Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP

Patrick Lajoie, Erik Lee Snapp

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

Aberrant expansion of the number of polyglutamine (polyQ) repeats in mutant proteins is the hallmark of various diseases. These pathologies include Huntington's disease (HD), a neurological disorder caused by expanded polyQ stretch within the huntingtin (Htt) protein. The expansions increase the propensity of the Htt protein to oligomerize. In the cytoplasm of living cells, the mutant form of Htt (mHtt) is present as soluble monomers and oligomers as well as insoluble aggregates termed inclusion bodies (IBs). Detecting and assessing the relative toxicity of these various forms of mHtt has proven difficult. To enable direct visualization of mHtt soluble oligomers in living cells, we established a split superfolder green fluorescent protein (sfGFP) complementation assay. In this assay, exon 1 variants of Htt (Httex1) containing non-pathological or HD-associated polyQ lengths were fused to two different nonfluorescent fragments of sfGFP. If the Htt proteins oligomerize and the sfGFP fragments come into close proximity, they can associate and complement each other to form a complete and fluorescent sfGFP reporter. Importantly, the irreversible nature of the split-sfGFP complementation allowed us to trap otherwise transient interactions and artificially increase mHtt oligomerization. When coupled with a fluorescent apoptosis reporter, this assay can correlate soluble mHtt oligomer levels and cell death leading to a better characterization of the toxic potential of various forms of mHtt in living cells.

Original languageEnglish (US)
Title of host publicationTandem Repeats in Genes, Proteins, and Disease
Subtitle of host publicationMethods and Protocols
Pages229-239
Number of pages11
DOIs
StatePublished - Sep 3 2013

Publication series

NameMethods in Molecular Biology
Volume1017
ISSN (Print)1064-3745

Keywords

  • Cell death
  • Huntingtin exon 1
  • Oligomers
  • Split-GFP

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Lajoie, P., & Snapp, E. L. (2013). Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP. In Tandem Repeats in Genes, Proteins, and Disease: Methods and Protocols (pp. 229-239). (Methods in Molecular Biology; Vol. 1017). https://doi.org/10.1007/978-1-62703-438-8-17