[Differences of Nonsense-Mediated mRNA Degradation Activity in Mammalian Cell Lines Revealed by a Fluorescence Reporter].

Activity of nonsense-mediated mRNA degradation (NMD) was studied in several mammalian cell cultures using recently developed genetically encoded fluorescence sensor [Pereverzev et al., Sci. Rep., 2015, vol. 5, p. 7729]. This NMD reporter enables measurement of NMD activity in single live cells using ratio of green and red fluorescent proteins signals. The following cell lines were analyzed: mouse colon carcinoma CT26, mouse Lewis lung carcinoma LLC, human T-cell leukemia Jurkat, and spontaneously immortalized human keratinocytes HaCaT. These cell lines demonstrated very different NMD activities. In CT26, NMD activity was low, whereas in LLC it was high (8.5-fold higher than in CT26). Jurkat and HaCaT cells possessed strong heterogeneity and consisted of two cell subpopulations with high and low NMD activities. In addition, we detected high NMD activity in primary culture of mouse embryonic hippocampal neurons.

Fiber-optic control and thermometry of single-cell thermosensation logic.

Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen--vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels.