20210611T133520210611T1405America/DetroitPoster Session I (+Lunch and Office Hours)PostersNIH Common Fund's 2021 High-Risk, High-Reward Research Symposiumbecky.miller2@nih.gov
Observation of extracellular and intracellular conformational coupling in membrane proteins using single-molecule FRET and nanodiscs
Molecular and Cellular Biology01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Approximately 30% of the genes in most genomes encode membrane proteins and membrane proteins are 60% of drug targets. Despite their importance, information about membrane protein function and interaction has been limited due to the challenges associated with maintaining their mixed hydrophobic/hydrophilic environment while conducting experiments. By combining nanodiscs and single-molecule Förster resonance energy transfer (FRET), we observe conformational coupling across the plasma membrane in two transmembrane receptors, the bacterial chemoreceptor and the mammalian epidermal growth factor receptor (EGFR). In chemoreceptors, periplasmic ligand binding changes the packing and dynamics of the cytoplasmic four-helix coiled-coil bundle > 200 Å away. The observed changes are consistent with a transition from a modestly to extended rhomboid organization of the cytoplasmic bundle, which may be a feature shared with other coiled-coil signaling proteins. In monomeric EGFR, extracellular ligand binding induces an intracellular conformational reorganization that is specific to the ligand and lipid composition. These findings demonstrate that the single a-helix of EGFR, and potentially other single-pass membrane proteins, can serve as a minimal yet sufficient system for signal transduction. These examples of transmembrane conformational signaling inform on the mechanistic underpinnings by which cells detect and respond to the fluctuating environment of biological systems.
Highly multiplexed spatial mapping of microbial communities
High-Throughput and Integrative Biology01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Mapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density and rich diversity of species in environmental microbiomes and the limitations of optical imaging technology. In this presentation, I will discuss High Phylogenetic Resolution microbiome mapping by Fluorescence in situ Hybridization (HiPRFISH), a versatile technology developed under with the support of a DP2 award that uses binary encoding, spectral imaging, and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. We have demonstrated the ability of 10-bit HiPR-FISH to distinguish 1023 E. coli isolates, each fluorescently labeled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and single-cell image analysis, revealed the disruption of spatial networks in the mouse gut microbiome in response to antibiotic treatment and the longitudinal stability of spatial architectures in the human oral plaque microbiome. Combined with super-resolution imaging, HiPR-FISH revealed the diverse ribosome organization strategies of human oral microbial taxa. HiPR-FISH provides a framework for analyzing the spatial ecology of environmental microbial communities at single-cell resolution.
Reorganization of cortical activity patterns supports learning of tactile shape discrimination
Neuroscience01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Tactile shape perception is critical for object recognition and tool use. Three-dimensional shape of an object is defined by the contour of object surface, which can be perceived by integrating object surface angles from multiple contact points. Thus, surface angle information is fundamental to object shape perception. We investigated how object surface angle is perceived during active touch using mouse whisker system. Head-fixed mice were trained to discriminate object surface angle using a single whisker. Before and after learning, we tested mice with 7 angles (45° to 135°) to quantify their resolution in angle discrimination. Across training, we recorded the same set of thousands of excitatory neurons in the primary somatosensory cortex (S1) to examine cortical representation of object angle and its change across angle discrimination learning. Mice learned to discriminate 2 angles in about 2 weeks, and their angle discrimination resolution was at least 15°. Using generalized linear models we extracted the most important sensory inputs in discriminating object angles: vertical bending and slide distance along the object. Encoding of these two sensory inputs were combined to generate object-angle tuning in majority (~80%) of touch-responsive excitatory neurons in S1, both before and after learning. We observed that only about 40% of neurons remained active across learning. Despite this high activity turnover rate, object-angle tuning remained stable in both population distribution and persistently active individual neurons. Sensory inputs as well as whisker movements also remained unchanged across learning. However, population activity patterns became better at discriminating object angles after learning, partly due to increased encoding of task-relevant sensory input, vertical bending, in newly recruited touch-responsive neurons. Collectively, the results suggest how primary sensory cortex can adapt to support learned behavior while maintaining a faithful representation of outside world during active sensation.
Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing
Molecular and Cellular Biology01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Keratinocytes, the predominant cell type of the epidermis, migrate to reinstate the epithelial barrier during wound healing. Mechanical cues are known to regulate keratinocyte re-epithelization and wound healing however, the underlying molecular transducers and biophysical mechanisms remain elusive. Here, we show through molecular, cellular and organismal studies that the mechanically-activated ion channel PIEZO1 regulates keratinocyte migration and wound healing. Epidermal-specific Piezo1 knockout mice exhibited faster wound closure while gain-of-function mice displayed slower wound closure compared to littermate controls. By imaging the spatiotemporal localization dynamics of endogenous PIEZO1 channels we find that channel enrichment in sub-cellular regions induces a localized cellular retraction that slows keratinocyte migration. Our findings suggest a potential pharmacological target for wound treatment. More broadly, we show that nanoscale spatiotemporal dynamics of Piezo1 channels can control tissue-scale events, a finding with implications beyond wound healing to processes as diverse as development, homeostasis, disease and repair.
Model for Postnatal Interneuron Migration in the Gyrencephalic Brain
Neuroscience01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Inhibitory neurons (interneurons) are a neuronal subpopulation implicated in the pathogenesis of neurodevelopmental disorders (NDDs) such as Epilepsy and Autism Spectrum Disorder (ASD). Interneurons originate in a fetal structure, the ganglionic eminence, which is further divided into medial, lateral, and caudal subregions (MGE, LGE, and CGE, respectively). Each area generates distinct interneuron subtypes with unique functions. We have previously shown that doublecortin (DCX)+ interneurons migrate through a ventricular structure, called the Arc, to areas of the frontal lobe for several months after birth. However, the extent of cortical migration in the infant brain and molecular profile of these neurons remains unknown. We use the piglet brain as a model for human postnatal cortical development given its anatomical and developmental similarities to the human brain. We map distribution of DCX+ neurons in the postnatal day 0 (P0) piglet cortex and identify additional cortical targets for migratory neurons in the postnatal cortex, including the insula and temporal lobe. We also quantify the composition of migratory DCX+ cells in the Embryonic day 89 (E89), E100, and P0 piglet cortex, using regional transcription factors such as LHX6 and NKX2.1 for the MGE and SP8 and COUPTFII for the CGE. Although a subset of these interneurons are derived from the MGE, the piglet Arc contains a majority of interneurons that originated in the CGE; the human Arc had a similar composition. Thus, CGE-derived neurons are the primary contributors to this wave of "late cortical migration" in the human and piglet brains. This study shows that the cellular composition of the postnatal cortex remains dynamic and supports that protracted interneuron development is conserved in gyrencephalic brains. It also provides a novel platform to understand the emergence of NDDs and reveal ways to therapeutically influence how neurons organize themselves,even after a child is born.
Live-cell transcriptomics via virus-like particles
High-Throughput and Integrative Biology01:35 PM - 02:05 PM (America/Detroit) 2021/06/11 17:35:00 UTC - 2021/06/11 18:05:00 UTC
Transcriptional information grants insight into the biological states and responses of living systems. However, available measurement approaches are destructive and prevent continuous retrieval and monitoring of transcriptome-wide RNA information from living cells. We overcame this limitation by repurposing the Gag polyprotein from murine leukemia virus (MLV) to package host transcripts and cause their secretion from living cells in virus-like particles (VLPs). With this RNA self-reporting approach, we collected quantitative transcriptome-wide RNA information from human HT1080 and HEK293 cells and iPSCs, preparing RNA-seq libraries that performed similarly to standard RNA-seq libraries produced from cellular lysates. Further, we engineered Gag with poly(A)-binding domains to enhance RNA detection, as well as orthogonal affinity purification tags to enable multiplexed RNA detection in co-cultures. Finally, we demonstrated that live-cell transcriptome measurements through RNA self-reporting could time-dependent transcriptional state changes in individual samples responding to TNFα stimulation. RNA self-reporting with VLPs enables faithful live-cell, transcriptome-wide measurements from the same biological samples over time using standard RNA-seq library construction and sequencing procedures.