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Pfeiffer, T.

Publications and source records attributed to Pfeiffer, T..

6 recordsLinked to original sources

Intracellular binding pocket revealed in the human bitter taste receptor TAS2R14

Bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs) expressed orally and extraorally, elicit signaling in response to a large set of ligands. Among the 25 functional TAS2Rs encoded in the human genome, TAS2R14 is the most promiscuous, and responds to hundreds of chemically diverse agonists. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human TAS2R14 (hTAS2R14) in complex with its cognate signaling partner gustducin, and bound to flufenamic acid (FFA), a clinically approved nonsteroidal anti-inflammatory drug. The structure reveals an unusual binding mode for FFA, where two copies are bound at distinct binding pockets: one at the canonical GPCR site within the trans-membrane bundle, and the other in the intracellular facet, bridging the receptor with gustducin. Combined with site-directed mutagenesis and the design of a fluorescent FFA derivative for pocket-specific ligand binding BRET assays, our studies support a dual binding mode for FFA in TAS2R14. These results fill a gap in the understanding of bitter taste signaling and provide tools for guided design of TAS2R-targeted compounds.

biochemistry↗

Imaging dendritic spines in the hippocampus of a living mouse by 3D-STED microscopy

STED microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples like cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging. In previous work, we established chronic STED imaging in the hippocampus in vivo but the gain in spatial resolution was restricted to the lateral plane. In this study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampus in vivo. The approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. Moreover, we corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser, which is required for 3D-STED microscopy. In summary, we present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampus in vivo, facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.

neuroscience↗

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and of chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/517821v4_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@30bd42org.highwire.dtl.DTLVardef@1d94fbforg.highwire.dtl.DTLVardef@2858borg.highwire.dtl.DTLVardef@18d9af2_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

Shadow imaging for panoptical visualisation of living brain tissue

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation, do not have cellular resolution or only give a fragmented view. Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue.

neuroscience↗

The Ca2+-gated Cl- channel TMEM16A amplifies capillary pericyte contraction reducing cerebral blood flow after ischemia

Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activates chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplifies the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slows the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction and pericyte death, reduces neutrophil stalling and improves cerebrovascular reperfusion. Genetic analysis implicates altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function, and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimers disease and vascular dementia.

physiology↗

A comprehensive framework for handling location error in animal tracking data

Animal tracking data are being collected more frequently, in greater detail, and on smaller taxa than ever before. These data hold the promise to increase the relevance of animal movement for understanding ecological processes, but this potential will only be fully realized if their accompanying location error is properly addressed. Historically, coarsely-sampled movement data have proved invaluable for understanding large scale processes (e.g., home range, habitat selection, etc.), but modern fine-scale data promise to unlock far more ecological information. While GPS location error can often be ignored in coarsely sampled data, fine-scale data require more care, and tools to do this have not kept pace. Current approaches to dealing with location error largely fall into two categories--either discarding the least accurate location estimates prior to analysis or simultaneously fitting movement and error parameters in a hidden-state model. In some cases these approaches can provide a level of correction, but they have known limitations, and in some cases they can be worse than doing nothing. Here, we provide a general framework to account for location error in the analysis of triangulated and trilatcralizcd animal tracking data, which includes GPS, Argos Doppler-shift, triangulated VHF, trilatcralized acoustic and cellular location data. We apply our error-modelselection framework to 190 GPS, cellular, and acoustic devices representing 27 models from 14 manufacturers. Collectively, these devices were used to track a wide range of taxa comprising birds, fish, reptiles, and mammals of different sizes and with different behaviors, in urban, suburban, and wild settings. In almost half of the tested device models, error-model selection was necessary to obtain the best performing error model, and in almost a quarter of tested device models, the reported DOP values were actually misinformative. Then, using empirical tracking data from multiple species, we provide an overview of modern, error-informed movement analyses, including continuous-time path reconstruction, home-range distribution, home-range overlap, speed, and distance estimation. Adding to these techniques, we introduce new error-informed estimators for outlier detection and autocorrelation visualization. Because error-induced biases depend on many factors--sampling schedule, movement characteristics, tracking device, habitat, etc.--differential bias can easily confound biological inference and lead researchers to draw false conclusions. We demonstrate how error-informed analyses on calibrated tracking data can provide more accurate estimates are that are insensitive to location error, and allow researchers to use all of their data.

ecology↗