208 research outputs found

    Programmable RNA-binding protein composed of repeats of a single modular unit

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    The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby–RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.United States. National Institutes of Health (1R01NS075421)National Science Foundation (U.S.) (1344219)United States. National Institutes of Health (1U01MH106011)United States. National Institutes of Health (1R01MH103910)United States. National Institutes of Health (1DP1NS087724

    A fully genetically encoded protein architecture for optical control of peptide ligand concentration

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    Ion channels are among the most important proteins in biology, regulating the activity of excitable cells and changing in diseases. Ideally it would be possible to actuate endogenous ion channels, in a temporally precise and reversible manner, and without requiring chemical cofactors. Here we present a modular protein architecture for fully genetically encoded, light-modulated control of ligands that modulate ion channels of a targeted cell. Our reagent, which we call a lumitoxin, combines a photoswitch and an ion channel-blocking peptide toxin. Illumination causes the photoswitch to unfold, lowering the toxin’s local concentration near the cell surface, and enabling the ion channel to function. We explore lumitoxin modularity by showing operation with peptide toxins that target different voltage-dependent K+ channels. The lumitoxin architecture may represent a new kind of modular protein-engineering strategy for designing light-activated proteins, and thus may enable development of novel tools for modulating cellular physiology.National Institutes of Health (U.S.) (grant NIH 1DP2OD002002)National Institutes of Health (U.S.) (grant NIH 1R01DA029639)National Institutes of Health (U.S.) (grant NIH 1R01NS075421)National Institutes of Health (U.S.) (grant NIH 1RC1MH088182)National Science Foundation (U.S.) (NSF CAREER Award CBET 1053233)United States. Defense Advanced Research Projects Agency (DARPA Living Foundries, Contract HR0011-12-C-0068)New York Stem Cell Foundation (Robertson Investigator Award)Damon Runyon Cancer Research Foundation (DRG 2095-11)Fannie and John Hertz FoundationNational Science Foundation (U.S.) (Graduate Research Fellowship under grant no. 1122374)Massachusetts Institute of Technology. Synthetic Intelligence Laboratory (project

    Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing

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    High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP) on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases – Dpo4 and Klenow exo[subscript −] – obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn[superscript 2+] with a misincorporation rate gain of ~2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn[superscript 2+] and Mg[superscript 2+] change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.Damon Runyon Cancer Research FoundationNational Institutes of Health (U.S.)National Science Foundation (U.S.)McGovern Institute for Brain Research at MITMassachusetts Institute of Technology. Media LaboratoryNew York Stem Cell Foundation (Robertson Neuroscience Investigator Award)Paul G. Allen Family Foundation (Distinguished Investigator in Neuroscience Award

    Hearing the light: neural and perceptual encoding of optogenetic stimulation in the central auditory pathway

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    Optogenetics provides a means to dissect the organization and function of neural circuits. Optogenetics also offers the translational promise of restoring sensation, enabling movement or supplanting abnormal activity patterns in pathological brain circuits. However, the inherent sluggishness of evoked photocurrents in conventional channelrhodopsins has hampered the development of optoprostheses that adequately mimic the rate and timing of natural spike patterning. Here, we explore the feasibility and limitations of a central auditory optoprosthesis by photoactivating mouse auditory midbrain neurons that either express channelrhodopsin-2 (ChR2) or Chronos, a channelrhodopsin with ultra-fast channel kinetics. Chronos-mediated spike fidelity surpassed ChR2 and natural acoustic stimulation to support a superior code for the detection and discrimination of rapid pulse trains. Interestingly, this midbrain coding advantage did not translate to a perceptual advantage, as behavioral detection of midbrain activation was equivalent with both opsins. Auditory cortex recordings revealed that the precisely synchronized midbrain responses had been converted to a simplified rate code that was indistinguishable between opsins and less robust overall than acoustic stimulation. These findings demonstrate the temporal coding benefits that can be realized with next-generation channelrhodopsins, but also highlight the challenge of inducing variegated patterns of forebrain spiking activity that support adaptive perception and behavior

    Optogenetic stimulation of the cochlear nucleus using channelrhodopsin-2 evokes activity in the central auditory pathways

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    Optogenetics has become an important research tool and is being considered as the basis for several neural prostheses. However, few studies have applied optogenetics to the auditory brainstem. This study explored whether optical activation of the cochlear nucleus (CN) elicited responses in neurons in higher centers of the auditory pathway and whether it elicited an evoked response. Viral-mediated gene transfer was used to express channelrhodopsin-2 (ChR2) in the mouse CN. Blue light was delivered via an optical fiber placed near the surface of the infected CN and recordings were made in higher-level centers. Optical stimulation evoked excitatory multiunit spiking activity throughout the tonotopic axis of the central nucleus of the inferior colliculus (IC) and the auditory cortex (Actx). The pattern and magnitude of IC activity elicited by optical stimulation was comparable to that obtained with a 50 dB SPL acoustic click. This broad pattern of activity was consistent with histological confirmation of green fluorescent protein (GFP) label of cell bodies and axons throughout the CN. Increasing pulse rates up to 320 Hz did not significantly affect threshold or bandwidth of the IC responses, but rates higher than 50 Hz resulted in desynchronized activity. Optical stimulation also evoked an auditory brainstem response, which had a simpler waveform than the response to acoustic stimulation. Control cases showed no responses to optical stimulation. These data suggest that optogenetic control of central auditory neurons is feasible, but opsins with faster channel kinetics may be necessary to convey information at rates typical of many auditory signals

    Memory consolidation in the cerebellar cortex

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    Several forms of learning, including classical conditioning of the eyeblink, depend upon the cerebellum. In examining mechanisms of eyeblink conditioning in rabbits, reversible inactivations of the control circuitry have begun to dissociate aspects of cerebellar cortical and nuclear function in memory consolidation. It was previously shown that post-training cerebellar cortical, but not nuclear, inactivations with the GABA(A) agonist muscimol prevented consolidation but these findings left open the question as to how final memory storage was partitioned across cortical and nuclear levels. Memory consolidation might be essentially cortical and directly disturbed by actions of the muscimol, or it might be nuclear, and sensitive to the raised excitability of the nuclear neurons following the loss of cortical inhibition. To resolve this question, we simultaneously inactivated cerebellar cortical lobule HVI and the anterior interpositus nucleus of rabbits during the post-training period, so protecting the nuclei from disinhibitory effects of cortical inactivation. Consolidation was impaired by these simultaneous inactivations. Because direct application of muscimol to the nuclei alone has no impact upon consolidation, we can conclude that post-training, consolidation processes and memory storage for eyeblink conditioning have critical cerebellar cortical components. The findings are consistent with a recent model that suggests the distribution of learning-related plasticity across cortical and nuclear levels is task-dependent. There can be transfer to nuclear or brainstem levels for control of high-frequency responses but learning with lower frequency response components, such as in eyeblink conditioning, remains mainly dependent upon cortical memory storage

    Expansion microscopy of zebrafish for neuroscience and developmental biology studies

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    Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.National Institutes of Health (U.S.) (Grant 1R01EB024261)National Institutes of Health (U.S.) (Grant 1R01MH110932)National Institutes of Health (U.S.) (Grant 2R01DA029639)National Institutes of Health (U.S.) (Grant 1R01NS087950)National Institutes of Health (U.S.) (Grant 1U01MH106011
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