Methylene blue- and thiol-based oxygen depletion for super-resolution imaging.

Anaerobic conditions are often required in solution-based bionanotechnological applications. Efficient oxygen depletion is essential for increasing photostability, optimizing fluorescence signals, and adjusting kinetics of fluorescence intermittency in single-molecule fluorescence spectroscopy/microscopy, particularly for super-resolution imaging techniques. We characterized methylene blue (MB)- and thiol-based redox reactions with the aim of designing an oxygen scavenger system as an alternative to the established enzyme-based oxygen scavenging systems or purging procedures. Redox reactions of the chromophore methylene blue in aqueous solution, commonly visualized in the blue bottle experiment, deplete molecular oxygen as long as a sacrificial reduction component is present in excess concentrations. We demonstrate that methylene blue in combination with reducing compounds such as ß-mercaptoethylamine (MEA) can serve as fast and efficient oxygen scavenger. Efficient oxygen scavenging in aqueous solution is also possible with mere ß-mercaptoethylamine at mM concentrations. We present kinetic parameters of the relevant reactions, pH-stability of the MB/MEA-oxygen scavenging system, and its application in single-molecule based super-resolution imaging.

Acidosis-induced activation of anion channel SLAH3 in the flooding-related stress response of Arabidopsis.

Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

Light-induced cell damage in live-cell super-resolution microscopy.

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

Loss of Nogo receptor homolog NgR2 alters spine morphology of CA1 neurons and emotionality in adult mice.

Molecular mechanisms which stabilize dendrites and dendritic spines are essential for regulation of neuronal plasticity in development and adulthood. The class of Nogo receptor proteins, which are critical for restricting neurite outgrowth inhibition signaling, have been shown to have roles in developmental, experience and activity induced plasticity. Here we investigated the role of the Nogo receptor homolog NgR2 in structural plasticity in a transgenic null mutant for NgR2. Using Golgi-Cox staining to analyze morphology, we show that loss of NgR2 alters spine morphology in adult CA1 pyramidal neurons of the hippocampus, significantly increasing mushroom-type spines, without altering dendritic tree complexity. Furthermore, this shift is specific to apical dendrites in distal CA1 stratum radiatum (SR). Behavioral alterations in NgR2(-/-) mice were investigated using a battery of standardized tests and showed that whilst there were no alterations in learning and memory in NgR2(-/-) mice compared to littermate controls, NgR2(-/-) displayed reduced fear expression in the contextual conditioned fear test, and exhibited reduced anxiety- and depression-related behaviors. This suggests that the loss of NgR2 results in a specific phenotype of reduced emotionality. We conclude that NgR2 has role in maintenance of mature spines and may also regulate fear and anxiety-like behaviors.