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Red-shifted voltage-sensitive fluorescent proteins.

ABSTRACT: Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically encoded voltage-sensitive fluorescent proteins by molecular fusion of the voltage-sensing domain of Ci-VSP (Ciona intestinalis voltage sensor-containing phosphatase) to red-shifted fluorescent protein operands. We show how these indicator proteins convert voltage-dependent structural rearrangements into a modulation of fluorescence output and demonstrate their applicability for optical recording of individual or simultaneous electrical signals in cultured hippocampal neurons at single-cell resolution without temporal averaging.

SUBMITTER: Perron A

PROVIDER: S-EPMC2818747 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Red-shifted voltage-sensitive fluorescent proteins.

Perron Amelie A Mutoh Hiroki H Launey Thomas T Knöpfel Thomas T

Chemistry & biology 20091201 12

Electrical signals generated by nerve cells provide the basis of brain function. Whereas single or small numbers of cells are easily accessible using microelectrode recording techniques, less invasive optogenetic methods with spectral properties optimized for in vivo imaging are required for elucidating the operation mechanisms of neuronal circuits composed of large numbers of neurons originating from heterogeneous populations. To this end, we generated and characterized a series of genetically ...[more]

PMID: 20064437

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Project description:BackgroundHuman African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.Methodology/principal findingsHere, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5'-variant surface glycoprotein untranslated region (UTR) and a downstream 3'-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter.Conclusions/significanceThe optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

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Red-shifted voltage-sensitive fluorescent proteins.

Publications

Red-shifted voltage-sensitive fluorescent proteins.

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