The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

Department of Biological and Medical Sciences Oxford Brookes University Oxford United Kingdom

Department of Parasitology Charles University Prague Czech Republic

Peter Medawar Building for Pathogen Research University of Oxford Oxford United Kingdom

Zobrazit více v PubMed

Gossage S, Rogers M, Bates P. Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle. International journal for parasitology [Internet]. 2003. Sep 15 [cited 2023 Aug 6];33(10). Available from: https://pubmed.ncbi.nlm.nih.gov/13129524/ doi: 10.1016/s0020-7519(03)00142-5 PubMed DOI PMC

Burza S, Croft S, Boelaert M. Leishmaniasis. Lancet (London, England) [Internet]. 2018. Sep 15 [cited 2023 Aug 6];392(10151). Available from: https://pubmed.ncbi.nlm.nih.gov/30126638/ PubMed

Ambit A, Woods K, Cull B, Coombs G, Mottram J. Morphological Events during the Cell Cycle of Leishmania major. Eukaryot Cell. 2011. Nov;10(11):1429–38. doi: 10.1128/EC.05118-11 PubMed DOI PMC

Wheeler R, Gluenz E, Gull K. The cell cycle of Leishmania: morphogenetic events and their implications for parasite biology. Mol Microbiol. 2011. Feb;79(3):647–62. PubMed PMC

Sunter Jack, and Gull Keith. 2017. ‘Shape, Form, Function and Leishmania Pathogenicity: From Textbook Descriptions to Biological Understanding’. Open Biology 7 (9): 170165. doi: 10.1098/rsob.170165 PubMed DOI PMC

Sunter J, Yanase R, Wang Z, Catta-Preta C, Moreira-Leite F, Myskova J, et al.. Leishmania flagellum attachment zone is critical for flagellar pocket shape, development in the sand fly, and pathogenicity in the host. Proc Natl Acad Sci U S A. 2019. Mar 26;116(13):6351–60. doi: 10.1073/pnas.1812462116 PubMed DOI PMC

Wheeler Richard, Gluenz Eva, and Gull Keith. 2013. ‘The Limits on Trypanosomatid Morphological Diversity’. PLoS ONE 8 (11): e79581. doi: 10.1371/journal.pone.0079581 PubMed DOI PMC

Wheeler R, Sunter J, Gull K. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone. J Cell Sci. 2016. Feb 15;129(4):854–67. doi: 10.1242/jcs.183152 PubMed DOI PMC

Sunter J, Gull K. The Flagellum Attachment Zone: ‘The Cellular Ruler’ of Trypanosome Morphology. Trends Parasitol. 2016. Apr;32(4):309–24. doi: 10.1016/j.pt.2015.12.010 PubMed DOI PMC

Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol. 1995. Mar;128(6):1163–72. doi: 10.1083/jcb.128.6.1163 PubMed DOI PMC

Sherwin T, Gull K. The cell division cycle of Trypanosoma brucei brucei: timing of event markers and cytoskeletal modulations. Philos Trans R Soc Lond B Biol Sci. 1989. Jun 12;323(1218):573–88. doi: 10.1098/rstb.1989.0037 PubMed DOI

Abeywickrema M, Vachova H, Farr H, Mohr T, Wheeler R, Lai DH, et al.. Non-equivalence in old- and new-flagellum daughter cells of a proliferative division in Trypanosoma brucei. Molecular Microbiology. 2019;112(3):1024–40. doi: 10.1111/mmi.14345 PubMed DOI PMC

Campbell P, de Graffenried CL. Morphogenesis in Trypanosoma cruzi epimastigotes proceeds via a highly asymmetric cell division [Internet]. bioRxiv; 2023. [cited 2023 Aug 7]. p. 2023.05.24.542100. Available from: https://www.biorxiv.org/content/10.1101/2023.05.24.542100v2 PubMed DOI PMC

Rudzinska M, D’alesandro P, Trager W. The Fine Structure of Leishmania donovani and the Role of the Kinetoplast in the Leishmania-Leptomonad Transformation*. The Journal of Protozoology. 1964;11(2):166–91. PubMed

Aleman C. Finestructure of cultured Leishmania brasiliensis. Experimental Parasitology. 1969. Apr 1;24(2):259–64. doi: 10.1016/0014-4894(69)90163-5 PubMed DOI

Minocha N, Kumar D, Rajanala K, Saha S. Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani. J Eukaryot Microbiol. 2011;58(3):249–53. doi: 10.1111/j.1550-7408.2011.00539.x PubMed DOI

Wang Z, Wheeler RJ, Sunter JD. Lysosome assembly and disassembly changes endocytosis rate through the Leishmania cell cycle. Microbiologyopen. 2019. Nov 19;9(2):e969. doi: 10.1002/mbo3.969 PubMed DOI PMC

