Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

Charles University Faculty of Science Albertov 6 Prague 128 00 Czech Republic

IMCF at BIOCEV Faculty of Science Charles University Průmyslová 595 252 50 Vestec Czech Republic

Laboratory of Adaptive Immunity Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czech Republic

Laboratory of Cell Motility Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 Prague 14220 Czech Republic

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Matthews KR. 2005. The developmental cell biology of Trypanosoma brucei. J. Cell Sci. 118, 283-290. (10.1242/jcs.01649) PubMed DOI PMC

Gull K. 1999. The cytoskeleton of trypanosomatid parasites. Annu. Rev. Microbiol. 53, 629-655. (10.1146/annurev.micro.53.1.629) PubMed DOI

Hayes P, Varga V, Olego-Fernandez S, Sunter J, Ginger ML, Gull K. 2014. Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology. J. Cell Biol. 206, 377-384. (10.1083/jcb.201312067) PubMed DOI PMC

Kohl L, Robinson D, Bastin P. 2003. Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes. EMBO J. 22, 5336-5346. (10.1093/emboj/cdg518) PubMed DOI PMC

Sherwin T, Gull K. 1989. The cell division cycle of Trypanosoma brucei brucei: timing of event markers and cytoskeletal modulations. Phil. Trans. R. Soc. Lond. B 323, 573-588. (10.1098/rstb.1989.0037) PubMed DOI

Tam J, Merino D. 2015. Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods. J. Neurochem. 135, 643-658. (10.1111/jnc.13257) PubMed DOI

Lacomble S, Vaughan S, Gadelha C, Morphew MK, Shaw MK, Mcintosh JR, Gull K. 2009. Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography. J. Cell Sci. 122, 1081-1090. (10.1242/jcs.045740) PubMed DOI PMC

Wheeler RJ, Sunter JD, Gull K. 2016. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone. J. Cell Sci. 129, 854-867. PubMed PMC

Chen F, Tillberg PW, Boyden ES. 2015. Expansion microscopy. Science 347, 543-548. (10.1126/science.1260088) PubMed DOI PMC

Gambarotto D, 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

Ku T, et al. 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

Tillberg PW, et al. 2016. Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies. Nat. Biotechnol. 34, 987-992. (10.1038/nbt.3625) PubMed DOI PMC

Zhao Y, et al. 2017. Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy. Nat. Biotechnol. 35, 757-764. (10.1038/nbt.3892) 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, 12 677-12 686. (10.1021/acsnano.7b07200) PubMed DOI PMC

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. (10.1371/journal.pbio.3001020) PubMed DOI PMC

Amodeo S, Kalichava A, Fradera-Sola A, Bertiaux-Lequoy E, Guichard P, Butter F, Ochsenreiter T. 2021. Characterization of the novel mitochondrial genome segregation factor TAP110 in Trypanosoma brucei. J. Cell Sci. 134, jcs254300. (10.1242/jcs.254300) PubMed DOI PMC

Büttner M, Lagerholm CB, Waithe D, Galiani S, Schliebs W, Erdmann R, Eggeling C, Reglinski K. 2020. Challenges of using expansion microscopy for super-resolved imaging of cellular organelles. ChemBioChem cbic 22, 686-693. (doi:0.1002/cbic.202000571) PubMed PMC

Pernal SP, et al. 2020. Nanoscale imaging using differential expansion microscopy. Histochem. Cell Biol. 153, 469-480. (10.1007/s00418-020-01869-7) PubMed DOI

Woods A, Sherwin T, Sasse R, Macrae TH, Baines AJ, Gull K. 1989. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93, 491-500. (10.1242/jcs.93.3.491) PubMed DOI

Birkett CR, Foster KE, Johnson L, Gull K. 1985. Use of monoclonal antibodies to analyse the expression of a multi-tubulin family. FEBS Lett. 187, 211-218. (10.1016/0014-5793(85)81244-8) PubMed DOI

Kohl L, Sherwin T, Gull K. 1999. Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle. J. Eukaryot. Microbiol. 46, 105-109. (10.1111/j.1550-7408.1999.tb04592.x) PubMed DOI

