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Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy

Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy
Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy
Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm-1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm-1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm-1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.
1932-6203
1-9
Perney, Nicolas M.
169bd490-31cb-4379-b25b-ae3e7880081b
Braddick, Lucy
c43b91b2-a10a-414c-a73e-ed26c62c8797
Jurna, Martin
f54c009d-a025-4252-a9dd-d335302e3cbb
Garbacik, Erik T.
c7ba8694-72f1-4d99-8363-2b383f1d7dfe
Offerhaus, Herman L.
d7748841-61eb-4055-bb95-e7d5f5916552
Serpell, Louise C.
3a51f573-781c-4c80-bb36-bfc6c8f73c10
Blanch, Ewan
5149101e-190e-4bf8-8bbb-509224713c7c
Holden-Dye, Lindy
8032bf60-5db6-40cb-b71c-ddda9d212c8e
Brocklesby, William S.
c53ca2f6-db65-4e19-ad00-eebeb2e6de67
Melvin, Tracy
fd87f5eb-2bb9-48fa-b7be-7100ace9c50f
Perney, Nicolas M.
169bd490-31cb-4379-b25b-ae3e7880081b
Braddick, Lucy
c43b91b2-a10a-414c-a73e-ed26c62c8797
Jurna, Martin
f54c009d-a025-4252-a9dd-d335302e3cbb
Garbacik, Erik T.
c7ba8694-72f1-4d99-8363-2b383f1d7dfe
Offerhaus, Herman L.
d7748841-61eb-4055-bb95-e7d5f5916552
Serpell, Louise C.
3a51f573-781c-4c80-bb36-bfc6c8f73c10
Blanch, Ewan
5149101e-190e-4bf8-8bbb-509224713c7c
Holden-Dye, Lindy
8032bf60-5db6-40cb-b71c-ddda9d212c8e
Brocklesby, William S.
c53ca2f6-db65-4e19-ad00-eebeb2e6de67
Melvin, Tracy
fd87f5eb-2bb9-48fa-b7be-7100ace9c50f

Perney, Nicolas M., Braddick, Lucy, Jurna, Martin, Garbacik, Erik T., Offerhaus, Herman L., Serpell, Louise C., Blanch, Ewan, Holden-Dye, Lindy, Brocklesby, William S. and Melvin, Tracy (2012) Polyglutamine aggregate structure in vitro and in vivo; new avenues for coherent anti-Stokes Raman scattering microscopy. PLoS ONE, 7 (7), 1-9, [e40536]. (doi:10.1371/journal.pone.0040536).

Record type: Article

Abstract

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D2Q15K2) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm-1. CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm-1) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm-1) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.

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Published date: 20 July 2012
Organisations: Optoelectronics Research Centre, Electronics & Computer Science

Identifiers

Local EPrints ID: 341467
URI: http://eprints.soton.ac.uk/id/eprint/341467
ISSN: 1932-6203
PURE UUID: 6e90b094-e594-4cf3-b1a1-712b0fd0a5ac
ORCID for Lindy Holden-Dye: ORCID iD orcid.org/0000-0002-9704-1217
ORCID for William S. Brocklesby: ORCID iD orcid.org/0000-0002-2123-6712

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Date deposited: 27 Jul 2012 08:44
Last modified: 08 Apr 2020 00:24

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