Hughes L, Borrett S, Towers K, Starborg T, Vaughan S. Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy. J Cell Sci. 2017. Feb 1;130(3):637–47. doi: 10.1242/jcs.198887 PubMed DOI

Chang PCH. The Ultrastructure of Leishmania donovani. The Journal of Parasitology. 1956;42(2):126–36. PubMed

Pan AA, Pan SC. Leishmania mexicana: Comparative fine structure of amastigotes and promastigotes in vitro and in vivo. Experimental Parasitology. 1986. Oct 1;62(2):254–65. doi: 10.1016/0014-4894(86)90030-5 PubMed DOI

Wheeler R. Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles. Mol Biol Cell. 2015. Nov 5;26(22):3898–903. doi: 10.1091/mbc.E15-03-0151 PubMed DOI PMC

Lacomble S, Vaughan S, Gadelha C, Morphew MK, Shaw MK, McIntosh JR, et al.. Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei. J Cell Sci. 2010. Sep 1;123(17):2884–91. doi: 10.1242/jcs.074161 PubMed DOI PMC

Vickerman K. The mechanism of cyclical development in trypanosomes of the Trypanosoma brucei sub-group: An hypothesis based on ultrastructural observations. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1962. Nov 1;56(6):487–95. doi: 10.1016/0035-9203(62)90072-x PubMed DOI

Halliday C, de Castro-Neto A, Alcantara CL, Cunha-e-Silva NL, Vaughan S, Sunter JD. Trypanosomatid Flagellar Pocket from Structure to Function. Trends in Parasitology. 2021. Apr 1;37(4):317–29. doi: 10.1016/j.pt.2020.11.005 PubMed DOI

Absalon S, Blisnick T, Kohl L, Toutirais G, Doré G, Julkowska D, et al.. Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes. Mol Biol Cell. 2008. Mar;19(3):929–44. doi: 10.1091/mbc.e07-08-0749 PubMed DOI PMC

Sunter JD, Moreira-Leite F, Gull K. Dependency relationships between IFT-dependent flagellum elongation and cell morphogenesis in Leishmania. Open Biol. 2018. Nov 21;8(11):180124. doi: 10.1098/rsob.180124 PubMed DOI PMC

Soares H, Carmona B, Nolasco S, Viseu Melo L, Gonçalves J. Cilia Distal Domain: Diversity in Evolutionarily Conserved Structures. Cells. 2019. Feb 14;8(2):160. doi: 10.3390/cells8020160 PubMed DOI PMC

Woolley D, Gadelha C, Gull K. Evidence for a sliding-resistance at the tip of the trypanosome flagellum. Cell Motil Cytoskeleton. 2006. Dec;63(12):741–6. doi: 10.1002/cm.20159 PubMed DOI

Wheeler R, Gluenz E, Gull K. Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum. Nat Commun. 2015. Dec 15;6(1):8964. PubMed PMC

Lacomble S, Vaughan S, Deghelt M, Moreira-Leite FF, Gull K. A Trypanosoma brucei protein required for maintenance of the flagellum attachment zone and flagellar pocket ER domains. Protist. 2012. Jul;163(4):602–15. doi: 10.1016/j.protis.2011.10.010 PubMed DOI PMC

DiMaio J, Ruthel G, Cannon JJ, Malfara MF, Povelones ML. The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network. PLoS One. 2018;13(12):e0202711. doi: 10.1371/journal.pone.0202711 PubMed DOI PMC

Wheeler R, Scheumann N, Wickstead B, Gull K, Vaughan S. Cytokinesis in Trypanosoma brucei differs between bloodstream and tsetse trypomastigote forms: implications for microtubule-based morphogenesis and mutant analysis. Mol Microbiol. 2013. Dec;90(6):1339–55. doi: 10.1111/mmi.12436 PubMed DOI PMC

Yanase R, Moreira-Leite F, Rea E, Wilburn L, Sádlová J, Vojtkova B, et al.. Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector. Zamboni DS, Akhmanova A, editors. eLife. 2023. May 10;12:e84552. doi: 10.7554/eLife.84552 PubMed DOI PMC

Bates P. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. International journal for parasitology. 2007. Sep 1;37:1097–106. PubMed PMC

Rogers ME, Chance ML, Bates PA. The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis. Parasitology. 2002. May;124(Pt 5):495–507. doi: 10.1017/s0031182002001439 PubMed DOI

Inbar E, Hughitt VK, Dillon LAL, Ghosh K, El-Sayed NM, Sacks DL. The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector. mBio. 2017. Apr 4;8(2):e00029–17. doi: 10.1128/mBio.00029-17 PubMed DOI PMC

Wheeler RJ, Gull K, Sunter JD. Coordination of the Cell Cycle in Trypanosomes. Annu Rev Microbiol. 2019. Sep 8;73:133–54. doi: 10.1146/annurev-micro-020518-115617 PubMed DOI

