TagsBooks chloride ChRs Cryptochrome Dimerizers Electroporation Epilepsy Expression fiberoptics FRET GECI GEVI Highlighters Holography hVoS Indicators LED Lines LOV Opsins Optics Optrodes Phototropin prosthetics protons Pumps Recordings Reporter Retina Reviews Scattering Therapeutics Thoughts Toxicity Viruses Voltage waveguides
Category Archives: Uncategorized
In a recent study published in in ACS Chemical Neuroscience, Campbell, Li, Nagai and coworkers report the development of a new series of orange and red genetically encoded Ca2+ indicators with improved sensitivity. To expand the color palette of genetically encoded Ca2+ indicators, semi-rational design and directed evolution were used to explore different chromophore structures and to modulate the environment adjacent to the chromophore of a previously reported red Ca2+ indicator, R-GECO1. These efforts lead to the identification of O-GECO (blue shifted), R-GECO1.2 and CAR-GECO1 (red shifted emission) with Ca2+ dependent intensiometric signal changes of 14600%, 3300% and 2700%, respectively (see figure 1 below). The authors go on to describe a troublesome photoactivation phenomenon that was discovered when these new indicators were used in conjunction with ChR2. Specifically, the fluorescence signals of these orange and red Ca2+ sensors exhibit a reversible increase with the intense blue light illumination used for ChR2 activation, even when there is no change in the Ca2+ concentration (see figure 2 below). By carrying out extensive in vitro and tissue-based characterizations, the authors showed that using an appropriate intensity of blue light could minimize this photoactivation problem and allowed these new orange and red Ca2+ indicators … Continue reading
If we could control the activity of any intracellular protein of interest with light, it would revolutionize how we study biology and engineer gene- or cell-based therapies. For instance, optical protein control would allow testing of protein functions in cells with high spatiotemporal precision, so we can observe how protein activation at specific times and places affects downstream biochemical reactions and cellular behavior, without secondary effects from chronic or ubiquitous activation. Optical protein control would also allow four-dimensional regulation of cellular proliferation, survival, or differentiation in tissues or animals for basic research or therapy. Thus controlling a wide variety of proteins with light is one of the major long-term goals of optogenetics.
Meeting this challenge will require the development of easily generalizable methods for optical control of protein activity. Considerable efforts have been made to adapt natural light-responsive signaling proteins to regulate specific proteins of interest. In recent years, phytochrome-PIF, cryptochrome-CIB, and FKF-gigantea light-dependent interactions from plants have been adapted to control heterodimerization of proteins in mammalian cells. The LOV domain from phototropin has also been used to create single-chain photoactivatable proteins via light-modulated allosteric mechanisms. However, these methods have certain disadvantages. Light-controlled heterodimerization can not effectively control activities of … Continue reading