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. 2019 Mar 14;9:4466. doi: 10.1038/s41598-019-41084-6

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© The Author(s) 2019

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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Organisation of cytoskeleton and motor protein myosin in Acanthamoeba quina cysts revealed by confocal laser scanning microscopy. (A–L) Immature cyst double-labelled with anti-α-tubulin antibody and phalloidin-TRITC showing contractile vacuole (arrow, G) and formed ectocyst (arrowheads, E and H). (A–C) Confocal sections through a cell labelled for α-tubulin, showing an irregular microtubular network. (D) Distribution of microtubules in immature cyst. Merged data showing FITC (anti-α-tubulin) and UV (Hoechst) fluorescence channels. (E–G) Confocal sections through a cell with TRITC-phalloidin labelling displaying a diffuse and patchy distribution of F-actin throughout the entire cytoplasm. (H) Distribution of F-actin in an immature cyst. Merged data showing TRITC (phalloidin) and UV (Hoechst) fluorescence channels. (I–K) Merged confocal sections through a cell showing FITC (anti-α-tubulin), TRITC (phalloidin) and UV (Hoechst) fluorescence channels. (L) Distribution of microtubules and F-actin in an immature cyst. Merged data showing FITC (anti-α-tubulin), TRITC (phalloidin) and UV (Hoechst) fluorescence channels. (M–P) Mature cyst with completed cyst wall (arrowheads, M–P), double-labelled with anti-α-tubulin antibody and phalloidin-TRITC. (M) Merged confocal sections through a mature cyst showing patchy presence of F-actin. TRITC (phalloidin) fluorescence channel. (N) Merged confocal sections through a mature cyst showing absence of microtubules. FITC (anti-α-tubulin) fluorescence channel. (O) Merged confocal sections through a mature cyst showing FITC (anti-α-tubulin) and TRITC (phalloidin) fluorescence channels. (P) Distribution of F-actin in a mature cyst with negative labelling with anti-α-tubulin, showing FITC (anti-α-tubulin), TRITC (phalloidin) and UV (Hoechst) fluorescence channels. (Q–T) Mature cyst (arrowheads) double-labelled with anti-actin and anti-myosin antibodies. (Q) Mature cyst (arrowhead) lacking the monomeric form of actin, which is present in a trophozoites (arrows). Merged confocal sections showing FITC (anti-actin) fluorescence channel. (R) Mature cyst (arrowhead) with absence of myosin, which is present in a trophozoites (arrows). Merged confocal sections showing TRITC (anti-myosin) fluorescence channel. (S) Merged confocal sections through a mature cyst (arrowhead) and trophozoites (arrows) showing FITC (anti-actin) and TRITC (anti-myosin) fluorescence channels. (T) Double-labelled cyst (arrowhead) showing the absence of both myosin and monomeric form of actin, both of which are demonstrated in Acanthamoeba trophozoites (arrows), showing FITC (anti-actin), TRITC (anti-myosin) and UV (Hoechst) fluorescence channels. In figures (D,H,I–L,P,T) the localisation of nuclei is displayed by Hoechst. The distance between the confocal sections in (A–C,E–G) and (I–K) is 0.5 μm. The accumulation of data in each of the figures (D,H,L) originated from 30 optical sections taken at distances of 0.5 μm. The accumulation of data in each of the figures (M–T) originated from 15 optical sections taken at distances of 1.0 μm. Bars, 10 μm.