Advertisement

Defining Assembly Pathways by Fluorescence Microscopy

Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

Bacterial secretion systems are among the largest protein complexes in prokaryotes and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish building these large functional complexes but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within bacteria, and even functional insights. The emergence of fluorescent proteins has provided a new powerful tool for biological imaging, and the use of fluorescently labeled components presents an interesting method to accurately define the biogenesis of macromolecular complexes. Here we describe the use of this method to decipher the assembly pathway of bacterial secretion systems.

Key words

Fluorescence microscopy Biogenesis Secretion systems Fluorescently labeled proteins Macromolecular complexes Epistasis experiments Subcellular localization 

References

  1. 1.
    Tseng T-T, Tyler B, Setubal J (2009) Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol 9:S2CrossRefGoogle Scholar
  2. 2.
    Costa TRD, Felisberto-Rodrigues C, Meir A, Prevost MS, Redzej A, Trokter M, Waksman G (2015) Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13:343–359CrossRefGoogle Scholar
  3. 3.
    Kimbrough TG, Miller SI (2000) Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc Natl Acad Sci U S A 97:11008–11013CrossRefGoogle Scholar
  4. 4.
    Sukhan A, Kubori T, Wilson J, Galán JE (2001) Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex. J Bacteriol 183:1159–1167CrossRefGoogle Scholar
  5. 5.
    Kimbrough TG, Miller SI (2002) Assembly of the type III secretion needle complex of Salmonella typhimurium. Microbes Infect 4:75–82CrossRefGoogle Scholar
  6. 6.
    Ogino T, Ohno R, Sekiya K, Kuwae A, Matsuzawa T, Nonaka T, Fukuda H, Imajoh-Ohmi S, Abe A (2006) Assembly of the type III secretion apparatus of enteropathogenic Escherichia coli. J Bacteriol 188:2801–2811CrossRefGoogle Scholar
  7. 7.
    Fronzes R, Schäfer E, Wang L, Saibil H, Orlova E, Waksman G (2009) Structure of a type IV secretion system core complex. Science 323:266–268CrossRefGoogle Scholar
  8. 8.
    Schraidt O, Lefebre MD, Brunner MJ, Schmied WH, Schmidt A, Radics J, Mechtler K, Galán JE, Marlovits TC (2010) Topology and organization of the Salmonella typhimurium type III secretion needle complex components. PLoS Pathog 6:e1000824CrossRefGoogle Scholar
  9. 9.
    Reichow SL, Korotkov KV, Hol WGJ, Gonen T (2010) Structure of the cholera toxin secretion channel in its closed state. Nat Struct Mol Biol 17:1226–1232CrossRefGoogle Scholar
  10. 10.
    Chandran Darbari V, Waksman G (2015) Structural biology of bacterial type IV secretion systems. Annu Rev Biochem 84:603–629CrossRefGoogle Scholar
  11. 11.
    Rose P, Fröbel J, Graumann PL, Müller M (2013) Substrate-dependent assembly of the Tat translocase as observed in live Escherichia coli cells. PLoS One 8:e69488CrossRefGoogle Scholar
  12. 12.
    Alcock F, Baker MAB, Greene NP, Palmer T, Wallace MI, Berks BC (2013) Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system. Proc Natl Acad Sci U S A 110:3650–3659CrossRefGoogle Scholar
  13. 13.
    Lybarger S, Johnson TL, Gray M, Sikora A, Sandkvist M (2009) Docking and assembly of the type II secretion complex of Vibrio cholerae. J Bacteriol 191:3149–3161CrossRefGoogle Scholar
  14. 14.
    Johnson TL, Sikora AE, Zielke RA, Sandkvist M (2013) Fluorescence microscopy and proteomics to investigate subcellular localization, assembly, and function of the type II secretion system. Methods Mol Biol 966:157–172CrossRefGoogle Scholar
  15. 15.
    Diepold A, Amstutz M, Abel S, Sorg I, Jenal U, Cornelis GR (2010) Deciphering the assembly of the Yersinia type III secretion injectisome. EMBO J 29:1928–1940CrossRefGoogle Scholar
  16. 16.
    Diepold A, Wiesand U, Cornelis GR (2011) The assembly of the export apparatus (YscR,S,T,U,V) of the Yersinia type III secretion apparatus occurs independently of other structural components and involves the formation of an YscV oligomer. Mol Microbiol 82:502–514CrossRefGoogle Scholar
  17. 17.
    Aguilar J, Zupan J, Cameron TA, Zambryski PC (2010) Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells. Proc Natl Acad Sci U S A 107:3758–3763CrossRefGoogle Scholar
  18. 18.
    Durand E, Nguyen VS, Zoued A, Logger L, Péhau-Arnaudet G, Aschtgen M-S, Spinelli S, Desmyter A, Bardiaux B, Dujeancourt A, Roussel A, Cambillau C, Cascales E, Fronzes R (2015) Biogenesis and structure of a type VI secretion membrane core complex. Nature 523:555–560CrossRefGoogle Scholar
  19. 19.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefGoogle Scholar
  20. 20.
    Köhler A (1893) Ein neues Beleuchtungsverfahren für mikrophotographische Zwecke. Z Wiss Mikrosk 10:433–440Google Scholar
  21. 21.
    Paintdakhi A, Parry B, Campos M, Irnov I, Elf J, Surovtsev I, Jacobs-Wagner C (2015) Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Mol Microbiol 99:767–777CrossRefGoogle Scholar
  22. 22.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909CrossRefGoogle Scholar
  23. 23.
    Adams S, Campbell R, Gross L, Martin B, Walkup G, Yao Y, Llopis J, Tsien RY (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 124:6063–6076CrossRefGoogle Scholar
  24. 24.
    Andresen M, Schmitz-Salue R, Jakobs S (2004) Short tetracysteine tags to beta-tubulin demonstrate the significance of small labels for live cell imaging. Mol Biol Cell 15:5616–5622CrossRefGoogle Scholar
  25. 25.
    Enninga J, Mounier J, Sansonetti P, Tran Van Nhieu G, Van Nhieu GT (2005) Secretion of type III effectors into host cells in real time. Nat Methods 2:959–965CrossRefGoogle Scholar
  26. 26.
    Diepold A, Kudryashev M, Delalez NJ, Berry RM, Armitage JP (2015) Composition, formation, and regulation of the cytosolic C-ring, a dynamic component of the type III secretion injectisome. PLoS Biol 13:e1002039CrossRefGoogle Scholar
  27. 27.
    Poulter NS, Pitkeathly WTE, Smith PJ, Rappoport JZ (2015) In: Verveer PJ (ed) Advanced fluorescence microscopy. Springer, New YorkGoogle Scholar
  28. 28.
    MacDonald L, Baldini G, Storrie B (2015) Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization? Methods Mol Biol 1270:255–275CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Laboratoire d’Ingénierie des Systèmes MacromoléculairesAix-Marseille UniversitéMarseille Cedex 20France
  2. 2.Department of BiochemistryUniversity of OxfordOxfordUK
  3. 3.Division of Infectious Diseases and Harvard Medical School, Department of Microbiology and ImmunobiologyHoward Hughes Medical Institute, Brigham and Women’s HospitalBostonUSA
  4. 4.Department of Ecophysiology, Max Planck Institute for Terrestrial MicrobiologyKarl-von-Frisch-Str.10, 35043 MarburgGermany

Personalised recommendations