This article explores the use of expansion microscopy, a technique that enhances resolution in fluorescence microscopy, on the autotrophic protist Euglena gracilis A modified protocol was developed to preserve the cell structures during fixation. Using antibodies against key cytoskeletal and organelle markers, α-tubulin, β-ATPase, and Rubisco activase, the microtubular structures, mitochondria, and chloroplasts were visualised. The organisation of the cytoskeleton corresponded to the findings from electron microscopy while allowing for the visualisation of the flagellar pocket in its entirety and revealing previously unnoticed details. This study offered insights into the shape and development of mitochondria and chloroplasts under varying conditions, such as culture ages and light cycles. This work demonstrated that expansion microscopy is a robust tool for visualising cellular structures in E. gracilis, an organism whose internal structures cannot be stained using standard immunofluorescence because of its complex pellicle. This technique also serves as a complement to electron microscopy, facilitating tomographic reconstructions in a routine fashion.

https ror org 024d6js02 Faculty of Science Department of Parasitology BIOCEV Charles University Vestec Czech Republic

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Bertiaux E, Balestra AC, Bournonville L, Louvel V, Maco B, Soldati-Favre D, Brochet M, Guichard P, Hamel V (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria parasites including the conservation of a conoid. PLoS Biol 19: e3001020–e3001022. 10.1371/journal.pbio.3001020 PubMed DOI PMC

Bricheux G, Brugerolle G (1987) The pellicular complex of euglenoids: II. A biochemical and immunological comparative study of major epiplasmic proteins. Protoplasma 140: 43–54. 10.1007/BF01273254 DOI

Cavalier-Smith T (2017) Euglenoid pellicle morphogenesis and evolution in light of comparative ultrastructure and trypanosomatid biology: Semi-conservative microtubule/strip duplication, strip shaping and transformation. Eur J Protistol 61: 137–179. 10.1016/j.ejop.2017.09.002 PubMed DOI

Chen F, Tillberg PW, Boyden ES (2015) Optical imaging. Expansion microscopy. Science 347: 543–548. 10.1126/SCIENCE.1260088 PubMed DOI PMC

Chen Z, Dong Y, Duan S, He J, Qin H, Bian C, Chen Z, Liu C, Zheng C, Du M, et al. (2024) A chromosome-level genome assembly for the paramylon-producing microalga Euglena gracilis. Sci Data 11: 780. 10.1038/s41597-024-03404-y PubMed DOI PMC

Cordoba J, Perez E, Van Vlierberghe M, Bertrand AR, Lupo V, Cardol P, Baurain D (2021) De novo transcriptome meta-assembly of the mixotrophic freshwater microalga Euglena gracilis. Genes 12: 842. 10.3390/genes12060842 PubMed DOI PMC

Cramer M, Myers J (1952) Growth and photosynthetic characteristics of Euglena gracilis. Arch Mikrobiol 17: 384–402. 10.1007/BF00410835 DOI

Dubreuil RR, Bouck GB (1988) Interrelationships among the plasma membrane, the membrane skeleton and surface form in a unicellular flagellate. Protoplasma 143: 150–164. 10.1007/BF01291159 DOI

Ebenezer TE, Zoltner M, Burrell A, Nenarokova A, Novák Vanclová AMG, Prasad B, Soukal P, Santana-Molina C, O’Neill E, Nankissoor NN, et al. (2019) Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol 17: 11–23. 10.1186/s12915-019-0626-8 PubMed DOI PMC

Ebenezer TE, Low RS, O’Neill EC, Huang I, DeSimone A, Farrow SC, Field RA, Ginger ML, Guerrero SA, Hammond M, et al. (2022) Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world. Biol Open 11: bio059561. 10.1242/BIO.059561 PubMed DOI PMC

Esson HJ, Leander BS (2006) A model for the morphogenesis of strip reduction patterns in phototrophic euglenids: Evidence for heterochrony in pellicle evolution. Evol Dev 8: 378–388. 10.1111/j.1525-142X.2006.00110.x PubMed DOI

