Attachment to the intestinal epithelium is critical to the lifestyle of the ubiquitous parasite Giardia lamblia. The ventrolateral flange is a sheet-like membrane protrusion at the interface between parasites and attached surfaces. This structure has been implicated in attachment, but its role has been poorly defined. Here, we identified a novel actin associated protein with putative WH2-like actin binding domains we named Flangin. Flangin complexes with Giardia actin (GlActin) and is enriched in the ventrolateral flange making it a valuable marker for studying the flanges' role in Giardia biology. Live imaging revealed that the flange grows to around 1 μm in width after cytokinesis, then remains uniform in size during interphase, grows in mitosis, and is resorbed during cytokinesis. A flangin truncation mutant stabilizes the flange and blocks cytokinesis, indicating that flange disassembly is necessary for rapid myosin-independent cytokinesis in Giardia. Rho family GTPases are important regulators of membrane protrusions and GlRac, the sole Rho family GTPase in Giardia, was localized to the flange. Knockdown of Flangin, GlActin, and GlRac result in flange formation defects. This indicates a conserved role for GlRac and GlActin in forming membrane protrusions, despite the absence of canonical actin binding proteins that link Rho GTPase signaling to lamellipodia formation. Flangin-depleted parasites had reduced surface contact and when challenged with fluid shear force in flow chambers they had a reduced ability to remain attached, confirming a role for the flange in attachment. This secondary attachment mechanism complements the microtubule based adhesive ventral disc, a feature that may be particularly important during mitosis when the parental ventral disc disassembles in preparation for cytokinesis. This work supports the emerging view that Giardia's unconventional actin cytoskeleton has an important role in supporting parasite attachment.

Bioengineering University of Washington Seattle Washington United States of America

Center for Cardiovascular Biology University of Washington Seattle Washington United States of America

Department of Biochemistry University of Washington Seattle Washington United States of America

Department of Biology University of Washington Seattle Washington United States of America

Department of Chemistry University of Washington Seattle Washington United States of America

Department of Mechanical Engineering University of Washington Seattle Washington United States of America

Department of Physiology and Biophysics University of Washington Seattle Washington United States of America

Institute for Stem Cell and Regenerative Medicine University of Washington Seattle Washington United States of America

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Prague Czech Republic

Lab Medicine and Pathology University of Washington Seattle Washington United States of America

Zobrazit více v PubMed

Lane S, Lloyd D. Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol. 2002;28(2):123–47. Epub 2002/07/12. doi: 10.1080/1040-840291046713 . PubMed DOI

Baldursson S, Karanis P. Waterborne transmission of protozoan parasites: review of worldwide outbreaks—an update 2004–2010. Water Res. 2011;45(20):6603–14. doi: 10.1016/j.watres.2011.10.013 . PubMed DOI

Dawson SC, Paredez AR. Alternative cytoskeletal landscapes: cytoskeletal novelty and evolution in basal excavate protists. Curr Opin Cell Biol. 2013;25(1):134–41. doi: 10.1016/j.ceb.2012.11.005 . PubMed DOI PMC

Nosala C, Hagen KD, Dawson SC. ’Disc-o-Fever’: Getting Down with Giardia’s Groovy Microtubule Organelle. Trends Cell Biol. 2018;28(2):99–112. Epub 2017/11/21. doi: 10.1016/j.tcb.2017.10.007 . PubMed DOI PMC

Steele-Ogus MC, Johnson RS, MacCoss MJ, Paredez AR. Identification of Actin Filament-Associated Proteins in Giardia lamblia. Microbiol Spectr. 2021:e0055821. Epub 2021/07/22. doi: 10.1128/Spectrum.00558-21 . PubMed DOI PMC

Steele-Ogus MC, Obenaus AM, Sniadecki NJ, Paredez AR. Disc and Actin-Associated Protein 1 Influences Attachment in the Intestinal Parasite Giardia lamblia. bioRxiv. 2021:2021.08.06.455446. doi: 10.1101/2021.08.06.455446 PubMed DOI PMC

Lynch M, Field MC, Goodson HV, Malik HS, Pereira-Leal JB, Roos DS, et al.. Evolutionary cell biology: two origins, one objective. Proc Natl Acad Sci U S A. 2014;111(48):16990–4. Epub 2014/11/19. doi: 10.1073/pnas.1415861111 ; PubMed Central PMCID: PMC4260604. PubMed DOI PMC

Velle KB, Fritz-Laylin LK. Diversity and evolution of actin-dependent phenotypes. Current opinion in genetics & development. 2019;58–59:40–8. Epub 2019/08/30. doi: 10.1016/j.gde.2019.07.016 . PubMed DOI

Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P. Toward automatic reconstruction of a highly resolved tree of life. Science. 2006;311(5765):1283–7. doi: 10.1126/science.1123061 . PubMed DOI

Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, et al.. Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science. 2007;317(5846):1921–6. Epub 2007/09/29. doi: 10.1126/science.1143837 . PubMed DOI

Hampl V, Hug L, Leigh JW, Dacks JB, Lang BF, Simpson AG, et al.. Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic "supergroups". Proc Natl Acad Sci U S A. 2009;106(10):3859–64. doi: 10.1073/pnas.0807880106 ; PubMed Central PMCID: PMC2656170. PubMed DOI PMC

Sousa MC, Gonçalves CA, Bairos VA, Poiares-Da-Silva J. Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin Diagn Lab Immunol. 2001;8(2):258–65. doi: 10.1128/CDLI.8.2.258-265.2001 ; PubMed Central PMCID: PMC96046. PubMed DOI PMC

Elmendorf HG, Dawson SC, McCaffery JM. The cytoskeleton of Giardia lamblia. Int J Parasitol. 2003;33(1):3–28. doi: 10.1016/s0020-7519(02)00228-x . PubMed DOI

Paredez AR, Assaf ZJ, Sept D, Timofejeva L, Dawson SC, Wang CJ, et al.. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins. Proc Natl Acad Sci U S A. 2011;108(15):6151–6. doi: 10.1073/pnas.1018593108 ; PubMed Central PMCID: PMC3076823. PubMed DOI PMC

Hardin WR, Li R, Xu J, Shelton AM, Alas GCM, Minin VN, et al.. Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking. Proc Natl Acad Sci U S A. 2017;114(29):E5854–E63. Epub 2017/07/07. doi: 10.1073/pnas.1705096114 ; PubMed Central PMCID: PMC5530689. PubMed DOI PMC

Paredez AR, Nayeri A, Xu JW, Krtkova J, Cande WZ. Identification of obscure yet conserved actin associated proteins in Giardia lamblia. Eukaryot Cell. 2014;13(6):776–84. Epub 2014/04/15. doi: 10.1128/EC.00041-14 ; PubMed Central PMCID: PMC4054273. PubMed DOI PMC

Krtkova J, Xu J, Lalle M, Steele-Ogus M, Alas GCM, Sept D, et al.. 14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia. mSphere. 2017;2(5). doi: 10.1128/mSphere.00248-17 ; PubMed Central PMCID: PMC5597967. PubMed DOI PMC

Carlier MF, Hertzog M, Didry D, Renault L, Cantrelle FX, van Heijenoort C, et al.. Structure, function, and evolution of the beta-thymosin/WH2 (WASP-Homology2) actin-binding module. Annals of the New York Academy of Sciences. 2007;1112:67–75. Epub 2007/10/20. doi: 10.1196/annals.1415.037 . PubMed DOI

Paunola E, Mattila PK, Lappalainen P. WH2 domain: a small, versatile adapter for actin monomers. Febs Lett. 2002;513(1):92–7. doi: 10.1016/s0014-5793(01)03242-2 WOS:000174208800015. PubMed DOI

Dominguez R. The WH2 Domain and Actin Nucleation: Necessary but Insufficient. Trends Biochem Sci. 2016;41(6):478–90. Epub 2016/04/14. doi: 10.1016/j.tibs.2016.03.004 ; PubMed Central PMCID: PMC4884163. PubMed DOI PMC

Fletcher DA, Mullins RD. Cell mechanics and the cytoskeleton. Nature. 2010;463(7280):485–92. Epub 2010/01/30. doi: 10.1038/nature08908 ; PubMed Central PMCID: PMC2851742. PubMed DOI PMC

Brayford S, Bryce NS, Schevzov G, Haynes EM, Bear JE, Hardeman EC, et al.. Tropomyosin Promotes Lamellipodial Persistence by Collaborating with Arp2/3 at the Leading Edge. Current biology: CB. 2016;26(10):1312–8. Epub 2016/04/27. doi: 10.1016/j.cub.2016.03.028 . PubMed DOI PMC

Fritz-Laylin LK, Prochnik SE, Ginger ML, Dacks JB, Carpenter ML, Field MC, et al.. The genome of Naegleria gruberi illuminates early eukaryotic versatility. Cell. 2010;140(5):631–42. doi: 10.1016/j.cell.2010.01.032 . PubMed DOI

