Abstract
The ESCRT machinery mediates scission of the intercellular bridge that connects two daughter cells at the end of cytokinesis. Structured illumination microscopy (SIM) and cryo-soft-X-ray tomography (cryo-SXT) have been used in recent years to study the topology of ESCRT-driven cytokinetic abscission. These studies revealed that the intercellular bridge is occupied by cortical rings and spiral-like filaments and that ESCRTs form ring-like structures in this region during abscission. In this chapter, we provide two protocols: a protocol for determining the spatial organization of specific ESCRT components at the intercellular bridge using SIM and a protocol for resolving the ultrastructural organization of cortical filaments at the intercellular bridge using cryo-SXT.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Elia N, Sougrat R, Spurlin TA, Hurley JH, Lippincott-Schwartz J (2011) Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proc Natl Acad Sci U S A 108(12):4846–4851. https://doi.org/10.1073/pnas.1102714108
Elia N, Ott C, Lippincott-Schwartz J (2013) Incisive imaging and computation for cellular mysteries: lessons from abscission. Cell 155(6):1220–1231. https://doi.org/10.1016/j.cell.2013.11.011
Mierzwa BE, Chiaruttini N, Redondo-Morata L, Moser von Filseck J, Konig J, Larios J, Poser I, Muller-Reichert T, Scheuring S, Roux A, Gerlich DW (2017) Dynamic subunit turnover in ESCRT-III assemblies is regulated by Vps4 to mediate membrane remodelling during cytokinesis. Nat Cell Biol 19(7):787–798. https://doi.org/10.1038/ncb3559
Guizetti J, Schermelleh L, Mantler J, Maar S, Poser I, Leonhardt H, Muller-Reichert T, Gerlich DW (2011) Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331(6024):1616–1620. https://doi.org/10.1126/science.1201847
Van Engelenburg SB, Shtengel G, Sengupta P, Waki K, Jarnik M, Ablan SD, Freed EO, Hess HF, Lippincott-Schwartz J (2014) Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science 343(6171):653–656. https://doi.org/10.1126/science.1247786
Goliand I, Nachmias D, Gershony O, Elia N (2014) Inhibition of ESCRT-II-CHMP6 interactions impedes cytokinetic abscission and leads to cell death. Mol Biol Cell 25(23):3740–3748. https://doi.org/10.1091/mbc.E14-08-1317
Elia N, Fabrikant G, Kozlov M, Lippincott-Schwartz J (2012) Computational model of cytokinetic abscission driven by ESCRT-III polymerization and remodeling. Biophys J 102:2309–2320
Heintzmann R, Cremer CG (1999) Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc SPIE 3568(1):185–196
Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(Pt 2):82–87
Schneider G, Guttmann P, Heim S, Rehbein S, Mueller F, Nagashima K, Heymann JB, Muller WG, McNally JG (2010) Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nat Methods 7(12):985–987. https://doi.org/10.1038/nmeth.1533
Do M, Isaacson SA, McDermott G, Le Gros MA, Larabell CA (2015) Imaging and characterizing cells using tomography. Arch Biochem Biophys 581:111–121. https://doi.org/10.1016/j.abb.2015.01.011
Hagen C, Guttmann P, Klupp B, Werner S, Rehbein S, Mettenleiter TC, Schneider G, Grunewald K (2012) Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J Struct Biol 177(2):193–201. https://doi.org/10.1016/j.jsb.2011.12.012
Elad N, Abramovitch S, Sabanay H, Medalia O (2011) Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography. J Cell Sci 124(Pt 2):207–215. https://doi.org/10.1242/jcs.073486
Sherman S, Kirchenbuechler D, Nachmias D, Tamir A, Werner S, Elbaum M, Elia N (2016) Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography. Sci Rep 6:27629. https://doi.org/10.1038/srep27629
Varsano N, Dadosh T, Kapishnikov S, Pereiro E, Shimoni E, Jin X, Kruth HS, Leiserowitz L, Addadi L (2016) Development of correlative cryo-soft X-ray tomography and stochastic reconstruction microscopy. A study of cholesterol crystal early formation in cells. J Am Chem Soc 138(45):14931–14940. https://doi.org/10.1021/jacs.6b07584
Gershony O, Sherman S, Adar S, Segal I, Nachmias D, Goliand I, Elia N (2017) Measuring abscission spatiotemporal dynamics using quantitative high-resolution microscopy. Methods Cell Biol 137:205–224. https://doi.org/10.1016/bs.mcb.2016.03.032
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71–76. https://doi.org/10.1006/jsbi.1996.0013
Heymann JB, Belnap DM (2007) Bsoft: image processing and molecular modeling for electron microscopy. J Struct Biol 157(1):3–18. https://doi.org/10.1016/j.jsb.2006.06.006
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675
Agulleiro JI, Fernandez JJ (2011) Fast tomographic reconstruction on multicore computers. Bioinformatics 27(4):582–583. https://doi.org/10.1093/bioinformatics/btq692
Messaoudii C, Boudier T, Sanchez Sorzano CO, Marco S (2007) TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy. BMC Bioinformatics 8:288. https://doi.org/10.1186/1471-2105-8-288
Sorzano CO, Messaoudi C, Eibauer M, Bilbao-Castro JR, Hegerl R, Nickell S, Marco S, Carazo JM (2009) Marker-free image registration of electron tomography tilt-series. BMC Bioinformatics 10:124. https://doi.org/10.1186/1471-2105-10-124
Acknowledgments
We thank the all members of our laboratory and especially Dr. Dikla Nachmias for contributing technical tips. We acknowledge the Helmholtz-Zentrum Berlin–electron storage ring BESSY II for the allocation of synchrotron radiation beamtime at beamline U41-FSGM. This work is funded in part by the Instruct R&D pilot award for integrating biology, the Israeli Science Foundation (ISF) grant no. 455/13 (to N.E.) and 1285/14 (to M.E.), and the Marie Curie Integration Grant (CIG) (to N.E.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Adar-Levor, S., Goliand, I., Elbaum, M., Elia, N. (2019). Studying the Spatial Organization of ESCRTs in Cytokinetic Abscission Using the High-Resolution Imaging Techniques SIM and Cryo-SXT. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9492-2_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9491-5
Online ISBN: 978-1-4939-9492-2
eBook Packages: Springer Protocols