Farmer MA, Triemer RE (1988) Flagellar systems in the euglenoid flagellates. BioSystems 21: 283–291. 10.1016/0303-2647(88)90024-X PubMed DOI

Gambarotto D, Zwettler FU, Guennec ML, Schmidt-Cernohorska M, Fortun D, Borgers S, Heine J, Schloetel JG, Reuss M, Unser M, et al. (2019) Imaging cellular ultrastructures using expansion microscopy (U- ExM). Nat Methods 16: 71–74. 10.1038/s41592-018-0238-1 PubMed DOI PMC

Gorilak P, Pružincová M, Vachova H, Olšinová M, Schmidt Cernohorska M, Varga V (2021) Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites. Open Biol 11: 210131. 10.1098/rsob.210131 PubMed DOI PMC

Halpern AR, Alas GCM, Chozinski TJ, Paredez AR, Vaughan JC (2017) Hybrid structured illumination expansion microscopy reveals microbial cytoskeleton organization. ACS Nano 11: 12677–12686. 10.1021/acsnano.7b07200 PubMed DOI PMC

Hammond MJ, Nenarokova A, Butenko A, Zoltner M, Dobáková EL, Field MC, Lukeš J (2020) A uniquely complex mitochondrial proteome from Euglena gracilis. Mol Biol Evol 37: 2173–2191. 10.1093/molbev/msaa061 PubMed DOI PMC

Hammond M, Zoltner M, Garrigan J, Butterfield E, Varga V, Lukeš J, Field MC (2021) The distinctive flagellar proteome of Euglena gracilis illuminates the complexities of protistan flagella adaptation. New Phytol 232: 1323–1336. 10.1111/nph.17638 PubMed DOI

Heiss AA, Fang Y, Peña-Diaz P, Zítek J, Novák J, Šmída A, Zelená M, Švagr E, Hampl V (2024) Striated fibre assemblins localise in the feeding groove of the ‘typical excavate’ Paratrimastix pyriformis. BioRxiv. 10.1101/2024.03.29.587336 (Preprint posted March 29, 2024). DOI

Hümpfer N, Thielhorn R, Ewers H (2024) Expanding boundaries - a cell biologist’s guide to expansion microscopy. J Cell Sci 137: jcs260765. 10.1242/jcs.260765 PubMed DOI PMC

Hutner SH, Zahalsky AC, Aaronson S, Baker H, Frank O (1966) Chapter 8 culture media for Euglena gracilispter. Methods Cell Biol 2: 217–228. 10.1016/S0091-679X(08)62140-8 DOI

Kivic PA, Walne PL (1984) An evaluation of a possible phylogenetic relationship between the euglenophyta and kinetoplastida. Orig Life 13: 269–288. 10.1007/BF00927177 DOI

Ku T, Swaney J, Park JY, Albanese A, Murray E, Cho JH, Park YG, Mangena V, Chen J, Chung K (2016) Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues. Nat Biotechnol 34: 973–981. 10.1038/nbt.3641 PubMed DOI PMC

Leander BS, Esson HJ, Breglia SA (2007) Macroevolution of complex cytoskeletal systems in euglenids. Bioessays 29: 987–1000. 10.1002/bies.20645 PubMed DOI

Makki A, Kereïche S, Le T, Kučerová J, Rada P, Žárský V, Hrdý I, Tachezy J (2024) A hybrid TIM complex mediates protein import into hydrogenosomes of Trichomonas vaginalis. BMC Biol 22: 130. 10.1186/s12915-024-01928-8 PubMed DOI PMC

Mermelstein CS, Rodrigues APM, Einicker-Lamas M, Navarrete REDB, Farina M, Costa ML (1998) Distribution of F-actin, alpha-actinin, tropomyosin, tubulin and organelles in Euglena gracilis by immunofluorescence microscopy. Tissue Cell 30: 545–553. 10.1016/S0040-8166(98)80035-9 PubMed DOI

