Although most proteins conform to the classical one-structure/one-function paradigm, an increasing number of proteins with dual structures and functions are emerging. These fold-switching proteins remodel their secondary structures in response to cellular stimuli, fostering multi-functionality and tight cellular control. Accurate predictions of fold-switching proteins could both suggest underlying mechanisms for uncharacterized biological processes and reveal potential drug targets. Previously, we developed a prediction method for fold-switching proteins based on secondary structure predictions and structure-based thermodynamic calculations. Given the large number of genomic sequences without homologous experimentally characterized structures, however, we sought to predict fold-switching proteins from their sequences alone. To do this, we leveraged state-of-the-art secondary structure predictions, which require only amino acid sequences but are not currently designed to identify structural duality in proteins. Thus, we hypothesized that incorrect and inconsistent secondary structure predictions could be good initial predictors of fold-switching proteins. We found that secondary structure predictions of fold-switching proteins with solved structures are indeed less accurate than secondary structure predictions of non-fold-switching proteins with solved structures. These inaccuracies result largely from the conformations of fold-switching proteins that are underrepresented in the Protein Data Bank (PDB), and, consequently, the training sets of secondary structure predictors. Given that secondary structure predictions are homology-based, we hypothesized that decontextualizing the inaccurately-predicted regions of fold-switching proteins could weaken the homology relationships between these regions and their overpopulated structural representatives. Thus, we reran secondary structure predictions on these regions in isolation and found that they were significantly more inconsistent than in regions of non-fold-switching proteins. Thus, inconsistent secondary structure predictions can serve as a preliminary marker of fold switching. These findings have implications for genomics and the future development of secondary structure predictors.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

Down syndrome (DS) is a genetic disorder that causes cognitive impairment. The staggering effects associated with an extra copy of human chromosome 21 (HSA21) complicates mechanistic understanding of DS pathophysiology. We examined the neuron-astrocyte interplay in a fully recapitulated HSA21 trisomy cellular model differentiated from DS-patient-derived induced pluripotent stem cells (iPSCs). By combining calcium imaging with genetic approaches, we discovered the functional defects of DS astroglia and their effects on neuronal excitability. Compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous calcium fluctuations, which reduced the excitability of co-cultured neurons. Furthermore, suppressed neuronal activity could be rescued by abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-mediated signaling or genetically by knockdown of inositol triphosphate (IP3) receptors or S100B, a calcium binding protein coded on HSA21. Our results suggest a mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal excitability.

A central tenet of biology is that globular proteins have a unique 3D structure under physiological conditions. Recent work has challenged this notion by demonstrating that some proteins switch folds, a process that involves remodeling of secondary structure in response to a few mutations (evolved fold switchers) or cellular stimuli (extant fold switchers). To date, extant fold switchers have been viewed as rare byproducts of evolution, but their frequency has been neither quantified nor estimated. By systematically and exhaustively searching the Protein Data Bank (PDB), we found ∼100 extant fold-switching proteins. Furthermore, we gathered multiple lines of evidence suggesting that these proteins are widespread in nature. Based on these lines of evidence, we hypothesized that the frequency of extant fold-switching proteins may be underrepresented by the structures in the PDB. Thus, we sought to identify other putative extant fold switchers with only one solved conformation. To do this, we identified two characteristic features of our ∼100 extant fold-switching proteins, incorrect secondary structure predictions and likely independent folding cooperativity, and searched the PDB for other proteins with similar features. Reassuringly, this method identified dozens of other proteins in the literature with indication of a structural change but only one solved conformation in the PDB. Thus, we used it to estimate that 0.5-4% of PDB proteins switch folds. These results demonstrate that extant fold-switching proteins are likely more common than the PDB reflects, which has implications for cell biology, genomics, and human health.

Understanding how activity patterns in specific neural circuits coordinate an animal's behavior remains a key area of neuroscience research. Genetic tools and a brain of tractable complexity make a premier model organism for these studies. Here, we review the wealth of reagents available to map and manipulate neuronal activity with light.