Friend DS. The fine structure of Giardia muris. The Journal of cell biology. 1966;29(2):317–32. Epub 1966/05/01. doi: 10.1083/jcb.29.2.317 ; PubMed Central PMCID: PMC2106914. PubMed DOI PMC

Holberton DV. Attachment of Giardia-a hydrodynamic model based on flagellar activity. The Journal of experimental biology. 1974;60(1):207–21. Epub 1974/02/01. doi: 10.1242/jeb.60.1.207 . PubMed DOI

Hemphill A, Müller N, Müller J. Comparative Pathobiology of the Intestinal Protozoan Parasites Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum. Pathogens. 2019;8(3). Epub 20190729. doi: 10.3390/pathogens8030116 ; PubMed Central PMCID: PMC6789772. PubMed DOI PMC

House SA, Richter DJ, Pham JK, Dawson SC. Giardia flagellar motility is not directly required to maintain attachment to surfaces. PLoS Pathog. 2011;7(8):e1002167. Epub 2011/08/11. doi: 10.1371/journal.ppat.1002167 ; PubMed Central PMCID: PMC3150270. PubMed DOI PMC

Erlandsen SL, Russo AP, Turner JN. Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia. The Journal of eukaryotic microbiology. 2004;51(1):73–80. Epub 2004/04/08. doi: 10.1111/j.1550-7408.2004.tb00165.x . PubMed DOI

Campellone KG, Welch MD. A nucleator arms race: cellular control of actin assembly. Nature reviews Molecular cell biology. 2010;11(4):237–51. doi: 10.1038/nrm2867 ; PubMed Central PMCID: PMC2929822. PubMed DOI PMC

Pan S, Wang R, Zhou X, He G, Koomen J, Kobayashi R, et al.. Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly. The Journal of biological chemistry. 2006;281(45):34640–50. Epub 2006/09/13. doi: 10.1074/jbc.M602263200 . PubMed DOI

Cabezas A, Bache KG, Brech A, Stenmark H. Alix regulates cortical actin and the spatial distribution of endosomes. J Cell Sci. 2005;118(Pt 12):2625–35. Epub 2005/05/26. doi: 10.1242/jcs.02382 . PubMed DOI

Gourguechon S, Cande WZ. Rapid tagging and integration of genes in Giardia intestinalis. Eukaryot Cell. 2011;10(1):142–5. doi: 10.1128/EC.00190-10 ; PubMed Central PMCID: PMC3019802. PubMed DOI PMC

Chen F, Tillberg PW, Boyden ES. Optical imaging. Expansion microscopy. Science. 2015;347(6221):543–8. Epub 2015/01/17. doi: 10.1126/science.1260088 ; PubMed Central PMCID: PMC4312537. PubMed DOI PMC

Chozinski TJ, Halpern AR, Okawa H, Kim HJ, Tremel GJ, Wong RO, et al.. Expansion microscopy with conventional antibodies and fluorescent proteins. Nature methods. 2016;13(6):485–8. Epub 2016/04/12. doi: 10.1038/nmeth.3833 ; PubMed Central PMCID: PMC4929147. PubMed DOI PMC

Picou TJ III. The Hydrodynamic Model of Giardia lamblia Attachment [Ph.D.]. Ann Arbor: Georgetown University; 2017.

Jones RD, Lemanski CL, Jones TJ. Theory of attachment in Giardia. Biophysical Journal. 1983;44(2):185–90. doi: 10.1016/S0006-3495(83)84290-8 PubMed DOI PMC

Lenaghan SC, Davis CA, Henson WR, Zhang Z, Zhang M. High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites. Proc Natl Acad Sci U S A. 2011;108(34):E550–8. Epub 2011/08/03. doi: 10.1073/pnas.1106904108 ; PubMed Central PMCID: PMC3161553. PubMed DOI PMC

Nohynkova E, Tumova P, Kulda J. Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell. Eukaryot Cell. 2006;5(4):753–61. doi: 10.1128/EC.5.4.753-761.2006 ; PubMed Central PMCID: PMC1459668. PubMed DOI PMC

Tumova P, Kulda J, Nohynkova E. Cell division of Giardia intestinalis: Assembly and disassembly of the adhesive disc, and the cytokinesis. Cell motility and the cytoskeleton. 2007;64(4):288–98. doi: 10.1002/cm.20183 WOS:000244943600005. PubMed DOI

Poirier CC, Ng WP, Robinson DN, Iglesias PA. Deconvolution of the Cellular Force-Generating Subsystems that Govern Cytokinesis Furrow Ingression. Plos Computational Biology. 2012;8(4). doi: 10.1371/journal.pcbi.1002467 WOS:000303440400019. PubMed DOI PMC