Akiyoshi B. Analysis of a Mad2 homolog in Trypanosoma brucei provides possible hints on the origin of the spindle checkpoint [Internet]. bioRxiv; 2020. [cited 2023 Aug 6]. p. 2020.12.29.424754. Available from: https://www.biorxiv.org/content/10.1101/2020.12.29.424754v1 DOI

Marques CA, Ridgway M, Tinti M, Cassidy A, Horn D. Genome-scale RNA interference profiling of Trypanosoma brucei cell cycle progression defects. Nat Commun. 2022. Sep 10;13(1):5326. doi: 10.1038/s41467-022-33109-y PubMed DOI PMC

Simpson L, Kretzer F. The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion. Mol Biochem Parasitol. 1997. Jul;87(1):71–8. doi: 10.1016/s0166-6851(97)00044-3 PubMed DOI

da Silva MS, Monteiro JP, Nunes VS, Vasconcelos EJ, Perez AM, de Freitas-Júnior L H, et al.. Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle. PLOS ONE. 2013. Nov 21;8(11):e81397. doi: 10.1371/journal.pone.0081397 PubMed DOI PMC

Bianchi L, Rondanelli EG, Carosi G, Gerna G. Endonuclear Mitotic Spindle in the Leptomonad of Leishmania tropica. The Journal of Parasitology. 1969;55(5):1091–2.

Triemer RE, Fritz LM, Herman R. Ultrastructural features of mitosis inLeishmania adleri. Protoplasma. 1986. Jun 1;134(2):154–62.

Varberg J, Unruh J, Bestul A, Khan A, Jaspersen S. Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe. Life Sci Alliance. 2022. Jul;5(7):e202201423. doi: 10.26508/lsa.202201423 PubMed DOI PMC

Billington K, Halliday C, Madden R, Dyer P, Barker AR, Moreira-Leite F, et al.. Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei. Nat Microbiol. 2023. Mar;8(3):533–47. doi: 10.1038/s41564-022-01295-6 PubMed DOI PMC

Sherwin T, Gull K. Visualization of detyrosination along single microtubules reveals novel mechanisms of assembly during cytoskeletal duplication in trypanosomes. Cell. 1989. Apr 21;57(2):211–21. doi: 10.1016/0092-8674(89)90959-8 PubMed DOI

Sheriff O, Lim LF, He CY. Tracking the biogenesis and inheritance of subpellicular microtubule in Trypanosoma brucei with inducible YFP-α-tubulin. Biomed Res Int. 2014;2014:893272. PubMed PMC

Sinclair A, de Graffenried C. More than microtubules: The structure and function of the subpellicular array in trypanosomatids. Trends Parasitol. 2019. Oct;35(10):760–77. doi: 10.1016/j.pt.2019.07.008 PubMed DOI PMC

Powell CD, Quain DE, Smart KA. Chitin scar breaks in aged Saccharomyces cerevisiae. Microbiology. 2003;149(11):3129–37. doi: 10.1099/mic.0.25940-0 PubMed DOI

Dostálová A, Volf P. Leishmania development in sand flies: parasite-vector interactions overview. Parasit Vectors. 2012. Dec 3;5:276. doi: 10.1186/1756-3305-5-276 PubMed DOI PMC

Saxena A, Lahav T, Holland N, Aggarwal G, Anupama A, Huang Y, et al.. Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation. Mol Biochem Parasitol. 2007. Mar;152(1):53–65. doi: 10.1016/j.molbiopara.2006.11.011 PubMed DOI PMC

Cantacessi C, Dantas-Torres F, Nolan MJ, Otranto D. The past, present, and future of Leishmania genomics and transcriptomics. Trends Parasitol. 2015. Mar;31(3):100–8. doi: 10.1016/j.pt.2014.12.012 PubMed DOI PMC

McConville MJ, Saunders EC, Kloehn J, Dagley MJ. Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine? F1000Res. 2015. Oct 1;4(F1000 Faculty Rev):938. doi: 10.12688/f1000research.6724.1 PubMed DOI PMC

Iudin A, Korir PK, Somasundharam S, Weyand S, Cattavitello C, Fonseca N, et al.. EMPIAR: the Electron Microscopy Public Image Archive. Nucleic Acids Research. 2023. Jan 6;51(D1):D1503–11. doi: 10.1093/nar/gkac1062 PubMed DOI PMC

Sádlová J, Yeo M, Seblova V, Lewis MD, Mauricio I, Volf P, et al.. Visualisation of Leishmania donovani Fluorescent Hybrids during Early Stage Development in the Sand Fly Vector. PLoS ONE [Internet]. 2011. [cited 2023 Oct 24];6. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019851 PubMed PMC

Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. doi: 10.1006/jsbi.1996.0013 PubMed DOI

Sádlová J, Price HP, Smith BA, Votýpka J, Volf P, Smith DF. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi. Cellular Microbiology. 2010;12(12):1765–79. doi: 10.1111/j.1462-5822.2010.01507.x PubMed DOI PMC