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. 2020 Jan 17;9:445. doi: 10.3389/fcimb.2019.00445

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Copyright © 2020 Chasen, Coppens and Etheridge.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Overexpressed CP1-mNeon-Ty Localizes to the SPC. (A) Vector map of the CP1-pTREX overexpression plasmid with a common detrimental mutation of the neomycin cassette restored (i). This fixed neomycin cassette allows for harsh G418 selection of epimastigotes at up to 2,000 μg/mL. (B) SR-SIM microscopy of a CP1-mNeon epimastigote after the endocytosis of BSA-Rhodamine shows that BSA and CP1-mNeon label the same endocytic pathway. (C) Treating epimastigotes with cytochalasin B during the assay prevented endocytosis via the SPC, causing BSA to strongly label the pre-oral ridge in front of the cytostome (arrow). (D) SR-SIM of amastigotes expressing CP1-mNeon-Ty shows SPC labeling. (E) SR-SIM images of epimastigotes expressing CP1-mNeon-Ty, showing SPC labeling. Labeling at 4°C with Concanavalin A - Rhodamine was used to identify the entrance to the SPC (arrow). Scale bars: 2 μm.