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Connecting Female Mating Decisions and Male Courtship Strategies in Drosophila
No full textIn many species, males have evolved elaborate behavioral patterns to attract females or repel rival males. The neural mechanisms mediating these male behavioral traits and how females use them to guide their mating decisions remain elusive. In this thesis,I present our efforts to uncover the neural logic underlying mate selection in Drosophila melanogaster. Unlike males who display overt courtship behaviors in pursuit of a female mate, including chasing her and singing a courtship song to her, female behaviors during courtship are subtle, making it challenging to infer how a female\u27s sexual receptivity fluctuates based on the male\u27s performance.To gain insight into dynamic changes in female sexual receptivity, we explored two distinct female behaviors displayed during courtship. First, we characterized the \u27avoidance\u27 behaviors displayed by females in response to pursuit by an inappropriate mate, where a fast approaching visual target elicits rapid turning and acceleration but can be suppressed by male courtship song. Neural circuits underlying avoidance behavior appear to interface with those governing a female\u27s sexual receptivity, providing an indirect reflection of her mating preferences. Second, we analyzed female vaginal plate opening, a final motor program indicative of her sexual acceptance of a male, and how it is elicited in response to male performance. We reveal that the probability that a male\u27s song triggers females to open their vaginal plates varies over time, which likely reflects dynamic fluctuations in a female\u27s sexual receptivity. Beyond exploring how females respond to the courtship performance of individual males, we also considered how they assess and choose between alternate mates. Indeed, in the wild, Drosophila congregate on their host plants, creating a complex social environment where females are often pursued by multiple males concurrently. We demonstrate that when two males compete for a female, they rapidly alternate between courtship behaviors towards the female and agonistic behaviors towards their rival, characterized by performance of bilateral wing flicks. We show that agonistic wing flicks produce distinct acoustic signals that jam the auditory pathways of the female, impeding her detection of the advertising courtship song produced by rival males. Consequently, females are less likely to show receptive behaviors when serenaded by multiple males. Moreover, agonistic wing flicks serve to physically repel a male\u27s rivals, enabling him to overcome the acoustic interference from his competitors and gain sole access to the female. Reproductive success therefore requires that males persistently court females while also executing agonistic wing flicks to drive their competitors away. Combining optogenetic perturbations and calcium imaging in tethered males interacting with virtual fly targets, we gained insight into the circuit architecture that mediates the rapid alternation between courtship and agonistic behaviors, revealing how sex-specific sensory signals allow males to modulate their behavior on a moment- to-moment timescale and flexibly navigate complex social scenes. Together, these studies reveal the neural circuit logic underlying female mating decisions and how males adapt their courtship strategies accordingly to achieve reproductive success
Structural Mechanisms of F-actin Mechanochemical Regulation and Flexible Network Assembly
Full text linkThe actin cytoskeleton integrates physical and chemical cues from a cell\u27s environment to instruct its behavior, coordinating processes including cell migration, organelle dynamics, and endocytosis. Actin-binding proteins (ABPs) specify actin filament (F-actin) organization to build micron-scale networks which power these cell dynamics. Interactions between ABPs and F-actin are regulated by both biochemical processes and mechanical forces. The mechanisms mediating this interplay remain unclear at the protein structural level. Newly polymerized F-actin hydrolyzes bound ATP to produce metastable ADP-Pi–F-actin, which persists for minutes before phosphate release to yield long-lived ADP–F-actin. I determined cryo-EM structures of both ADP–F-actin and ADP-Pi–F-actin with sufficient resolution (~2.4 Å) to confidently build atomic models including solvent, which showed the two states to be essentially identical at the protein backbone level. This led me to hypothesize that differences in bound small molecules and solvent could alter the energetic landscape of filament bending, producing structural differences in bent states which could be discriminated by ABPs. By developing a neural-network–based particle picking approach and using methods to handle flexible heterogeneity in cryo-electron microscopy (cryo-EM) maps, I determined structures of bent ADP–F-actin and ADP-Pi–F-actin, revealing that individual