In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell.

Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an \~{}16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.

We have developed a series of yellow genetically encoded Ca indicators for optical imaging (Y-GECOs) with inverted responses to Ca and apparent dissociation constants (K') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca indicators, we expressed the four highest affinity variants (K's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower K' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 μM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

Dendritic ion channels play a critical role in shaping synaptic input and are fundamentally important for synaptic integration and plasticity. In the hippocampal region CA1, somato-dendritic gradients of AMPA receptors and the hyperpolarization-activated cation conductance (I(h)) counteract the effects of dendritic filtering on the amplitude, time-course, and temporal integration of distal Schaffer collateral (SC) synaptic inputs within stratum radiatum (SR). While ion channel gradients in CA1 distal apical trunk dendrites within SR have been well characterized, little is known about the patterns of ion channel expression in the distal apical tuft dendrites within stratum lacunosum moleculare (SLM) that receive distinct input from the entorhinal cortex via perforant path (PP) axons. Here, we measured local ion channels densities within these distal apical tuft dendrites to determine if the somato-dendritic gradients of I(h) and AMPA receptors extend into distal tuft dendrites. We also determined the densities of voltage-gated sodium channels and NMDA receptors. We found that the densities of AMPA receptors, I(h,) and voltage-gated sodium channels are similar in tuft dendrites in SLM when compared with distal apical dendrites in SR, while the ratio of NMDA receptors to AMPA receptors increases in tuft dendrites relative to distal apical dendrites within SR. These data indicate that the somato-dendritic gradients of I(h) and AMPA receptors in apical dendrites do not extend into the distal tuft, and the relative densities of voltage-gated sodium channels and NMDA receptors are poised to support nonlinear integration of correlated SC and PP input.

From patch-clamp techniques to recombinant DNA technologies, three-dimensional protein modeling, and optogenetics, diverse and sophisticated methods have been used to study ion channels and how they determine the electrical properties of cells.

The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.

Chemotherapy is often combined with immune checkpoint inhibitor (ICIs) to enhance immunotherapy responses. Despite the approval of chemo-immunotherapy in multiple human cancers, many immunologically cold tumors remain unresponsive. The mechanisms determining the immunogenicity of chemotherapy are elusive. Here, we identify the ER stress sensor IRE1α as a critical checkpoint that restricts the immunostimulatory effects of taxane chemotherapy and prevents the innate immune recognition of immunologically cold triple-negative breast cancer (TNBC). IRE1α RNase silences taxane-induced double-stranded RNA (dsRNA) through regulated IRE1-dependent decay (RIDD) to prevent NLRP3 inflammasome-dependent pyroptosis. Inhibition of IRE1α in Trp53 TNBC allows taxane to induce extensive dsRNAs that are sensed by ZBP1, which in turn activates NLRP3-GSDMD-mediated pyroptosis. Consequently, IRE1α RNase inhibitor plus taxane converts PD-L1-negative, ICI-unresponsive TNBC tumors into PD-L1 immunogenic tumors that are hyper-sensitive to ICI. We reveal IRE1α as a cancer cell defense mechanism that prevents taxane-induced danger signal accumulation and pyroptotic cell death.

Rat or mouse liver is the most frequently used tissue for mitochondrial preparations because it is readily available, easy to homogenize, and replete with mitochondria. A motor-driven Teflon and glass Potter-Elvehjem homogenizer is the best choice for homogenizing liver, but if one is not available, this tissue is soft enough that a Dounce homogenizer with a loose (A) pestle can also be used. The yield and purity of the mitochondrial preparation will be influenced by the method and speed of preparation and the age and physiological condition of the animal.