA (green) and anti-RON4 (red). Scale bar, 5 μm. (G and H) (G) RiprloxCre and (H) CyRPAloxCre schizonts (+R) or (−R) rapamycin. Expression of PfRipr or CyRPA was quantitated by determining mean pixel intensity. n = 40 for each track. ∗∗∗p < 0.001. Error bars indicate SD. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 PfRipr and CyRPA Are Required for P. falciparum Growth (A) Growth rate of Riprlox compared to RiprloxCre in rapamycin. Error bars indicate SD. (B) Growth rate of CyRPAloxCre in rapamycin or DMSO. Error bars indicate SD. (C) Invasion rate of merozoites from RiprloxCre compared to Riprlox, in rapamycin (Ripr-deficient). Invasion rate of CyRPAloxCre ± rapamycin (CyRPA-deficient). Error bars indicate SD. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Loss of PfRipr or CyRPA Function Blocks Merozoite Invasion (A) Time-lapse image of invasion following egress. Arrowhead indicates invading merozoite. Time in seconds. (B) Time-lapse images of PfRipr-deficient merozoites. A merozoite, indicated by arrowhead, in close proximity to an erythrocyte failed to invade (1, 2, and 3 are assigned to the same three merozoites). (C) Time-lapse image of CyRPA-deficient merozoites attempting to invade. Merozoite, indicated by arrowhead, in close proximity to an erythrocyte failed to invade (1, 2, 3, and 4 are assigned to the same four merozoites). (D) Percentage of Riprlox merozoites (+ rapamycin) that interact then invade erythrocytes (black). Of 196 merozoites followed 35 successfully invaded. Percentage of RiprloxCre merozoites (+ rapamycin) that interact and invade erythrocytes. 0/100, bottom number is total merozoites followed and top number the subset that invaded. Percentage of CyRPAloxCre merozoites (+ rapamycin) that interact and invade. 1/40, bottom number refers to total number of merozoites followed and top number subset invaded. (E) Percentage of merozoites that deformed the erythrocyte in merozoites contacting erythrocytes. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 PfRipr and CyRPA Function Are Required Downstream of Erythrocyte Deformation (A) Representative images of 3D7 merozoites invading erythrocytes loaded with Fluo-4AM and Draq5. Images are DIC with fluorescence overlayed. Blue, Draq5. Green, Flou-4AM. Merozoite deforms erythrocyte followed by a flash that spreads through the host cell. Shown are invasions occurring (in seconds). (B) Images of 3D7 merozoite invading Fluo-4AM-loaded erythrocyte. Erythrocyte membrane labeled with Bodipy TR Ceramide (red). Events are above image (secondns). 1 and 2 label two merozoites as they invade. (C) Images of CyRPAloxCre merozoite attempting to invade a Fluo-4AM-loaded erythrocyte. Erythrocyte membrane labeled with Bodipy TR Ceramide (red). Events are above each image (seconds). 1 shows merozoite attempting to invade. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 PfRipr and CyRPA Function Are Required for Ca2+ Flux during Merozoite Invasion (A) Plot of Ca2+ mean intensity versus time for 3D7 merozoite and erythrocyte. Ca2+ fluorescence in merozoite and erythrocyte are indiscernible at phase 2. Intensity normalized to compare across microscope platforms. (B) Plot of Ca2+ mean intensity over time for invading 3D7 (black) and RiprloxCre + rapamycin (yellow) and the erythrocyte. (C) Plot of Ca2+ mean intensity over time for 3D7 (black) and CyRPAloxCre + rapamycin (yellow) and the erythrocyte. (D) Percent inhibition of Ca2+ flux from merozoites attempting invasion. Percent Ca2+ flux shown for merozoites in presence of anti-PfRipr, anti-PfRh5, anti-basigin, soluble basigin, and R1 peptide. Shown are data for merozoites RiprloxCre, CyRPAloxCre, and AMA1loxCre that lack PfRipr, CyRPA, and AMA1 expression, respectively. Numbers are merozoites causing visible Ca2+ flux above the total number visualized. (E) Inhibition of invasion by anti-PfRh5, anti-PfRipr, anti-basigin, soluble basigin, and R1 peptide. Shown are normal IgG and PBS. (F) Decreased AMA1 expression in AMA1loxCre parasites + rapamycin. HSP70 expression levels are shown as control. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Subcellular Localization of PfRipr, CyRPA, and PfRh5 in Schizonts and Invading Merozoites (A) Top panel, overlay of CyRPA (green) and PfRipr (red) with DAPI and DIC fields. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI and DIC fields. Lower panel, overlay of PfRipr (green) and PfRh5 (red) with DAPI and DIC images. (B) Merozoites were mixed with erythrocytes and CyRPA, PfRipr, and PfRh5 visualized. Top panel, colocalization of CyRPA (green) and PfRipr (red) with DAPI and DIC images. Two merozoites invading a single erythrocyte. Middle panel, overlay of CyRPA (green) and PfRh5 (red) with DAPI and DIC fields with a single merozoite. Lower panel, overlay of PfRipr (green) and PfRh5 (red) with DAPI and DIC images. White bar indicates 5 μm. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 STED Super-Resolution Imaging of PfRipr, CyRPA, and PfRh5 on Merozoites Invading Erythrocytes (A) 2D STED of merozoites interacting with erythrocyte. Overlays of PfRh5 (green), PfRipr (green), CyRPA (green) colocalized with RON4 (red). DAPI is blue. RON4, marker of the apical end of merozoite. Erythrocyte membrane stained with WGA (gray). White bar indicates 1 μm. (B) Localization of PfRipr on merozoites invading an erythrocyte. Erythrocyte membrane stained with WGA (green). First panel, confocal image; second panel, visualization of PfRipr using 2D STED (maximum intensity projection) and overlay with the membrane marker. DAPI (blue). White bar indicates 1 μm. (C) Colocalization of PfRh5 (green), PfRipr (red), and CyRPA (green) on invading merozoites (z stacks). Erythrocyte membrane stained with WGA (gray). White bar indicates 1 μm. (D) Pearson’s correlation coefficient for colocalization of Ron4/PfRipr, PfRh5/PfRipr, and CyRPA/PfRipr. (E) Percent increase of colocalization at the apical end of the merozoite for PfRh5/PfRipr and CyRPA/PfRipr. Cell Host & Microbe 2016 20, 60-71DOI: (10.1016/j.chom.2016.06.004) Copyright © 2016 Elsevier Inc. Terms and Conditions