actin subunits were distorted in nucleotide-state dependent patterns. Although ADP–F-actin and ADP-Pi–F-actin have essentially identical protein structures in their ground states, the release of phosphate and associated changes in the solvent content of ADP–F-actin\u27s nucleotide cleft renders its subunits more deformable. This decreased rigidity at the subunit level allows ADP–F-actin to bend more readily. I summarized these findings in a steric boundaries model for mechanical regulation: Lattice architectural rearrangements remodel the physical space available for a protomer to occupy, thereby inducing the protomer to deform to minimize steric clashes. I continued my explorations of the mechanobiology of F-actin by investigating the effects of tethered myosin motor systems on F-actin structure. Previous members of the Alushin lab had identified nanoscale domains of oscillating curvature, but these structures were refractory to traditional averaging approaches. I updated the tools developed to investigate the nucleotide state dependence of F-actin bending mechanics to these structures. These tools enabled 3Dreconstruction of these structures. From these reconstructions, the myosin motor-evoked filament lattice deformations were elucidated. The remodeled filament lattice is detected byα-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we found that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by α-catenin, providing a direct structural glimpse at force transduction through the cytoskeleton. I next studied how ABPs integrate actin filaments into functional ensembles, focusing on the structural basis for F-actin bundling by ABPs in filopodia. Filopodia are actin-based cellular protrusions localized at the leading edge of migrating cells, which play a prominent role in the dissemination of metastatic cancer cells. I first focused on T-plastin, an ABP that loosely bundles F-actin at the roots of filipodia. By adapting my neural-network–based picking scheme to identify bundled filaments while excluding single filaments, I determined structures of T-plastin bridging filaments in two configurations: parallel and antiparallel. By comparing these structures with a structure of T- plastin bound to a single filament, I was able to reconstruct a sequential bundling pathway, in which T-plastin\u27s conformation changes upon initial binding to a single actin filament, priming it to engage another filament in either one of these two distinct configurations. Most recently, I have focused on fascin, the protein primarily responsible for tightly bundling F-actin in filopodia. Our structure of fascin bridging filaments explains the mechanism of action of a fascin inhibitor currently in phase 3 clinical trials as a cancer therapeutic, and heterogeneity analysis revealed substantial crossbridge flexibility which mediates crosslinking helical actin filaments into hexagonal bundles with mismatched symmetry. I also adapted my neural network denoising approach to analyze cryo-electron tomograms of fascin-bundled F-actin. This enabled me to localize tens of thousands of individual proteins across dozens of bundled filaments with unprecedented accuracy, allowing unambiguous assignment of fascin positions and orientations without averaging. From these data, I discovered geometric rules which produce emergent patterns of fascin decoration within F-actin bundles and impose constraints on bundle growth. Altogether, I elucidated how individual fascin crossbridge flexibility at the nanoscale supports large flexible bundle construction at the mesoscale. The research presented in this thesis lays the groundwork for future work that will integrate these computational approaches for visualizing F-actin networks and force-induced deformations of filaments. Specifically, branched actin networks at the leading edges of migrating cells and bundled actin networks such as those at focal adhesions are force responsive and good targets for these studies. These investigations will probe the structural basis for the cellular mechano-response through the actin cytoskeleton in its native context
Elucidating the Role of Phospholipid Scramblase 1 in Antiviral Innate Immunity
No full textHosts possess an array of defenses against pathogens, some of which form a baseline protective barrier against infection. This baseline resistance, or intrinsic immunity, is the first layer of cellular defense that a virus must evade or antagonize in order to infect a cell. Intrinsic immunity also forms an important barrier against zoonosis—the transmission of a virus to new species outside its normal host range. Understanding these intrinsic restriction factors and their mechanisms of action can help us understand how pathogens evolve to circumvent or oppose these factors, and identify those pathogens that might be poised to infect new species.In this thesis, I detail our findings that phospholipid scramblase 1 (PLSCR1) works independently of innate immune signaling to oppose SARS-CoV-2 infection. SARS-CoV-2 