Extant fold-switching proteins are widespread Porter, Lauren L.; Looger, Loren L.
Proceedings of the National Academy of Sciences - PNAS,
06/2018, Letnik:
115, Številka:
23
Journal Article
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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.
Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also ...be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.
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•AAV can be endowed with robust retrograde functionality through directed evolution•Up to two orders of magnitude increase in retrograde transport over existing variants•Efficiency comparable to synthetic tracers•Sufficient payload expression for circuit interrogation and gene manipulation
Projection neurons are a critical component of large-scale networks distributing the results of local circuit computations between distant brain regions, but their specific contribution is often hard to pinpoint because of the difficulty of gaining selective genetic access. Tervo et al. introduce a designer variant of adeno-associated virus, rAAV2-retro, that allows for efficient mapping, monitoring, and manipulation of projection neurons.
Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging ...are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.
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.
Astrocytes tile the entire CNS, but their functions within neural circuits in health and disease remain incompletely understood. We used genetically encoded Ca(2+)and glutamate indicators to explore ...the rules for astrocyte engagement in the corticostriatal circuit of adult wild-type (WT) and Huntington's disease (HD) model mice at ages not accompanied by overt astrogliosis (at approximately postnatal days 70-80). WT striatal astrocytes displayed extensive spontaneous Ca(2+)signals, but did not respond to cortical stimulation, implying that astrocytes were largely disengaged from cortical input in healthy tissue. In contrast, in HD model mice, spontaneous Ca(2+)signals were significantly reduced in frequency, duration, and amplitude, but astrocytes responded robustly to cortical stimulation with evoked Ca(2+)signals. These action-potential-dependent astrocyte Ca(2+)signals were mediated by neuronal glutamate release during cortical stimulation, accompanied by prolonged extracellular glutamate levels near astrocytes and tightly gated by Glt1 glutamate transporters. Moreover, dysfunctional Ca(2+)and glutamate signaling that was observed in HD model mice was largely, but not completely, rescued by astrocyte specific restoration of Kir4.1, emphasizing the important contributions of K(+)homeostatic mechanisms that are known to be reduced in HD model mice. Overall, our data show that astrocyte engagement in the corticostriatal circuit is markedly altered in HD. Such prodromal astrocyte dysfunctions may represent novel therapeutic targets in HD and other brain disorders.
We report how early-onset astrocyte dysfunction without detectable astrogliosis drives disease-related processes in a mouse model of Huntington's disease (HD). The cellular mechanisms involve astrocyte homeostasis and signaling mediated by Kir4.1, Glt1, and Ca(2+) The data show that the rules for astrocyte engagement in a neuronal circuit are fundamentally altered in a brain disease caused by a known molecular defect and that fixing early homeostasis dysfunction remedies additional cellular deficits. Overall, our data suggest that key aspects of altered striatal function associated with HD may be triggered, at least in part, by dysfunctional astrocytes, thereby providing details of an emerging striatal microcircuit mechanism in HD. Such prodromal changes in astrocytes may represent novel therapeutic targets.
The spatiotemporal activities of astrocyte Ca2+ signaling in mature neuronal circuits remain unclear. We used genetically encoded Ca2+ and glutamate indicators as well as pharmacogenetic and ...electrical control of neurotransmitter release to explore astrocyte activity in the hippocampal mossy fiber pathway. Our data revealed numerous localized, spontaneous Ca2+ signals in astrocyte branches and territories, but these were not driven by neuronal activity or glutamate. Moreover, evoked astrocyte Ca2+ signaling changed linearly with the number of mossy fiber action potentials. Under these settings, astrocyte responses were global, suppressed by neurotransmitter clearance, and mediated by glutamate and GABA. Thus, astrocyte engagement in the fully developed mossy fiber pathway was slow and territorial, contrary to that frequently proposed for astrocytes within microcircuits. We show that astrocyte Ca2+ signaling functionally segregates large volumes of neuropil and that these transients are not suited for responding to, or regulating, single synapses in the mossy fiber pathway.
•Astrocyte branches/territories were studied using optical and pharmacogenetic tools•Spontaneous astrocyte Ca2+ signals were not due to action potentials or glutamate•Evoked astrocyte Ca2+ signals were slow, territorial and triggered by spike trains•Astrocyte Ca2+ signaling was tightly gated by neurotransmitter clearance
Haustein et al. use optical and genetic methods to explore the rules under which astrocytes are engaged in a model neuronal circuit. The findings show that astrocytes listen to synapses during bursts of action potentials in mossy fiber axons.
Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and ...characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F
F
-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR
responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.
Temperature dependent sex determination (TSD) is the process by which the environmental temperature experienced during embryogenesis influences the sex of an organism, as in the red-eared slider ...turtle Trachemys scripta elegans. In accord with current paradigms of vertebrate sex determination, temperature is believed to exert its effects on sexual development in T. scripta entirely within the middle third of development, when the gonad is forming. However, whether temperature regulates the transcriptome in T. scripta early embryos in a manner that could influence secondary sex characteristics or establish a pro-male or pro-female environment has not been investigated. In addition, apart from a handful of candidate genes, very little is known about potential similarities between the expression cascade during TSD and the genetic cascade that drives mammalian sex determination. Here, we conducted an unbiased transcriptome-wide analysis of the effects of male- and female-promoting temperatures on the turtle embryo prior to gonad formation, and on the gonad during the temperature sensitive period. We found sexually dimorphic expression reflecting differences in steroidogenic enzymes and brain development prior to gonad formation. Within the gonad, we mapped a cascade of differential expression similar to the genetic cascade established in mammals. Using a Hidden Markov Model based clustering approach, we identified groups of genes that show heterochronic shifts between M. musculus and T. scripta. We propose a model in which multiple factors influenced by temperature accumulate during early gonadogenesis, and converge on the antagonistic regulation of aromatase to canalize sex determination near the end of the temperature sensitive window of development.
•TSD gene expression shows similarity to mammals with some differences in timing/order.•Some dimorphism occurs via temperature and independent of gonadal differentiation.•Multiple temperature inputs converge on hormonal signaling during sex determination.
Spatial navigation is often used as a behavioral task in studies of the neuronal circuits that underlie cognition, learning and memory in rodents. The combination of in vivo microscopy with ...genetically encoded indicators has provided an important new tool for studying neuronal circuits, but has been technically difficult to apply during navigation. Here we describe methods for imaging the activity of neurons in the CA1 region of the hippocampus with subcellular resolution in behaving mice. Neurons that expressed the genetically encoded calcium indicator GCaMP3 were imaged through a chronic hippocampal window. Head-restrained mice performed spatial behaviors in a setup combining a virtual reality system and a custom-built two-photon microscope. We optically identified populations of place cells and determined the correlation between the location of their place fields in the virtual environment and their anatomical location in the local circuit. The combination of virtual reality and high-resolution functional imaging should allow a new generation of studies to investigate neuronal circuit dynamics during behavior.
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.
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