Membrane-coated glass electrodes for stable, low-noise electrophysiology recordings in Drosophila central neurons.

BACKGROUND: Electrophysiological recording with glass electrodes is one of the best techniques to measure membrane potential dynamics and ionic currents of voltage-gated channels in neurons. However, artifactual variability of the biophysical state variables that determine recording quality can be caused by insufficient affinity between the electrode and cell membrane during the recording. NEW METHOD: We introduce a phospholipid membrane coating on glass electrodes to improve intracellular electrophysiology recording quality. Membrane-coated electrodes were prepared with a tip-dip protocol for perforated-patch, sharp-electrode current-clamp, and cell-attached patch-clamp recordings from specific circadian clock neurons in Drosophila. We perform quantitative comparisons based on the variability of functional biophysical parameters used in various electrophysiological methods, and advanced statistical comparisons based on the degree of stationariness and signal-to-noise ratio. RESULTS: Results indicate a dramatic reduction in artifactual variabilities of functional parameters from enhanced stability. We also identify significant exclusions of a statistically estimated noise component in a time series of membrane voltage signals, improving signal-to-noise ratio. COMPARISON WITH EXISTING METHODS: Compared to standard glass electrodes, using membrane-coated glass electrodes achieves improved recording quality in intracellular electrophysiology. CONCLUSIONS: Electrophysiological recordings from Drosophila central neurons can be technically challenging, however, membrane-coated electrodes will possibly be beneficial for reliable data acquisition and improving the technical feasibility of axonal intracellular activities measurements and single-channel recordings. The improved electrical stability of the recordings should also contribute to increased mechanical stability, thus facilitating long-term stable measurements of neural activity. Therefore, it is possible that membrane-coated electrodes will be useful for any model system.

Engineered ice-binding protein (FfIBP) shows increased stability and resistance to thermal and chemical denaturation compared to the wildtype.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing-thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.

Structural and functional characterization of sulfurtransferase from Frondihabitans sp. PAMC28461.

Sulfurtransferases transfer of sulfur atoms from thiols to acceptors like cyanide. They are categorized as thiosulfate sulfurtransferases (TSTs) and 3-mercaptopyruvate sulfurtransferases (MSTs). TSTs transfer sulfur from thiosulfate to cyanide, producing thiocyanate. MSTs transfer sulfur from 3-mercaptopyruvate to cyanide, yielding pyruvate and thiocyanate. The present study aimed to isolate and characterize the sulfurtransferase FrST from Frondihabitans sp. PAMC28461 using biochemical and structural analyses. FrST exists as a dimer and can be classified as a TST rather than an MST according to sequence-based clustering and enzyme activity. Furthermore, the discovery of activity over a wide temperature range and the broad substrate specificity exhibited by FrST suggest promising prospects for its utilization in industrial applications, such as the detoxification of cyanide.