is a highly contagious virus that was first discovered spreading in human populations in late 2019, having likely jumped from bats to humans. It rapidly became a pandemic, and since its emergence it has evolved to spread faster and more effectively in human populations. An arrayed CRISPR KO screen against SARS-CoV-2 identified many pro-and antiviral host factors, but the identification of PLSCR1 as an interferon (IFN) independent antiviral gene was curious, as no antiviral role for PLSCR1 outside of the IFN response had been identified before. Subsequent follow-up work revealed that, in the Huh-7.5 and A549-ACE2 cells used in this study, PLSCR1 was not required for the IFN response as had previously been reported in Hey1B cells. PLSCR1KO also did not substantially alter the transcriptional landscape of the cell, indicating that its mechanism of action was unrelated to its transcriptional activation activity. In a series of mechanistic studies, we found that PLSCR1 KO cells were more susceptible to SARS-CoV-2 infection, and that PLSCR1 inhibited virus entry. It did not inhibit both SARS-CoV-2 entry routes equally, primarily inhibiting endosomal entry by mechanisms that remain to be elucidated. In addition to our work elucidating where in the lifecycle of SARS-CoV-2 PLSCR1 acts, we also sought to understand how variation in SARS-CoV-2 and in PLSCR1 affected this dynamic. This line of investigation revealed that newer variants of SARS-CoV-2 are less susceptible to restriction by PLSCR1, perhaps linked to reported changes in the entry and tropism of Omicron and its descendant variants. Additionally, we found that the His262Tyr variant of PLSCR1, identified as a risk factor for severe SARS-2 infection, was hypomorphic and dominant in both our A549-ACE2 cells and immortalized cells from heterozygous patients. Taken together, this work identifies a previously uncharacterized role for PLSCR1 in the antiviral defense of the cell—a role that our research suggests shapes both SARS-CoV-2evolution and patient disease outcomes. This opens new avenues of investigation into the interplay between intrinsic and innate immunity, as well as further investigation into PLSCR1and its antiviral activities within and outside of the innate immune response
The Role of Gram - Negative Anaerobes in Hidradenitis Suppurativa
Full text linkHidradenitis Suppurativa is a chronic autoinflammatory disease with unknown triggers.New evidence in the last five years has begun to clarify two parts of the pathogenesis of the disease. These two parts are the fundamental role of keratinocyte inflammation from early through late-stage disease and the role of the microbiome as an antigenic and infectious trigger of pathogenesis. Gram-negative anaerobic bacteria (GNA) such as Prevotella, Porphyromonas, and Fusobacterium are commonly identified in HS lesions, and their prevalence in lesions is associated with HS disease severity. Additionally, newly epithelialized structures called dermal tunnels are found in severe disease and are associated with biofilm as well as the production of keratinocyte inflammatory cytokines. Our lab has previously defined the effects of gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes and found them to stimulate atopic dermatitis and psoriasis-like transcriptome profiles. In this thesis, using in vitro models of keratinocyte stimulation with heat-killed preparations of both gram-positive and gram-negative bacteria, we have explored a robust inflammatory profile generated in normal human keratinocytes by these GNAs. These GNAs elicit a strong IL17-dependent pathway response not seen in gram-positive bacteria and likely driven by IL-17C production. Overall, the magnitude of production of cytokines is much greater in all GNAs, with Fusobacterium nucleatum (FN) demonstrating the most intense activation that is mimicked to a lesser extent by Prevotella melaninogenica and Prevotella nigrescens. Significant differences in transcriptome profiles were found between all species, including members of the same species. We also find that this inflammatory response in GNAs is both TLR and JAK-dependent and that variation in cytokine production is likely driven by differential activation by TLR4 andTLR2 receptors. We then compare transcriptomes obtained from biopsies samples of HS patient lesional, perilesional, and nonlesional skin and from patients with psoriasis to GNA-keratinocyte profiles and IL17A stimulated keratinocyte profiles. We find that FN is most and significantly associated with HS gene transcriptomics. Our data is the first to provide a comprehensive profile of the effects of GNA stimulation on human keratinocytes that is more broadly applicable to innate immune epithelial defenses against GNAs. The identification of these bacteria as strong antigenic stimuli prioritizes targeting these bacteria in new therapies for HS, potentially through TLR4 or JAK inhibition
Revealing SMC5/6\u27s Dynamic Behavior on Diverse DNA Topologies Through Single-Molecule Methods
