Optogenetic reagents allow for depolarization and hyperpolarization of cells with light. This provides unprecedented spatial and temporal resolution to the control of neuronal activity both in vitro and in vivo. In the intact animal this requires strategies to deliver light deep into the highly scattering tissue of the brain. A general approach that we describe here is to implant optical fibers just above brain regions targeted for light delivery. In part due to the fact that expression of optogenetic proteins is accomplished by techniques with inherent variability (e.g., viral expression levels), it also requires strategies to measure and calibrate the effect of stimulation. Here we describe general procedures that allow one to simultaneously stimulate neurons and use photometry with genetically encoded activity indicators to precisely calibrate stimulation.

Whole-cell recording has been used to measure and manipulate a neuron's spiking and subthreshold membrane potential, allowing assessment of the cell's inputs and outputs as well as its intrinsic membrane properties. This technique has also been combined with pharmacology and optogenetics as well as morphological reconstruction to address critical questions concerning neuronal integration, plasticity, and connectivity. This protocol describes a technique for obtaining whole-cell recordings in awake head-fixed animals, allowing such questions to be investigated within the context of an intact network and natural behavioral states. First, animals are habituated to sit quietly with their heads fixed in place. Then, a whole-cell recording is obtained using an efficient, blind patching protocol. We have successfully applied this technique to rats and mice.

Nitrate (NO3-) uptake and distribution are critical to plant life. Although the upstream regulation of nitrate uptake and downstream responses to nitrate in a variety of cells have been well-studied, it is still not possible to directly visualize the spatial and temporal distribution of nitrate with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded biosensor, nlsNitraMeter3.0, for the quantitative visualization of nitrate distribution in Arabidopsis thaliana. The biosensor tracked the spatiotemporal distribution of nitrate along the primary root axis and disruptions by genetic mutation of transport (low nitrate uptake) and assimilation (high nitrate accumulation). The developed biosensor effectively monitors nitrate concentrations at cellular level in real time and spatiotemporal changes during the plant life cycle.

Much focus has shifted towards understanding how glial dysfunction contributes to age-related neurodegeneration due to the critical roles glial cells play in maintaining healthy brain function. Cell-cell interactions, which are largely mediated by cell-surface proteins, control many critical aspects of development and physiology; as such, dysregulation of glial cell-surface proteins in particular is hypothesized to play an important role in age-related neurodegeneration. However, it remains technically difficult to profile glial cell-surface proteins in intact brains. Here, we applied a cell-surface proteomic profiling method to glial cells from intact brains in Drosophila, which enabled us to fully profile cell-surface proteomes in-situ, preserving native cell-cell interactions that would otherwise be omitted using traditional proteomics methods. Applying this platform to young and old flies, we investigated how glial cell-surface proteomes change during aging. We identified candidate genes predicted to be involved in brain aging, including several associated with neural development and synapse wiring molecules not previously thought to be particularly active in glia. Through a functional genetic screen, we identified one surface protein, DIP-β, which is down-regulated in old flies and can increase fly lifespan when overexpressed in adult glial cells. We further performed whole-head single-nucleus RNA-seq, and revealed that DIP-β overexpression mainly impacts glial and fat cells. We also found that glial DIP-β overexpression was associated with improved cell-cell communication, which may contribute to the observed lifespan extension. Our study is the first to apply in-situ cell-surface proteomics to glial cells in Drosophila, and to identify DIP-β as a potential glial regulator of brain aging.Competing Interest StatementThe authors have declared no competing interest.The original mass spectra and the protein sequence databases used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) (username: MSV000099083; password: glial). These datasets will be made public upon acceptance of the manuscript. Original proteomic data prior to analyses is provided in the Supplementary Table 1. snRNA-seq data has been deposited to NCBI Gene Expression Omnibus (GSE308135).

Although most proteins conform to the classical one-structure/one-function paradigm, an increasing number of proteins with dual structures and functions have been discovered. In response to cellular stimuli, such proteins undergo structural changes sufficiently dramatic to remodel even their secondary structures and domain organization. This "fold-switching" capability fosters protein multi-functionality, enabling cells to establish tight control over various biochemical processes. Accurate predictions of fold-switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Recently, we developed a prediction method for fold-switching proteins using structure-based thermodynamic calculations and discrepancies between predicted and experimentally determined protein secondary structure. Here we seek to leverage the negative information found in these secondary structure prediction discrepancies. To do this, we quantified secondary structure prediction accuracies of 192 known fold-switching regions (FSRs) within solved protein structures found in the Protein Data Bank (PDB). We find that the secondary structure prediction accuracies for these FSRs vary widely. Inaccurate secondary structure predictions are strongly associated with fold-switching proteins compared to equally long segments of non-fold-switching proteins selected at random. These inaccurate predictions are enriched in helix-to-strand and strand-to-coil discrepancies. Finally, we find that most proteins with inaccurate secondary structure predictions are underrepresented in the PDB compared with their alternatively folded cognates, suggesting that unequal representation of fold-switching conformers within the PDB could be an important cause of inaccurate secondary structure predictions. These results demonstrate that inconsistent secondary structure predictions can serve as a useful preliminary marker of fold switching. This article is protected by copyright. All rights reserved.

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER “sheet” proliferation after overexpression of tubular junction–forming proteins.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson’s excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

SUMMARY: INFERNAL builds consensus RNA secondary structure profiles called covariance models (CMs), and uses them to search nucleic acid sequence databases for homologous RNAs, or to create new sequence- and structure-based multiple sequence alignments. AVAILABILITY: Source code, documentation and benchmark downloadable from http://infernal.janelia.org. INFERNAL is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X.