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. 2022 Aug 8;13:4644. doi: 10.1038/s41467-022-32151-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic representation of BCC0 protein features. b Endogenously BCC0 tagged with spaghetti monster Myc tag (smMyc) co-stained for the centrosome (Centrin antiserum). DNA is stained with DAPI (blue) and applies to all panels with blue stain. The cartoons provide annotation and whole parasite perspective. c BCC0-smMyc colocalization with ISP1 antiserum. BCC0 at the daughter bud precedes ISP1. When ISP1 is deposited, BCC0 starts to elongate basally. Dotted lines outline parasites. d SR-SIM of BCC0-smMyc co-expressing endogenously tagged YFP-MORN1. Upon MORN1 appearance, BCC0 is present as 5–6 dots. Boxed regions in left panel correspond with the zoom panels. Asterisks mark mother BC; arrowhead marks the other centrosome and daughter bud in the middle parasite corresponding with box #3; ‘cc’ marks the centrocones (spindle poles); ‘api’ marks the apical end of this bud. Cartoon 1’ provides annotation of panel 1. Supplementary Movie 1 provides a 3D rendered rotation. e Airyscan images of BCC0-smMyc parasites co-stained with AAP4 antiserum (apical annuli). The ‘guide’ panel outlines 3 parasites in dotted lines (shades of blue); white arrowheads mark mother’s apical annuli; yellow arrowheads or ‘c’ mark centrosomes. f Plaque assays of inducible knock-down of BCC0 (BCC0-iKD) demonstrate that BCC0 is essential. The endogenous promoter of BCC0 was replaced with a TetO7sag4 anhydrous tetracycline (ATc) regulatable promoter fused to a Ty tag. TaTi-ΔKu80 is the parent line. Representative of n = 3 biological replicates. g BCC0-iKD parasites co-stained with Ty (BCC0) and IMC3 (IMC scaffolds) antisera. BCC0 depletion results in disorganized daughter IMC. h BCC0-iKD parasites co-stained with AAP4 and IMC3 antisera. BCC0 depletion misorganizes daughter annuli. White and yellow arrowheads mark maternal and daughter apical annuli, respectively. i BCC0-iKD parasites co-stained with endogenously expressed YFP-MORN1. White arrows and yellow arrowheads mark maternal and daughter BCs, respectively; orange arrowheads mark partial/broken daughter BC. Teal arrowheads mark MORN1 in the centrocone. j BCC0-iKD parasites co-stained with SAG1 (plasma membrane) and IMC3 antisera. Parasites continue expansion, but lose their shape. Some parasite nuclei are not encased by IMC.