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. 2022 Sep 16;13:5445. doi: 10.1038/s41467-022-33108-z

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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 Live cell fluorescence microscopy of procyclic T. brucei AnTat 1.1E expressing FLAM8-YFP (green) and CARP3-mCherry (CARP3-mCh, red) (left), AnTat 1.1 carp3 KO cells expressing FLAM8-YFP (center) or AnTat 1.1E flam8 KO cells expressing CARP3-mCherry (right). Top: merge of fluorescent channels with DIC; middle: YFP; bottom: mCherry. b Indirect immunofluorescence analysis of CARP3 (green) in procyclic T. brucei AnTat 1.1E flam8 KO cells. DNA was stained with DAPI (blue), the axoneme is labeled in red (detected by the antibody mAB25). Top: merge of fluorescent channels; middle: CARP3 + DAPI; bottom: mAB25+DAPI. White arrowheads in (a) and (b) point towards flagellar tips; scale bars 5 µm. PALM imaging (c) and colocalization analysis (d) of CARP3-PAmCherry (PAmCh) and FLAM8-mNeonGreen (mNG) at a procyclic T. brucei AnTat 1.1E flagellar tip. CARP3 (d, left) and FLAM8 (d, right) localizations are color-coded according to their respective CBC values. e PALM imaging of CARP3-PAmCherry (PAmCh) and FLAM8-mNeonGreen (mNG) at a cross-section of a procyclic T. brucei AnTat 1.1E flagellar tip. Scale bars in (c) and (e) are 0.5 μm. f Colocalization analysis as in (d) for a flagellar tip cross section. g CBC histograms of CARP3-PAmCherry (red) and FLAM8-mNG (green) single molecule distributions calculated from the data shown in (c–f). n = 27 flagella. h SoMo assay of procyclic T. brucei AnTat 1.1E wild type (WT), flam8 KO (subclones A, B) or in situ FLAM8 rescue (resc) cells expressing a fluorescent triple marker⁹². i Distribution of CARP3 and FLAM8 during the parasite cycle in the tsetse fly. Immunofluorescence on methanol-fixed trypanosomes obtained from dissected tsetse tissues four weeks after infection. Anti-CARP3 (red), anti-FLAM8 (green), DAPI (blue). Flagellum regions in white boxes on bright field are magnified in the bottom panel. Scale bar 5 μm. PC procyclic, LT long mesocyclic trypomastigote, DE dividing epimastigote, LE long epimastigote, SE short epimastigote, AE attached epimastigote, MT metacyclic, OF old flagellum, NF new flagellum.