Advertisement

Aggregation Profiling of C9orf72 Dipeptide Repeat Proteins Transgenically Expressed in Drosophila melanogaster Using an Analytical Ultracentrifuge Equipped with Fluorescence Detection

  • Bashkim Kokona
  • Nicole R. Cunningham
  • Jeanne M. Quinn
  • Robert FairmanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2039)

Abstract

The recent development of a fluorescence detection system for the analytical ultracentrifuge has allowed for the characterization of protein size and aggregation in complex mixtures. Protocols are described here to analyze protein aggregation seen in various human neurodegenerative diseases as they are presented in transgenic animal model systems. Proper preparation of crude extracts in appropriate sample buffers is critical for success in analyzing protein aggregation using sedimentation velocity methods. Furthermore, recent advances in sedimentation velocity analysis have led to data collection using single multispeed experiments, which may be analyzed using a wide distribution analysis approach. In this chapter, we describe the use of these new sedimentation velocity methods for faster determination of a wider range of sizes. In Chapter 7 of this book, we describe how agarose gel electrophoresis can be used to complement the analytical ultracentrifugation work, often as a prelude to careful biophysical analysis to help screen conditions in order to improve the success of sedimentation velocity experiments.

Key words

Analytical ultracentrifugation Sedimentation velocity Protein aggregation Neurodegeneration Amyotrophic lateral sclerosis Frontotemporal dementia Drosophila melanogaster 

References

  1. 1.
    Kingsbury JS, Laue TM (2011) Fluorescence-detected sedimentation in dilute and highly concentrated solutions. Methods Enzymol 492:283–304CrossRefGoogle Scholar
  2. 2.
    Kroe RR, Laue TM (2009) NUTS and BOLTS: applications of fluorescence-detected sedimentation. Anal Biochem 390:1–13CrossRefGoogle Scholar
  3. 3.
    Kim SA, D'Acunto VF, Kokona B, Hofmann J, Cunningham NR, Bistline EM et al (2017) Sedimentation velocity analysis with fluorescence detection of mutant huntingtin exon 1 aggregation in Drosophila melanogaster and Caenorhabditis elegans. Biochemistry 56:4676–4688CrossRefGoogle Scholar
  4. 4.
    Kokona B, May CA, Cunningham NR, Richmond L, Garcia FJ, Durante JC et al (2015) Studying polyglutamine aggregation in Caenorhabditis elegans using an analytical ultracentrifuge equipped with fluorescence detection. Protein Sci 25:605–617CrossRefGoogle Scholar
  5. 5.
    Olshina MA, Angley LM, Ramdzan YM, Tang J, Bailey MF, Hill AF et al (2010) Tracking mutant huntingtin aggregation kinetics in cells reveals three major populations that include an invariant oligomer pool. J Biol Chem 285:21807–21816CrossRefGoogle Scholar
  6. 6.
    Xi W, Wang X, Laue TM, Denis CL (2016) Multiple discrete soluble aggregates influence polyglutamine toxicity in a Huntington's disease model system. Sci Rep 6:34916CrossRefGoogle Scholar
  7. 7.
    Runge MS, Laue TM, Yphantis DA, Lifsics MR, Saito A, Altin M et al (1981) ATP-induced formation of an associated complex between microtubules and neurofilaments. Proc Natl Acad Sci U S A 78:1431–1435CrossRefGoogle Scholar
  8. 8.
    Stafford WF, Braswell EH (2004) Sedimentation velocity, multi-speed method for analyzing polydisperse solutions. Biophys Chem 108:273–279CrossRefGoogle Scholar
  9. 9.
    DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256CrossRefGoogle Scholar
  10. 10.
    Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR et al (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72:257–268CrossRefGoogle Scholar
  11. 11.
    Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH et al (2015) GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature 525:129–133CrossRefGoogle Scholar
  12. 12.
    Haeusler AR, Donnelly CJ, Periz G, Simko EA, Shaw PG, Kim MS et al (2014) C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507:195–200CrossRefGoogle Scholar
  13. 13.
    Polling S, Hatters DM, Mok YF (2013) Size analysis of polyglutamine protein aggregates using fluorescence detection in an analytical ultracentrifuge. Methods Mol Biol 1017:59–71CrossRefGoogle Scholar
  14. 14.
    Stafford WF, Sherwood PJ (2004) Analysis of heterologous interacting systems by sedimentation velocity: curve fitting algorithms for estimation of sedimentation coefficients, equilibrium and kinetic constants. Biophys Chem 108:231–243CrossRefGoogle Scholar
  15. 15.
    Stafford WF 3rd (1992) Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 203:295–301CrossRefGoogle Scholar
  16. 16.
    Schuck P, Zhao H (2011) Editorial for the special issue of methods “modern analytical ultracentrifugation”. Methods 54:1–3CrossRefGoogle Scholar
  17. 17.
    Dam J, Velikovsky CA, Mariuzza RA, Urbanke C, Schuck P (2005) Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s). Biophys J 89:619–634CrossRefGoogle Scholar
  18. 18.
    Zhao H, Fu Y, Glasser C, Andrade Alba EJ, Mayer ML, Patterson G et al (2016) Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions. eLife 5:pii: e17812CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Bashkim Kokona
    • 1
  • Nicole R. Cunningham
    • 1
  • Jeanne M. Quinn
    • 1
  • Robert Fairman
    • 1
    Email author
  1. 1.Department of BiologyHaverford CollegeHaverfordUSA

Personalised recommendations