Pradel LC, Bonhivers M, Landrein N, Robinson DR. 2006. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei. J. Cell Sci. 119, 1852-1863. (10.1242/jcs.02900) PubMed DOI

Bringaud F, Robinson DR, Barradeau S, Biteau N, Baltz D, Baltz T. 2000. Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition. Mol. Biochem. Parasitol. 111, 283-297. (10.1016/S0166-6851(00)00319-4) PubMed DOI

Abeywickrema M, et al. 2019. Non-equivalence in old- and new-flagellum daughter cells of a proliferative division in Trypanosoma brucei. Mol. Microbiol. 112, 1024-1040. (10.1111/mmi.14345) PubMed DOI PMC

Alizadehrad D, Krüger T, Engstler M, Stark H. 2015. Simulating the complex cell design of Trypanosoma brucei and its motility. PLoS Comput. Biol. 11, e1003967. (10.1371/journal.pcbi.1003967) PubMed DOI PMC

Hell S, Reiner G, Cremer C, Stelzer EHK. 1993. Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. J. Microsc. 169, 391-405. (10.1111/j.1365-2818.1993.tb03315.x) DOI

Gao M, Maraspini R, Beutel O, Zehtabian A, Eickholt B, Honigmann A, Ewers H. 2018. Expansion stimulated emission depletion microscopy (ExSTED). ACS Nano 12, 4178-4185. (10.1021/acsnano.8b00776) PubMed DOI

Sunter JD, Gull K. 2016. The flagellum attachment zone: ‘the cellular ruler’ of trypanosome morphology. Trends Parasitol. 32, 309-324. (10.1016/j.pt.2015.12.010) PubMed DOI PMC

Moreira-Leite FF, Sherwin T, Kohl L, Gull K. 2001. A trypanosome structure involved in transmitting cytoplasmic information during cell division. Science 294, 610-612. (10.1126/science.1063775) PubMed DOI

Hughes L, Towers K, Starborg T, Gull K, Vaughan S. 2013. A cell-body groove housing the new flagellum tip suggests an adaptation of cellular morphogenesis for parasitism in the bloodstream form of Trypanosoma brucei. J. Cell Sci. 126, 5748-5757. PubMed PMC

Ambit A, Woods KL, Cull B, Coombs GH, Mottram JC. 2011. Morphological events during the cell cycle of Leishmania major. Eukaryot. Cell 10, 1429-1438. (10.1128/EC.05118-11) PubMed DOI PMC

Wheeler RJ, Gluenz E, Gull K. 2011. The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology: the cell cycle of Leishmania. Mol. Microbiol. 79, 647-662. (10.1111/j.1365-2958.2010.07479.x) PubMed DOI PMC

Gadelha C, Wickstead B, Mckean PG, Gull K. 2006. Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes. J. Cell Sci. 119, 2405-2413. (10.1242/jcs.02969) PubMed DOI

Pigino G, Bui KH, Maheshwari A, Lupetti P, Diener D, Ishikawa T. 2011. Cryoelectron tomography of radial spokes in cilia and flagella. J. Cell Biol. 195, 673-687. (10.1083/jcb.201106125) PubMed DOI PMC

Höög JL, Bouchet-Marquis C, Mcintosh JR, Hoenger A, Gull K. 2012. Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei. J. Struct. Biol. 178, 189-198. (10.1016/j.jsb.2012.01.009) PubMed DOI PMC

Weise F, Stierhof YD, Kühn C, Wiese M, Overath P. 2000. Distribution of GPI-anchored proteins in the protozoan parasite Leishmania, based on an improved ultrastructural description using high-pressure frozen cells. J. Cell Sci. 113(Pt 24), 4587-4603. (10.1242/jcs.113.24.4587) PubMed DOI

Ureña F. 1986. Three-dimensional reconstructions of the mitotic spindle and dense plaques in three species of Leishmania. Z. Parasitenkd. Parasitol. Res. 72, 299-306. (10.1007/BF00928739) PubMed DOI

Le Guennec M, et al. 2020. A helical inner scaffold provides a structural basis for centriole cohesion. Sci. Adv. 6, eaaz4137. (10.1126/sciadv.aaz4137) PubMed DOI PMC

Vaughan S, Kohl L, Ngai I, Wheeler RJ, Gull K. 2008. A repetitive protein essential for the flagellum attachment zone filament structure and function in Trypanosoma brucei. Protist 159, 127-136. (10.1016/j.protis.2007.08.005) PubMed DOI

Müller N, Hemphill A, Imboden M, Duvallet G, Dwinger RH, Seebeck T. 1992. Identification and characterization of two repetitive non-variable antigens from African trypanosomes which are recognized early during infection. Parasitology 104, 111-120. (10.1017/S0031182000060856) PubMed DOI

Dacheux D, Landrein N, Thonnus M, Gilbert G, Sahin A, Wodrich H, Robinson DR, Bonhivers M. 2012. A MAP6-related protein is present in protozoa and is involved in flagellum motility. PLoS ONE 7, e31344. (10.1371/journal.pone.0031344) PubMed DOI PMC

An T, Li Z. 2018. An orphan kinesin controls trypanosome morphology transitions by targeting FLAM3 to the flagellum. PLOS Pathog. 14, e1007101. (10.1371/journal.ppat.1007101) PubMed DOI PMC

Chen F, et al. 2016. Nanoscale imaging of RNA with expansion microscopy. Nat. Methods 13, 679-684. (10.1038/nmeth.3899) PubMed DOI PMC

Shah FA, Johansson BR, Thomsen P, Palmquist A. 2015. Ultrastructural evaluation of shrinkage artefacts induced by fixatives and embedding resins on osteocyte processes and pericellular space dimensions: ultrastructural evaluation of shrinkage artefacts. J. Biomed. Mater. Res. A 103, 1565-1576. (10.1002/jbm.a.35287) PubMed DOI

Kubalová I, Schmidt Černohorská M, Huranová M, Weisshart K, Houben A, Schubert V. 2020. Prospects and limitations of expansion microscopy in chromatin ultrastructure determination. Chromosome Res. 28, 355-368. (10.1007/s10577-020-09637-y) PubMed DOI PMC

Pesce L, Cozzolino M, Lanzanò L, Diaspro A, Bianchini P. 2019. Measuring expansion from macro- to nanoscale using NPC as intrinsic reporter. J. Biophotonics 12, e201900018. (10.1002/jbio.201900018) PubMed DOI PMC

Sarkar D, et al. 2020. Expansion revealing: decrowding proteins to unmask invisible brain nanostructures. bioRxiv 2020, 08.29.273540.

Chozinski TJ, Halpern AR, Okawa H, Kim HJ, Tremel GJ, Wong ROL, Vaughan JC. 2016. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat. Methods 13, 485-488. (10.1038/nmeth.3833) PubMed DOI PMC

Dean S, Sunter J, Wheeler RJ, Hodkinson I, Gluenz E, Gull K. 2015. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 5, 140197. (10.1098/rsob.140197) PubMed DOI PMC

Poon SK, Peacock L, Gibson W, Gull K, Kelly S. 2012. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor. Open Biol. 2, 110037. (10.1098/rsob.110037) PubMed DOI PMC

Brun R, Schönenberger M. 1979. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 36, 289-292. PubMed

Aslett M, et al. 2010. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 38, D457-D462. (10.1093/nar/gkp851) PubMed DOI PMC

Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N. 2010. Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol. 92, 14.20.1-14.20.17. (10.1002/0471142727.mb1420s92) PubMed DOI PMC

Schindelin J, et al. 2012. Fiji—an open source platform for biological image analysis. Nat. Methods 9, 676-682. (10.1038/nmeth.2019) PubMed DOI PMC

Thevenaz P, Ruttimann UE, Unser M. 1998. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27-41. (10.1109/83.650848) PubMed DOI

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