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SNAREs pp 361-377 | Cite as

Visualization of SNARE-Mediated Organelle Membrane Hemifusion by Electron Microscopy

  • Sevan Mattie
  • Tom Kazmirchuk
  • Jeannie Mui
  • Hojatollah Vali
  • Christopher Leonard Brett
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

Abstract

SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have revealed many of the fundamental mechanisms that drive the membrane fusion process, as well as features unique to organelle fusion. However, until recently, it has not been amenable to electron microscopy methods that have been invaluable for studying hemifusion in other model systems. Herein, we describe a method to visualize hemifusion intermediates during homotypic vacuole membrane fusion in vitro by transmission electron microscopy (TEM), electron tomography, and cryogenic electron microscopy (cryoEM). This method facilitates acquisition of invaluable ultrastructural data needed to comprehensively understand how fusogenic lipids and proteins contribute to SNARE-mediated membrane fusion-by-hemifusion and the unique features of organelle versus small-vesicle fusion.

Key words

Membrane fusion Hemifusion SNARE Lysosome Vacuole Transmission electron microscopy (TEM) Cryogenic electron microscopy (cryoEM) Tomography Lipid bilayer merger 

Notes

Acknowledgments

We thank K. Basu and staff members at the Facility for Electron Microscopy Research at McGill University (Montreal, Canada) for technical assistance. S.M. was supported by a Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Award and a Fonds de Recherche du Québec Summer Research Scholarship. This work was supported by Natural Sciences and Engineering Research Council of Canada grants RGPIN/403537-2011 and RGPIN/2017-06652 to C.L.B.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sevan Mattie
    • 1
    • 2
  • Tom Kazmirchuk
    • 1
  • Jeannie Mui
    • 3
  • Hojatollah Vali
    • 3
  • Christopher Leonard Brett
    • 1
  1. 1.Department of BiologyConcordia UniversityMontréalCanada
  2. 2.Montreal Neurological Hospital and InstituteMcGill UniversityMontréalCanada
  3. 3.Facility for Electron Microscopy Research, Department of Anatomy and Cell BiologyMcGill UniversityMontréalCanada

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