Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically-encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally-applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high-to-low calcium signal ratio of 10 after 10 minutes of stimulation. Channelrhodopsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.
Protein-chromophore interactions are a central component of a wide variety of critical biological processes, such as color vision and photosynthesis. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside the protein cavity, we have redesigned human Cellular Retinol Binding Protein II (hCRBPII) to fully encapsulate all-trans-retinal and form a covalent bond as a protonated Schiff base. Using this system, the absorption maximum of the pigment was regulated from 425 nm to 644 nm using rational mutagenesis designed to alter the electrostatic environment within the binding pocket of the host protein. Employing only 9 point mutations, the hCRBPII mutants induce a systematic shift in the absorption profile of all trans-retinal of over 200 nm across the visible spectrum.
Protein
reengineering of cellular retinoic acid binding protein
II (CRABPII) has yielded a genetically addressable system, capable
of binding a profluorophoric chromophore that results in fluorescent
protein/chromophore complexes. These complexes exhibit far-red emission,
with high quantum efficiencies and brightness and also exhibit excellent
pH stability spanning the range of 2–11. In the course of this
study, it became evident that single mutations of L121E and R59W were
most effective in improving the fluorescent characteristics of CRABPII
mutants as well as the kinetics of complex formation. The readily
crystallizable nature of these proteins was invaluable to provide
clues for the observed spectroscopic behavior that results from single
mutation of key residues.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.