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 Citations (Scopus)

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 publicationMethods in Molecular Biology
Pages229-239
Number of pages11
Volume1017
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume1017
ISSN (Print)10643745

Fingerprint

Green Fluorescent Proteins
Huntington Disease
Poisons
Inclusion Bodies
Mutant Proteins
Nervous System Diseases
Exons
Cytoplasm
Cell Death
polyglutamine
Apoptosis
Pathology
Huntingtin Protein

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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

Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP. / Lajoie, Patrick; Snapp, Erik Lee.

Methods in Molecular Biology. Vol. 1017 2013. p. 229-239 (Methods in Molecular Biology; Vol. 1017).

Research output: Chapter in Book/Report/Conference proceedingChapter

Lajoie, P & Snapp, EL 2013, Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP. in Methods in Molecular Biology. vol. 1017, Methods in Molecular Biology, vol. 1017, pp. 229-239. https://doi.org/10.1007/978-1-62703-438-8-17
Lajoie P, Snapp EL. Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP. In Methods in Molecular Biology. Vol. 1017. 2013. p. 229-239. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-62703-438-8-17
Lajoie, Patrick ; Snapp, Erik Lee. / Detecting soluble PolyQ oligomers and investigating their impact on living cells using split-GFP. Methods in Molecular Biology. Vol. 1017 2013. pp. 229-239 (Methods in Molecular Biology).
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