Browsing by Subject "Adaptation, Physiological"
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Item The athlete's heart: friend or foe?(2017-01-27) Levine, BenjaminItem Physiology and Circuitry of Bile Acid Detection Within the Accessory Olfactory System(2021-05-01T05:00:00.000Z) Wong, Wen Mai; Lai, Helen; Pfeiffer, Brad E.; Konopka, Genevieve; Meeks, Julian P.The mouse accessory olfactory system (AOS) supports social and reproductive behavior through the sensation of environmental chemosignals. These chemosensory cues provide a vast amount of information that the olfactory systems must then encode and translate into behaviorally relevant outputs. The initial detection of olfactory stimuli in the accessory olfactory system (AOS) is mediated by the vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO), which relay the signals to the accessory olfactory bulb (AOB). Impaired vomeronasal signaling results in deficits in social communication and other behaviors. Despite decades of research, our understanding of how odors are encoded and processed within the AOS is still severely limited. For example, the extent to which the AOS discriminates and encodes chemosensory information at the peripheral level of the VNO is currently unknown. Furthermore, how the tuning of VSNs is then translated into a behaviorally relevant representation in the brain is still unclear. Understanding how the AOS processes and encodes chemosensory information will advance our understanding of how external cues can generate internal chemosensory representations that are critical for survival. Sensory adaptation is a source of experience-dependent feedback that impacts responses to environmental cues. In the mammalian main olfactory system (MOS), adaptation influences sensory coding at its earliest processing stages. However, sensory adaptation in the accessory olfactory system (AOS) remains relatively controversial, leaving many aspects of the phenomenon unclear. Thus, I investigated sensory adaptation in vomeronasal sensory neurons (VSNs) using in situ Ca2+ imaging. I found evidence for sensory adaptation in response to the monomolecular ligands, cholic acid (CA) and deoxycholic acid (DCA). These Ca2+ imaging experiments also revealed the presence of a slower form of VSN adaptation that accumulated over dozens of stimulus presentations delivered over tens of minutes. These studies help establish the presence of VSN sensory adaptation and provide a foundation for future inquiries into the molecular and cellular mechanisms of this phenomenon and its impact on mammalian behavior. A growing number of excreted steroids have been shown to be potent AOS cues, including bile acids (BAs) found in feces. As is still the case with most AOS ligands, the specific receptors used by vomeronasal sensory neurons (VSNs) to detect BAs remain unknown. To identify VSN BA receptors, we first performed a deep analysis of VSN BA tuning using volumetric GCaMP6f/s Ca2+ imaging. These experiments revealed multiple distinct populations of BA-receptive VSNs with submicromolar sensitivities. I then developed a new physiology-forward approach for identifying AOS ligand-receptor interactions, which I term Fluorescence Live Imaging for Cell Capture and RNA sequencing, or FLICCR-seq. FLICCR-seq analysis revealed five specific V1R family receptors enriched in BA-sensitive VSNs. These studies introduce a powerful new approach for ligand-receptor matching and reveal biological mechanisms underlying mammalian BA chemosensation. Finally, I've spent the remainder of my thesis laying the groundwork for exploring BA-mediated behaviors and the circuitry of BA information as it travels through the AOS. Importantly, my work opens many avenues for future studies. Here, I show evidence that five specific V1R receptors are BA-sensitive. The identification of ligands that can modulate the activity of orphan VRs is paramount to understanding their function and in turn understanding chemosensation. We now have more tools in the toolbox to understanding and studying the activation of VRs, their signal transduction, and their function.Item The Structural Properties of Adaptation and Allostery in Proteins: A Case Study in a PDZ Domain(2017-03-08) Raman, Arjun Swaminathan; Rosen, Michael K.; Ranganathan, Rama; Rice, Luke M.Complex systems are present at all scales of biology spanning amino acid interactions in proteins to inter-species interactions in ecosystems. Despite their ubiquity, an understanding of how to study complex systems methodically is lacking. Fundamentally, this is because a procedure to discover the appropriate parametrization of such systems into relevant parts is absent. The Ranganathan Lab developed an evolutionary approach, Statistical Coupling Analysis (SCA), to understand how amino acid interactions within proteins give rise to basic protein characteristics such as fold and function. The result of this approach was a structural decomposition of proteins into units of coevolving residues termed protein 'sectors'. Is the transformation from amino acids to protein sectors a useful and relevant representation of the essence of proteins? Previous work has demonstrated that specifying co-evolution is sufficient to design synthetic natural-like proteins and encodes the information needed to execute a primary function. As evolved biological systems however, proteins must also also adapt quickly to new functions. In addition, a fundamental characteristic of many proteins is the ability to transmit information over a significant distance in the protein structure, a phenomenon known as allostery. Where in the protein do these characteristics lie? Here, we address the sequence origins and structural properties of a) the adaptive capacity of proteins and b) allosteric communication within proteins. Understanding how proteins can adapt quickly to new function is an outstanding question in biology, marked by failed attempts at engineering new or altered function guided by structural approaches focused on the importance of active site or binding pocket positions. It is often the case that mutations located distal to the active site harbor adaptive potential, a non-obvious observation given the crystal structure of a protein. To more fundamentally understand the adaptive process in proteins, here we examine a two-step mutational path to new specificity in a model protein PSD95pdz3 where one intermediate, a Glycine (G) to Threonine (T) mutation at position 330, removed from the binding site of the protein, maintains native function while simultaneously adapting the protein to an alternate function (termed a 'conditionally neutral' mutation) whereas the other intermediate, a Histidine (H) to Alanine (A) mutation at position 372, promotes a direct specificity switch, abrogating native function. Through a stochastic population dynamics simulation, we find the conditionally neutral intermediate promotes adaptation over a wide range of mutation rates and rates of environmental fluctuation while the direct specificity switching mutation facilitates adaptation only within a special regime of population dynamics parameters. We comprehensively identify the spatial distribution of all adaptive mutations in PSD95pdz3, finding that direct specificity switching mutations are found exclusively at sector positions directly at the binding site whereas conditionally neutral mutations are generally found distal to the binding site but connected to it through the protein sector. Crystal structures reveal how a mutation at position 330 creates plasticity at the binding site to create a dual function protein. Overall, these results illustrate the importance of a spatially distributed network of coupled residues for adapting in a fluctuating environment. Revealing paths of allostery in protein structures has been a central goal of structural biology. In PSD95pdz3, we find that the G330T mutation causes local backbone remodeling and structurally affects solely a distant conserved helix. Detecting structurally coupled residues in the protein reveals a network of connected residues spanning the 330 position to the distant helix propagating through the core of the protein sector. As a check of this allosteric path, we are able to abrogate the allosteric transmission through a mutation in the middle of the protein sector. Both the ability to adapt to new function and the property of allostery are found almost exclusively within the protein sector. The work highlighted here in addition to previous studies in the lab thus appear to suggest that the sector is a good descriptor and model for understanding how proteins work. It will therefore be important for future studies to determine if other methods of protein design such as Direct Coupling Analysis and Rosetta provide equivalently sufficient descriptions of proteins and what additional information, if any, these approaches reveal.Item Using Evolutionary Statistics to Understand Cellular Systems(2019-11-18) Schober, Andrew Frank, Jr.; Lin, Milo; Reynolds, Kimberly A.; Reese, Michael L.; Tu, BenjaminMetabolic enzyme function is dependent on the larger context of a biochemical pathway. Despite detailed characterization of the requisite molecular "parts," it remains difficult to predict the adaptive response to a simple perturbation. That is: if the activity or expression of a single enzyme is changed, what other proteins (if any) require compensatory mutation? Comparative genomics and experimental evolution provide two powerful approaches to begin addressing these questions. In my thesis work, I examined adaptive interactions with the essential enzyme dihydrofolate reductase (DHFR). Analyses of gene synteny and co-occurrence across 1445 bacterial genomes indicated that DHFR coevolves with thymidylate synthase (TYMS), but is relatively decoupled from the rest of the folate metabolic pathway (and genome). Through directed evolution of E. coli, I demonstrated that these two enzymes adapt cooperatively in response to antibiotic stress. An allele replacement experiment confirmed that a pair of mutations to DHFR and TYMS were sufficient to reconstitute the entire trimethoprim resistance phenotype, establishing that the two enzymes are capable of independently driving adaptation. In the final component of my thesis, I drew on the 'mirror-tree' method to define a new measure of residue-residue coevolution which corrects for the phylogenetic relationship among species. In summary, my results verify that small groups of genes within larger metabolic pathways can form adaptive modules that evolve as a unit in response to environmental or mutational stress. Moreover, my mirror-tree inspired analysis provides a path forward for understanding how coupled adaptation between genes manifests at the resolution of site specific constraints on the protein sequence.