Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage - PubMed (original) (raw)

. 2007 Dec;103(6):2529-39.

doi: 10.1111/j.1471-4159.2007.04941.x. Epub 2007 Oct 1.

Affiliations

• PMID: 17908237
• DOI: 10.1111/j.1471-4159.2007.04941.x

Free article

Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage

Wanjoo Chun et al. J Neurochem. 2007 Dec.

Free article

Abstract

To quantitatively measure tau aggregation in situ, we established a cell model system using a split green fluorescence protein (GFP) complementation assay. In this assay the more aggregated the protein of interest the lower the GFP fluorescence. Tau microtubule-binding domain constructs, whose aggregation characteristics have been described previously (Khlistunova et al. 2006), were used to validate the assay. The aggregation-prone construct exhibited the lowest GFP intensity whereas the aggregation-resistant construct showed the highest GFP intensity. To examine the role of glycogen synthase kinase 3beta (GSK3beta) activity and caspase 3 cleavage on tau aggregation, GFP complementation of full length (T4), caspase-cleaved (T4C3), and pseudophosphorylated at S396/S404 (T4-2EC) tau was examined in the presence of an active or a kinase-dead GSK3beta. Extensive phosphorylation of T4 by GSK3beta resulted in increased GFP intensity. T4C3 showed neither efficient phosphorylation nor a significant GFP intensity change by GSK3beta. The GFP intensity of T4-2EC was significantly reduced by GSK3beta accompanying its presence in the sarkosyl-insoluble fraction, thus demonstrating that T4-2EC was partitioning into aggregates. This indicates that if the majority of tau is phosphorylated at S396/S404, in combination with increased GSK3beta activity, tau aggregation is favored. These data demonstrate that split GFP complementation may be a valuable approach to determine the aggregation process in living cells.

PubMed Disclaimer

Similar articles

• Activation of glycogen synthase kinase 3beta promotes the intermolecular association of tau. The use of fluorescence resonance energy transfer microscopy.
  Chun W, Johnson GV. Chun W, et al. J Biol Chem. 2007 Aug 10;282(32):23410-7. doi: 10.1074/jbc.M703706200. Epub 2007 Jun 12. J Biol Chem. 2007. PMID: 17565981
• 14-3-3zeta facilitates GSK3beta-catalyzed tau phosphorylation in HEK-293 cells by a mechanism that requires phosphorylation of GSK3beta on Ser9.
  Li T, Paudel HK. Li T, et al. Neurosci Lett. 2007 Mar 13;414(3):203-8. doi: 10.1016/j.neulet.2006.11.073. Epub 2007 Feb 3. Neurosci Lett. 2007. PMID: 17317006
• Active c-jun N-terminal kinase induces caspase cleavage of tau and additional phosphorylation by GSK-3beta is required for tau aggregation.
  Sahara N, Murayama M, Lee B, Park JM, Lagalwar S, Binder LI, Takashima A. Sahara N, et al. Eur J Neurosci. 2008 Jun;27(11):2897-906. doi: 10.1111/j.1460-9568.2008.06258.x. Epub 2008 Jun 6. Eur J Neurosci. 2008. PMID: 18540881
• O-GlcNAc modification and the tauopathies: insights from chemical biology.
  Yuzwa SA, Vocadlo DJ. Yuzwa SA, et al. Curr Alzheimer Res. 2009 Oct;6(5):451-4. doi: 10.2174/156720509789207967. Curr Alzheimer Res. 2009. PMID: 19874270 Review.
• Interactions between Microtubule-Associated Protein Tau (MAPT) and Small Molecules.
  Rauch JN, Olson SH, Gestwicki JE. Rauch JN, et al. Cold Spring Harb Perspect Med. 2017 Jul 5;7(7):a024034. doi: 10.1101/cshperspect.a024034. Cold Spring Harb Perspect Med. 2017. PMID: 27940599 Free PMC article. Review.

Cited by

• The role of wild-type tau in Alzheimer's disease and related tauopathies.
  Lo CH, Sachs JN. Lo CH, et al. J Life Sci (Westlake Village). 2020 Dec;2(4):1-17. doi: 10.36069/jols/20201201. J Life Sci (Westlake Village). 2020. PMID: 33665646 Free PMC article.
• An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta.
  Han SW, Park CJ, Lee SW, Ronald PC. Han SW, et al. BMC Microbiol. 2008 Sep 30;8:164. doi: 10.1186/1471-2180-8-164. BMC Microbiol. 2008. PMID: 18826644 Free PMC article.
• Structure and mechanism of action of tau aggregation inhibitors.
  Cisek K, Cooper GL, Huseby CJ, Kuret J. Cisek K, et al. Curr Alzheimer Res. 2014;11(10):918-27. doi: 10.2174/1567205011666141107150331. Curr Alzheimer Res. 2014. PMID: 25387336 Free PMC article. Review.
• The Brucella TIR-like protein TcpB interacts with the death domain of MyD88.
  Chaudhary A, Ganguly K, Cabantous S, Waldo GS, Micheva-Viteva SN, Nag K, Hlavacek WS, Tung CS. Chaudhary A, et al. Biochem Biophys Res Commun. 2012 Jan 6;417(1):299-304. doi: 10.1016/j.bbrc.2011.11.104. Epub 2011 Nov 29. Biochem Biophys Res Commun. 2012. PMID: 22155231 Free PMC article.
• Recent Insights on Alzheimer's Disease Originating from Yeast Models.
  Seynnaeve D, Vecchio MD, Fruhmann G, Verelst J, Cools M, Beckers J, Mulvihill DP, Winderickx J, Franssens V. Seynnaeve D, et al. Int J Mol Sci. 2018 Jul 3;19(7):1947. doi: 10.3390/ijms19071947. Int J Mol Sci. 2018. PMID: 29970827 Free PMC article. Review.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Ovid Technologies, Inc.
  • Wiley

• Other Literature Sources

  • The Lens - Patent Citations Database

• Research Materials

  • NCI CPTC Antibody Characterization Program

• Miscellaneous

  • NCI CPTAC Assay Portal