No full textThe structural maintenance of chromosomes (SMC) protein complexes serve as critical molecular motors for maintaining genome stability and ensuring faithful genome propagation. As one of the three eukaryotic SMC complexes, the Smc5/6 complex maintains chromosomal stability by coordinating DNA repair processes, promoting successful genome duplication, and silencing viral extrachromosomal DNA. Although these main functionalities have been identified, there is little understanding of how Smc5/6 operates as a versatile molecular machine capable of undertaking different functionalities. And despite being intimately involved with DNA processing reactions, little is known about Smc5/6\u27s dynamic behavior on various DNA substrates, including double-stranded DNA (dsDNA), stretches of single-stranded DNA (ssDNA), chromatinized DNA, forked/junction DNA, and recombination intermediates. In this thesis, our approach was to utilize correlative single-molecule fluorescence and force microscopy, which combined optical tweezers, automated microfluidics, and multi-color confocal microscopy, to investigate the S. cerevisiae Smc5/6 complex\u27s dynamic behavior on a diverse array of DNA topologies. Firstly, we uncovered dynamic modes of association for Smc5/6 on linear dsDNA. For the octameric holo-complex that contains the Nse5-6 subunit, Smc5/6 demonstrates both an ATP-independent dsDNA association mode and an ATP- dependent mode that exhibits greatly enhanced dsDNA association. This is likely due to a conformational change in the ATP-bound state that results in a \u27clamped\u27 configuration around dsDNA, making Smc5/6 resistant to high-salt challenges on dsDNA. However, the Smc5/6 hexamer complex lacking Nse5-6 exhibited dramatically increased dsDNA association independently of ATP, and this hexamer binding mode was resistant to high salt without requiring ATP. This indicates a novel mode of dsDNA association that may play a role in Smc5/6\u27s ability to loop extrude. Additionally, the Smc5/6 complex showed a striking capacity to accumulate onto free ssDNA stretches that can inhibit reannealing of complementary ssDNA strands, which could contribute to Smc5/6\u27s roles in stabilizing replication forks and preventing spurious strand invasion during homologous recombination. We also show that Smc5/6 can stably bind to DNA junctions resembling replication forks as well as linear ss-dsDNA junctions—both at the 5\u27 and 3\u27 end junctions. However, ATP and the RPA-ssDNA complex can confer a polarity preference for Smc5/6. We show that different subunits of Smc5/6 contribute to different functions as the Nse1-3-4 subunit constitutively contributes to DNA binding while the Nse5-6 subunit performs a modulatory role. Lastly, Smc5/6 displays the surprising interaction of stably associating with nucleosomes, while exhibiting more competitive behaviors with PCNA on DNA. Overall, our findings on the intrinsic properties of the Smc5/6 complex contribute to a framework for understanding how Smc5/6 may target and associate with DNA in a diverse array of cellular contexts ranging from DNA repair and stressed replication forks, to telomeric heterochromatin and extrachromosomal viral DNA
Structual Visualization of Cytoskeletal Force Tranduction
Full text linkCells adhere to their surroundings, mechanically interfacing their intracellular actin cytoskeletons with their local extracellular environments. This enables contractile forces generated by myosin motors to mediate transduction of mechanical cues into biochemical signaling pathways by unclear mechanisms. In this thesis, I show that myosin forces elicit conformational transitions in actin filaments (F-actin) that modulate interactions between F-actin and the force-activated cell adhesion protein α-catenin.In vitro reconstitution and cryo-electron microscopy reveal myosin force-evoked superhelical F-actin spirals. Three-dimensional reconstruction and variability analysis uncover extensive asymmetric remodeling of F-actin\u27s helical lattice. This is recognized by α-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, I find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by α-catenin, providing a direct structural glimpse at force transduction through the cytoskeleton
Human Inborn Errors of Immunity: Tuberculosis, Autoimmunity, and Beyond