Titus MA, Goodson HV. An evolutionary perspective on cell migration: Digging for the roots of amoeboid motility. The Journal of cell biology. 2017;216(6):1509–11. Epub 2017/05/26. doi: 10.1083/jcb.201704112 ; PubMed Central PMCID: PMC5461037. PubMed DOI PMC

Fritz-Laylin LK, Lord SJ, Mullins RD. WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility. The Journal of cell biology. 2017;216(6):1673–88. Epub 2017/05/06. doi: 10.1083/jcb.201701074 ; PubMed Central PMCID: PMC5461030. PubMed DOI PMC

Kufareva I, Lenoir M, Dancea F, Sridhar P, Raush E, Bissig C, et al.. Discovery of novel membrane binding structures and functions. Biochem Cell Biol. 2014;92(6):555–63. Epub 2014/11/14. doi: 10.1139/bcb-2014-0074 ; PubMed Central PMCID: PMC4267288. PubMed DOI PMC

Kim J, Sitaraman S, Hierro A, Beach BM, Odorizzi G, Hurley JH. Structural basis for endosomal targeting by the Bro1 domain. Developmental cell. 2005;8(6):937–47. Epub 2005/06/07. doi: 10.1016/j.devcel.2005.04.001 ; PubMed Central PMCID: PMC2862258. PubMed DOI PMC

Ahmed S, Goh WI, Bu W. I-BAR domains, IRSp53 and filopodium formation. Semin Cell Dev Biol. 2010;21(4):350–6. Epub 2009/11/17. doi: 10.1016/j.semcdb.2009.11.008 . PubMed DOI

Douthwright S, Sluder G. Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it. Journal of cellular physiology. 2017;232(9):2461–8. Epub 2016/09/09. doi: 10.1002/jcp.25588 . PubMed DOI

Hennessey KM, Alas GCM, Rogiers I, Li R, Merritt EA, Paredez AR. Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia. Mol Biol Cell. 2020:mbcE19070406. doi: 10.1091/mbc.E19-07-0406 . PubMed DOI PMC

Ladwein M, Rottner K. On the Rho’d: The regulation of membrane protrusions by Rho-GTPases. Febs Lett. 2008;582(14):2066–74. doi: 10.1016/j.febslet.2008.04.033 ISI:000257387100016. PubMed DOI

Michaelson D, Silletti J, Murphy G, D’Eustachio P, Rush M, Philips MR. Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. The Journal of cell biology. 2001;152(1):111–26. Epub 2001/01/10. doi: 10.1083/jcb.152.1.111 ; PubMed Central PMCID: PMC2193662. PubMed DOI PMC

Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994;367(6458):40–6. Epub 1994/01/06. doi: 10.1038/367040a0 . PubMed DOI

Srinivasan S, Wang F, Glavas S, Ott A, Hofmann F, Aktories K, et al.. Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. The Journal of cell biology. 2003;160(3):375–85. doi: 10.1083/jcb.200208179 ; PubMed Central PMCID: PMC2172671. PubMed DOI PMC

Thomas EB, Sutanto R, Johnson RS, Shih HW, Alas GCM, Krtkova J, et al.. Staging Encystation Progression in Giardia lamblia Using Encystation-Specific Vesicle Morphology and Associating Molecular Markers. Frontiers in cell and developmental biology. 2021;9(789):662945. Epub 2021/05/15. doi: 10.3389/fcell.2021.662945 ; PubMed Central PMCID: PMC8111296. PubMed DOI PMC

Woessner DJ, Dawson SC. The Giardia median body protein is a ventral disc protein that is critical for maintaining a domed disc conformation during attachment. Eukaryot Cell. 2012;11(3):292–301. Epub 2012/01/17. doi: 10.1128/EC.05262-11 ; PubMed Central PMCID: PMC3294447. PubMed DOI PMC

Nosala C, Dawson SC. The ventral disc is a flexible microtubule organelle that depends on domed ultrastructure for functional attachment of Giardia lamblia. bioRxiv. 2017:213421. doi: 10.1101/213421 DOI

Benchimol M. Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms. Parasitology. 2020:1–11. Epub 2020/12/08. doi: 10.1017/S0031182020002292 . PubMed DOI PMC

Wu YI, Wang X, He L, Montell D, Hahn KM. Spatiotemporal control of small GTPases with light using the LOV domain. Methods in enzymology. 2011;497:393–407. Epub 2011/05/24. doi: 10.1016/B978-0-12-385075-1.00016-0 ; PubMed Central PMCID: PMC3407667. PubMed DOI PMC

