The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.

Pluripotent stem cells give rise to all cells of the adult organism, making them an invaluable tool in regenerative medicine. In response to differentiation cues, they can activate markedly distinct lineagespecific gene networks while turning off or rewiring pluripotency networks. Recent innovations in chromatin and nuclear structure analyses combined with classical genetics have led to novel insights into the transcriptional and epigenetic mechanisms underlying these networks. Here, we review these findings in relation to their impact on the maintenance of and exit from pluripotency and highlight the many factors that drive these processes, including histone modifying enzymes, DNA methylation and demethylation, nucleosome remodeling complexes and transcription factor-mediated enhancer switching.

MicroRNAs (miRNAs) are crucial regulators of myelination in the peripheral nervous system (PNS). However, the miRNAs species involved and the underlying mechanisms are largely unknown. We found that let-7 miRNAs are highly abundant during PNS myelination and that their levels are inversely correlated to the expression of lin28 homolog B (Lin28B), an antagonist of let-7 accumulation. Sustained expression of Lin28B and consequently reduced levels of let-7 miRNAs results in a failure of Schwann cell myelination in transgenic mouse models and in cell culture. Subsequent analyses revealed that let-7 miRNAs promote expression of the myelination-driving master transcription factor Krox20 (also known as Egr2) through suppression of myelination inhibitory Notch signalling. We conclude that the Lin28B/let-7 axis acts as a critical driver of PNS myelination, in particular by regulating myelination onset, identifying this pathway also as a potential therapeutic target in demyelinating diseases.