No full textTuberculosis (TB), a multi-organ infectious disease caused by virulent mycobacteria, most predominantly M. tuberculosis, remains one of the deadliest infectious diseases in human history. Bacillus Calmette-Guérin (BCG) vaccine, first implemented medically in 1921, has remained the only available vaccine for TB, although its protective effect is moderate. TB occurs in 5-10% of individuals exposed to those pathogens, while 90-95% remain asymptomatic (i.e.,latent infection or spontaneous clearance). This surprisingly large inter-individual variability has long attracted the attention of human geneticists. Classical twin-based genetic studies have demonstrated the presence of a substantial genetic risk of developing TB disease between monozygotic twins. Later, the clinical and genetic characterization of a rare and severe disease caused by weakly virulent and normally harmless mycobacteria, such as the BCG vaccine substrain, now recognized as Mendelian Susceptibility to Mycobacterial Disease (MSMD), demonstrated that IFN-γ is indispensable for antimycobacterial immunity in humans.Moreover, homozygosity for a common missense mutation P1104A in a protein TYK2 was discovered as the first genetic predisposing factor to TB in humans. TYK2 P1104A selectively impairs IL-23-dependent IFN-γ production by lymphocytes.In light of these findings, I hypothesized that a partial impairment of IFN-γ immunity due to known or novel genetic defects underlies vulnerability to TB in humans. I characterized three rare genetic etiologies of TB in children and young adults: inherited ITK, PD-1, and LY9 deficiencies.These rare genetic defects partially impair IFN-γ production by diverse T cell subsets through distinct mechanisms such as development, exhaustion, and epigenetic imprinting.Thus, the first part of my graduate research demonstrates that even partial impairment of T-cell IFN-γ production can underlie TB in young humans. PD-1 is an inhibitory checkpoint in T lymphocytes ligated with two known ligands, PD-L1 and PD-L2, to maintain self-tolerance. The PD-1-deficient proband had not only TB but also multi-organ autoimmunity and died of autoimmune pneumonitis at age 11 years. His older brother also had type 1 diabetes and died of pneumonitis of undocumented etiology at age 3 years. Moreover, I had an opportunity to study another two siblings with inherited PD-L1 deficiency, both of whom had early-onset type 1diabetes. Unlike the two PD-1-deficient siblings, neither of the PD-L1-deficient siblings developed any autoimmune phenotypes other than endocrinological autoimmunity. Consistent with their much more severe clinical manifestations, PD-1 deficiency triggered much more severe leukocyte dysregulation than PD-L1 deficiency. The discordant severity in clinical and leukocytic phenotypes in humans with PD-1 and PD-L1 deficiencies suggest that 1) PD-1:PD-L2 interaction can compensate, at least in part, for the absence of PD-1:PD-L1 signal to mitigate unrestrained leukocytic dysregulation in vivo; and that 2) PD-1:PD-L1 signal is nonetheless indispensable in preventing type 1 diabetes in humans. Finally, during these studies, I discovered that PD-1 and PD-L1 contribute to optimal memory B cell development and antibody responses. Surprisingly, PD-1:PD-L1 interactions promote immunoglobulin production in a B-cell-autonomous manner, and a newly established B-cell-specific PD-1 KO mouse line showed a severe contraction of almost all B cell compartments. In fact, the B-cell phenotype was much more severe in B-cell-specific PD-1 KO mice than in whole-body KO mice, suggesting that 1) PD-1 on B cells has a critically indispensable role in the homeostasis of B cells and 2) PD-1 on leukocytes other than B cells (probably T cells) has a unique role in mitigating the disturbance the B-cell compartment. Thus, the second part of my graduate research provides unique insights into the non-redundant roles of PD-1 and PD-L1 in maintaining self-tolerance and functional oral immunity in mice and humans
Discovery and Optimization of a Family of Naturally Encoded Antibiotic Compounds that Evade Antibiotic
Full text linkAntibacterial resistant infections are an ongoing global health emergency. To combat this,novel potent antibiotics with unique modes of action are required. The classic arsenal of antibiotics used in clinical settings are largely natural products discovered by bulk fermentation of bacterial species isolates that are then extracted and purified to yield a bioactive compound.Unfortunately, this discovery pipeline no longer produces novel molecules, thus new discovery methodologies are essential to continue to identify antibiotic clinical candidate molecules. In the modern era, coupling natural product discovery with sequencing technologies has proven to bean efficient and fruitful method of identifying secondary metabolite natural products with unique bioactivity that could not previously be accessed through fermentation-based methods. This novel approach presents a promising reinvigoration to the field of antibiotics discovery. In our lab, we have developed a discovery method by which sequenced bacterial genomes are analyzed using bioinformatic algorithms to identify biosynthetic gene clusters (BGCs) and to make a structural prediction of the molecular product of a given cluster. This molecular prediction can be built using synthetic chemistry and assayed for its biological activity. The name given to this method and the resulting products that are synthesized is synthetic bioinformatic natural products (synBNP). It is using this method that our lab previously discovered cilagicin, a lipopeptide natural product that shows robust Gram-positive antibiotic activity and evades antibiotic resistance even after prolonged pathogen exposure. This resistance evasion is attributed to a dual polyprenyl phosphate binding mechanism. In this thesis, I present discovery and optimization efforts to expand this promising novel class of antibiotic natural products as well as to develop a singular lead clinical drug candidate that displays optimal bioactivity and in vivo efficacy. In Chapter 2, I present an investigation of bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to identify previously uncharacterized antibiotics that may possess the same molecular targets and resistance evasion ability seen with cilagicin. This structure-based screen yields three BGCs predicted to produce natural analogs of cilagicin. synBNPs for the products of each of these three clusters are synthesized and the resulting molecules are assayed for their biological activity. These compounds, called paenilagicin, bacilagicin, and virgilagicin, are shown to be potent antibiotics against multidrug-resistant Gram-positive pathogens. Paenilagicin and virgilagicin are further shown to engage both of the same polyprenyl phosphate targets as cilagicin, and both also demonstrate the ability to evade antibiotic resistance. Bacilagicin is shown to only bind a single molecular target and is susceptible to antibiotic resistance development. This discovery project expands the members of this family of polyprenyl phosphate binding antibiotics, which allows us to identify a conserved peptide moiety that we suspect may play a role in target engagement. In Chapter 3, building upon the structural diversity identified among polyprenyl phosphate binding antibiotics, I discuss a structural optimization project in which we sought to design an improved version of the most potent antibiotic in this family, cilagicin. To achieve our goal of a molecule with strong antibiotic activity and low serum protein binding to preserve activity invivo, we conduct two regional analyses of the overall molecular structure. In the first, we investigate the effect of structurally diverse lipid tail substituents on bioactivity. In the second, we conduct a series of orthogonal scans on the peptide core to explore the impact of different peptide moieties with various chemical properties on bioactivity. Ultimately, we identify an optimized compound, called dodecacilagicin, that maintains high Gram-positive antibiotic activity, shows minimal serum protein binding, and also evades antibiotic resistance. Overall, the work presented in this thesis represents the application and expansion of the synBNP discovery method as an ever evolving and robust means to discover novel structurally diverse natural products with unique bioactivity that were previously inaccessible by culture-dependent methods
Cells that Kill: The Chemosensory Cell Types of Aedes aegypti Mosquitoes
Full text linkThe ability to detect and respond to environmental cues is the fundamental start of any brain function, computation, or behavior. For my doctoral work, I studied chemosensation in the Aedes aegypti mosquito, which can detect cues including human odor, carbon dioxide, and insect repellents. Mosquitoes are human\u27s deadliest predator, causing 700,000 deaths every year through the transmission of malaria parasites and arboviruses including dengue. My research has focused on the molecular underpinnings of mosquito chemosensory cell types. In olfaction, research following the cloning of the first odorant receptors in1991 has shown that each mouse olfactory sensory neuron (OSN) expresses a single odorant receptor. Early work in Drosophila melanogaster flies reported a similar organization, although some key differences were noted. Insects detect odorants with three families of ionotropic ligand-gated ion channels encoded by three large multi-gene families: Odorant Receptors (ORs), Ionotropic Receptors (IRs), and Gustatory Receptors (GRs) each of which form heteromultimeric ligand-gated ion channels composed of co-receptor and ligand-selective receptor subunits. These families were thought to be mutually exclusively expressed in non-overlapping OSNs. We developed techniques to extract chemosensory neurons from the two mosquito olfactory organs, the antenna, and the maxillary palp, for single nucleus RNA-sequencing (snRNA-seq). Performing careful analysis, we found that mosquitoes co-express multiple chemosensory receptor genes within individual OSNs. In the maxillary palp, nearly all the OSNs co-expressed multiple chemo receptor subunits from at least two of the three families of chemoreceptors. In the antenna, there were at least 35 chemosensory cell types with unique receptor profiles, containing two, three, or more receptors per cell. We concluded that the mosquito olfactory system represents a departure from the canonical understanding of olfaction as first established in the mouse. These studies established a foundational molecular understanding of mosquito sensory cell