Skip to main content
SpringerLink
Log in
Book cover

Yeast Cytokinesis pp 151–179Cite as

In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1369))

Abstract

Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) “bulk” pyrene actin assembly/disassembly assays, and (3) “bulk” sedimentation actin filament binding and bundling assays.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Blanchoin L et al (2014) Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev 94(1):235–263

    Article  CAS  PubMed  Google Scholar 

  2. Chhabra ES, Higgs HN (2007) The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol 9(10):1110–1121

    Article  CAS  PubMed  Google Scholar 

  3. Balasubramanian MK, Bi E, Glotzer M (2004) Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells. Curr Biol 14:R806–R818

    Article  CAS  PubMed  Google Scholar 

  4. Glotzer M (2005) The molecular requirements for cytokinesis. Science 307(5716):1735–1739

    Article  CAS  PubMed  Google Scholar 

  5. Pollard TD, Wu JQ (2010) Understanding cytokinesis: lessons from fission yeast. Nat Rev Mol Cell Biol 11(2):149–155

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Lee IJ, Coffman VC, Wu JQ (2012) Contractile-ring assembly in fission yeast cytokinesis: recent advances and new perspectives. Cytoskeleton (Hoboken) 69(10):751–763

    Article  CAS  Google Scholar 

  7. Spudich JA, Watt S (1971) The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem 246(15):4866–4871

    CAS  PubMed  Google Scholar 

  8. Kuhn JR, Pollard TD (2005) Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy. Biophys J 88(2):1387–1402

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Kudryashov DS, Reisler E (2003) Solution properties of tetramethylrhodamine-modified G-actin. Biophys J 85(4):2466–2475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Cooper JA, Walker SB, Pollard TD (1983) Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil 4(2):253–262

    Article  CAS  PubMed  Google Scholar 

  11. Kouyama T, Mihashi K (1981) Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem 114(1):33–38

    Article  CAS  PubMed  Google Scholar 

  12. Cleveland DW (1982) Treadmilling of tubulin and actin. Cell 28(4):689–691

    Article  CAS  PubMed  Google Scholar 

  13. Neuhaus JM et al (1983) Treadmilling of actin. J Muscle Res Cell Motil 4(5):507–527

    Article  CAS  PubMed  Google Scholar 

  14. Pollard TD (1986) Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol 103(6 Pt 2):2747–2754

    Article  CAS  PubMed  Google Scholar 

  15. Wegner A, Isenberg G (1983) 12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions. Proc Natl Acad Sci U S A 80(16):4922–4925

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Theriot JA, Mitchison TJ (1991) Actin microfilament dynamics in locomoting cells. Nature 352(6331):126–131

    Article  CAS  PubMed  Google Scholar 

  17. Axelrod D, Thompson NL, Burghardt TP (1983) Total internal inflection fluorescent microscopy. J Microsc 129(Pt 1):19–28

    Article  CAS  PubMed  Google Scholar 

  18. Skau CT et al (2011) Actin filament bundling by fimbrin is important for endocytosis, cytokinesis and polarization in fission yeast. J Biol Chem 286:26964

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Biochemical studies of cytokinesis ABPs in the Kovar lab is supported by NIH grant R01 GM079265 (to D.R.K.), ACS grant RSG-11-126-01-CSM (to D.R.K.), and DOD MURI grant W911NF-14-1-0403 (to D.R.K.). We thank Jenna Christensen, Katie Homa, and Tom Burke for helpful comments and suggestions.

Author information

Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, 60637, USA

    Dennis Zimmermann, Alisha N. Morganthaler, David R. Kovar & Cristian Suarez

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, 920 E. 58th St., Chicago, IL, 60637, USA

    David R. Kovar

Authors
  1. Dennis Zimmermann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alisha N. Morganthaler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David R. Kovar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cristian Suarez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David R. Kovar .

Editor information

Editors and Affiliations

  1. Departamento de Biología Molecular, Universidad de Cantabria, Facultad de Medicina, Cardenal Herrera Oria s/n, Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria, CSIC, Santander, Spain

    Alberto Sanchez-Diaz

  2. Universidad de Salamanca, Instituto de Biología Funcional y Genómica, CSIC, Salamanca, Spain

    Pilar Perez

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Zimmermann, D., Morganthaler, A.N., Kovar, D.R., Suarez, C. (2016). In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins. In: Sanchez-Diaz, A., Perez, P. (eds) Yeast Cytokinesis. Methods in Molecular Biology, vol 1369. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3145-3_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3145-3_12

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3144-6

  • Online ISBN: 978-1-4939-3145-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation