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Methodological improvements for fluorescence recordings in Xenopus laevis oocytes.

ABSTRACT: Xenopus laevis oocytes are a widely used model system because of their capacity to translate exogenous mRNA, but their high intrinsic background fluorescence is a disadvantage for fluorescence recordings. Here, we developed two distinct methods for improving fluorescence recordings from oocytes. One was a pharmacological method in which a small-molecule salt-inducible kinase inhibitor was co-injected with the mRNA of interest to stimulate melanin production. We interrogated the oocytes using cut-open voltage clamp with simultaneous fluorescence recording and found that by increasing the amount of light-absorbing melanin in these oocytes, we decreased their intrinsic background fluorescence. The treated oocytes produced fluorescence signals that were approximately four times larger. The second method consisted of direct injection of synthetic melanin. This method also significantly improved (doubled) fluorescence signals and allowed any oocyte to be used for fluorescence recording. These two methods provide significant improvements of the signal quality for fluorescent oocyte recordings and allow all healthy oocytes to be used for high-sensitivity recordings.

SUBMITTER: Lee EEL

PROVIDER: S-EPMC6363413 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Project description:BackgroundA new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target.ResultsTo test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential.ConclusionThe M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

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Methodological improvements for fluorescence recordings in Xenopus laevis oocytes.

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