Lawson CD, Ridley AJ. Rho GTPase signaling complexes in cell migration and invasion. The Journal of cell biology. 2018;217(2):447–57. Epub 2017/12/14. doi: 10.1083/jcb.201612069 ; PubMed Central PMCID: PMC5800797. PubMed DOI PMC

Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature. 2002;418(6899):790–3. Epub 2002/08/16. doi: 10.1038/nature00859 . PubMed DOI

Katelaris PH, Naeem A, Farthing MJ. Attachment of Giardia lamblia trophozoites to a cultured human intestinal cell line. Gut. 1995;37(4):512–8. doi: 10.1136/gut.37.4.512 ; PubMed Central PMCID: PMC1382903. PubMed DOI PMC

Pegado MG, de Souza W. Role of surface components in the process of interaction of Giardia duodenalis with epithelial cells in vitro. Parasitology research. 1994;80(4):320–6. doi: 10.1007/BF02351874 . PubMed DOI

Weiland ME, Palm JE, Griffiths WJ, McCaffery JM, Svärd SG. Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity. Int J Parasitol. 2003;33(12):1341–51. Epub 2003/10/07. doi: 10.1016/s0020-7519(03)00201-7 . PubMed DOI

Holberton DV. Fine structure of the ventral disk apparatus and the mechanism of attachment in the flagellate Giardia muris. J Cell Sci. 1973;13(1):11–41. Epub 1973/07/01. doi: 10.1242/jcs.13.1.11 . PubMed DOI

Pollard TD. Nine unanswered questions about cytokinesis. The Journal of cell biology. 2017;216(10):3007–16. Epub 2017/08/16. doi: 10.1083/jcb.201612068 ; PubMed Central PMCID: PMC5626534. PubMed DOI PMC

Figard L, Sokac AM. A membrane reservoir at the cell surface: unfolding the plasma membrane to fuel cell shape change. Bioarchitecture. 2014;4(2):39–46. Epub 2014/05/23. doi: 10.4161/bioa.29069 ; PubMed Central PMCID: PMC4199810. PubMed DOI PMC

Figard L, Wang M, Zheng L, Golding I, Sokac AM. Membrane Supply and Demand Regulates F-Actin in a Cell Surface Reservoir. Developmental cell. 2016;37(3):267–78. Epub 2016/05/12. doi: 10.1016/j.devcel.2016.04.010 ; PubMed Central PMCID: PMC5019494. PubMed DOI PMC

Sagolla MS, Dawson SC, Mancuso JJ, Cande WZ. Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J Cell Sci. 2006;119(Pt 23):4889–900. doi: 10.1242/jcs.03276 . PubMed DOI

Krtkova J, Thomas EB, Alas GC, Schraner EM, Behjatnia HR, Hehl AB, et al.. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion. mBio. 2016;7(4). Epub 2016/08/25. doi: 10.1128/mBio.01003-16 ; PubMed Central PMCID: PMC4999545. PubMed DOI PMC

Krtkova J, Paredez AR. Use of Translation Blocking Morpholinos for Gene Knockdown in Giardia lamblia. Methods in molecular biology (Clifton, NJ). 2017;1565:123–40. Epub 2017/04/02. doi: 10.1007/978-1-4939-6817-6_11 . PubMed DOI

Halpern AR, Alas GCM, Chozinski TJ, Paredez AR, Vaughan JC. Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization. ACS nano. 2017;11(12):12677–86. Epub 2017/11/23. doi: 10.1021/acsnano.7b07200 ; PubMed Central PMCID: PMC5752594. PubMed DOI PMC

Tůmová P, Nohýnková E, Klingl A, Wanner G. A rapid workflow for the characterization of small numbers of unicellular eukaryotes by using correlative light and electron microscopy. J Microbiol Methods. 2020;172:105888. Epub 2020/03/15. doi: 10.1016/j.mimet.2020.105888 . PubMed DOI

Tůmová P, Voleman L, Klingl A, Nohýnková E, Wanner G, Doležal P. Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle. BMC biology. 2021;19(1):193. doi: 10.1186/s12915-021-01129-7 PubMed DOI PMC

Tinevez JY, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, et al.. TrackMate: An open and extensible platform for single-particle tracking. Methods. 2017;115:80–90. Epub 2016/10/08. doi: 10.1016/j.ymeth.2016.09.016 . PubMed DOI

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature methods. 2012;9(7):671–5. Epub 2012/08/30. doi: 10.1038/nmeth.2089 . PubMed DOI PMC