types, the neurobiological substrate of human detection and host-seeking. We applied our approach to mosquito legs, which contain taste and other sensory neurons that are critical to support mutually exclusive behaviors related to important reproductive life stages of the adult female mosquito. These include assessing the skin of a human host prior to blood feeding, sensing the pheromones of a conspecific prior to copulation, and detecting freshwater appropriate for egg-laying. We characterized the neuron types in the mosquito leg and discovered that the number of neurons varies across the fore-, mid-, and hind-legs. We also showed that each of the legs contributes to contact chemo avoidance of DEET, the principal ingredient in most insect repellents. Moreover, we discovered that the avoidance of DEET is a distinct sensation from the avoidance of bitter, as demonstrated by mosquitoes not being deterred by high concentrations of bitter substances applied to skin. We were interested in the possibility that taste neuron activity is modulated to reflect the female\u27s physiological needs for food, mating, or depositing her offspring. Using snRNA-seq, we were able to classify leg sensory cell types and their receptors and discovered that mosquito leg neurons are polymodal, co-expressing sensory receptors that respond to diverse stimuli across sensory domains, including noxious heat, warmth, sugar, salt, water, and fatty acids. Strikingly, these cell types also express distinct profiles of biogenic amine, neuropeptide, and neurotransmitter receptors. This suggests the possibility that these sensory neurons are subject to local or top-down modulation that drives context- and state-relevant behaviors. We are now expanding on this work with the Mosquito Cell Atlas project, a large-scale snRNA-seq project of essentially every tissue from the adult Aedes aegypti mosquito. Many mosquito sensory organs are capable of gustation, including the legs, wings, mouth parts, and egg-laying organs, and taste is necessary for every stage of the mosquito\u27s life and reproductive cycle. Our goal is to provide a reliable cell atlas to the field, interpret the molecular mechanisms driving mosquito biology and behavior, and understand potential targets that will be invaluable for developing interventions to mitigate the spread of deadly pathogens from mosquitoes to humans
Single-Molecule Studies of CFTR Gating and Pharmacology
No full textThe cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed in the apical membrane of epithelial tissues. Alterations in CFTR that disrupt activity cause cystic fibrosis, a fatal disease which is characterized by systemic salt and fluid dysregulation. By contrast, hyperactivation of CFTR is central to pathogenesis in secretory diarrhea and autosomal dominant polycystic kidney disease. Electrophysiological properties of CFTR have been analyzed for decades. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Opening of CFTR\u27s pore is regulated by phosphorylation- and ATP-dependent dimerization of its two nucleotide-binding domains (NBDs), but how the dimerization process is coupled to pore opening remains contested. Whereas the binding sites for therapeutic potentiators of CFTR gating are known, the mechanism by which they increase channel open probability is not understood. In this thesis, I combined ensemble functional measurements, single-molecule fluorescence resonance energy transfer imaging, electrophysiology, electron microscopy and kinetic simulations to study human CFTR gating and conduction, as well as how these processes are altered by disease mutations and pharmacological modulation. I first established a model for gating by wild-type CFTR. I found that CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization and weaken coupling of the dimerization process to opening of the pore. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while the NBDs are dimerized. A second modulator, elexacaftor, was found to act additively with GLPG1837 or ivacaftor to further increase open probability of both the wild-type CFTR and disease-causing variants. The combined effects of elexacaftor andGLPG1837 stabilize the open pore regardless of nucleotide state in the NBDs and thereby decouplepore opening from the ATP hydrolysis cycle. Despite determination of CFTR structures, the ion conduction path remains elusive. Here, a density consistent with dehydrated chloride is directly observed within the pore of CFTR. Substitution of residues proximal to the putative chloride altered the conductance and selectivity properties of the pore. The inhibitor CFTRinh-172 was found to act by direct occlusion of this selectivity filter. Finally, knowledge of CFTR structure and mechanism was applied for structure-based discovery of small-molecule modulators that allosterically potentiate or inhibit CFTR gating. These molecules represent potential leads for drug development and demonstrate the feasibility of large-scale docking for ion channel drug discovery