Mikus F, Ramos AR, Shah H, Olivetta M, Borgers S, Hellgoth J, Saint-Donat C, Araújo M, Bhickta C, Cherek Pet al. (2024) Charting the landscape of cytoskeletal diversity in microbial eukaryotes. BioRxiv. 10.1101/2024.10.18.618984 (Preprint posted October 18, 2024). DOI

Nasir A, Le Bail A, Daiker V, Klima J, Richter P, Lebert M (2018) Identification of a flagellar protein implicated in the gravitaxis in the flagellate Euglena gracilis. Sci Rep 8: 7605. 10.1038/s41598-018-26046-8 PubMed DOI PMC

Novák Vanclová AMG, Zoltner M, Kelly S, Soukal P, Záhonová K, Füssy Z, Ebenezer TE, Lacová Dobáková E, Eliáš M, Lukeš J, et al. (2020) Metabolic quirks and the colourful history of the Euglena gracilis secondary plastid. New Phytol 225: 1578–1592. 10.1111/nph.16237 PubMed DOI

O’Neill EC, Trick M, Hill L, Rejzek M, Dusi RG, Hamilton CJ, Zimba PV, Henrissat B, Field RA (2015) The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry. Mol Biosyst 11: 2808–2820. 10.1039/c5mb00319a PubMed DOI

Pellegrini M (1980a) Three-dimensional reconstruction of organelles in Euglena gracilis Z. I. Qualitative and quantitative changes of chloroplasts and mitochondrial reticulum in synchronous photoautotrophic culture. J Cell Sci 43: 137–166. 10.1242/jcs.43.1.137 PubMed DOI

Pellegrini M (1980b) Three-dimensional reconstruction of organelles in Euglena gracilis Z. II. Qualitative and quantitative changes of chloroplasts and mitochondrial reticulum in synchronous cultures during bleaching. J Cell Sci 46: 313–340. 10.1242/jcs.46.1.313 PubMed DOI

Piccinni E, Mammi M (1978) Motor apparatus of Euglena gracilis: Ultrastructure of the basal portion of the flagellum and the paraflagellar body. Boll Zoologia 45: 405–414. 10.1080/11250007809440150 DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. (2012) Fiji: An open-source platform for biological-image analysis. Nat Methods 9: 676–682. 10.1038/NMETH.2019 PubMed DOI PMC

Schwartzbach SD, Shigeoka S (2017) Photo and Nutritional Regulation of Euglena Organelle Development, Vol. 979, pp 159–182. Cham: Springer. PubMed

Shin W, Brosnan S, Triemer RE (2002) Are cytoplasmic pockets (MTR/pocket) present in all photosynthetic euglenoid genera? J Phycology 38: 790–799. 10.1046/j.1529-8817.2002.01208.x DOI

Simpson AGB (1997) The identity and composition of the euglenozoa. Archiv für Protistenkunde 148: 318–328. 10.1016/S0003-9365(97)80012-7 DOI

Sommer JR (1965) The ultrastructure of the pellicle complex of Euglena gracilis. J Cell Biol 24: 253–257. 10.1083/jcb.24.2.253 PubMed DOI PMC

Surek B, Melkonian M (1986) A cryptic cytostome is present in Euglena. Protoplasma 133: 39–49. 10.1007/BF01293186 DOI

Willey RL, Wibel RG (1985) A cytostome/cytopharynx in green euglenoid flagellates (Euglenales) and its phylogenetic implications. BioSystems 18: 369–376. 10.1016/0303-2647(85)90036-X PubMed DOI

Yoshida Y, Tomiyama T, Maruta T, Tomita M, Ishikawa T, Arakawa K (2016) De novo assembly and comparative transcriptome analysis of Euglena gracilis in response to anaerobic conditions. BMC Genomics 17: 182. 10.1186/s12864-016-2540-6 PubMed DOI PMC