The Structural Logic of Dynamic Signaling in the Escherichia coli Serine Chemoreceptor.

The experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, the Escherichia coli serine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created a series of cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. An AlphaFold 3 model of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli probably inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby freezing kinase activity.

The Ca2+-activated Cl- channel TMEM16B shapes the response time course of olfactory sensory neurons.

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.

Human Transient Receptor Potential Ankyrin 1 Channel: Structure, Function, and Physiology.

The transient receptor potential ion channel TRPA1 is a Ca2+-permeable nonselective cation channel widely expressed in sensory neurons, but also in many nonneuronal tissues typically possessing barrier functions, such as the skin, joint synoviocytes, cornea, and the respiratory and intestinal tracts. Here, the primary role of TRPA1 is to detect potential danger stimuli that may threaten the tissue homeostasis and the health of the organism. The ability to directly recognize signals of different modalities, including chemical irritants, extreme temperatures, or osmotic changes resides in the characteristic properties of the ion channel protein complex. Recent advances in cryo-electron microscopy have provided an important framework for understanding the molecular basis of TRPA1 function and have suggested novel directions in the search for its pharmacological regulation. This chapter summarizes the current knowledge of human TRPA1 from a structural and functional perspective and discusses the complex allosteric mechanisms of activation and modulation that play important roles under physiological or pathophysiological conditions. In this context, major challenges for future research on TRPA1 are outlined.

Vibrio fischeri: a model for host-associated biofilm formation.

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

A bacterial sensor taxonomy across earth ecosystems for machine learning applications.

Microbial communities have evolved to colonize all ecosystems of the planet, from the deep sea to the human gut. Microbes survive by sensing, responding, and adapting to immediate environmental cues. This process is driven by signal transduction proteins such as histidine kinases, which use their sensing domains to bind or otherwise detect environmental cues and "transduce" signals to adjust internal processes. We hypothesized that an ecosystem's unique stimuli leave a sensor "fingerprint," able to identify and shed insight on ecosystem conditions. To test this, we collected 20,712 publicly available metagenomes from Host-associated, Environmental, and Engineered ecosystems across the globe. We extracted and clustered the collection's nearly 18M unique sensory domains into 113,712 similar groupings with MMseqs2. We built gradient-boosted decision tree machine learning models and found we could classify the ecosystem type (accuracy: 87%) and predict the levels of different physical parameters (R2 score: 83%) using the sensor cluster abundance as features. Feature importance enables identification of the most predictive sensors to differentiate between ecosystems which can lead to mechanistic interpretations if the sensor domains are well annotated. To demonstrate this, a machine learning model was trained to predict patient's disease state and used to identify domains related to oxygen sensing present in a healthy gut but missing in patients with abnormal conditions. Moreover, since 98.7% of identified sensor domains are uncharacterized, importance ranking can be used to prioritize sensors to determine what ecosystem function they may be sensing. Furthermore, these new predictive sensors can function as targets for novel sensor engineering with applications in biotechnology, ecosystem maintenance, and medicine.IMPORTANCEMicrobes infect, colonize, and proliferate due to their ability to sense and respond quickly to their surroundings. In this research, we extract the sensory proteins from a diverse range of environmental, engineered, and host-associated metagenomes. We trained machine learning classifiers using sensors as features such that it is possible to predict the ecosystem for a metagenome from its sensor profile. We use the optimized model's feature importance to identify the most impactful and predictive sensors in different environments. We next use the sensor profile from human gut metagenomes to classify their disease states and explore which sensors can explain differences between diseases. The sensors most predictive of environmental labels here, most of which correspond to uncharacterized proteins, are a useful starting point for the discovery of important environment signals and the development of possible diagnostic interventions.

Anion efflux mediates transduction in the hair cells of the zebrafish lateral line.

Hair cells are the principal sensory receptors of the vertebrate auditory system, where they transduce sounds through mechanically gated ion channels that permit cations to flow from the surrounding endolymph into the cells. The lateral line of zebrafish has served as a key model system for understanding hair cell physiology and development, often with the belief that these hair cells employ a similar transduction mechanism. In this study, we demonstrate that these hair cells are exposed to an unregulated external environment with cation concentrations that are too low to support transduction. Our results indicate that hair cell excitation is instead mediated by a substantially different mechanism involving the outward flow of anions. Further investigation of hair cell transduction in a diversity of sensory systems and species will likely yield deep insights into the physiology of these unique cells.

Clinical potential of sensory neurites in the heart and their role in decision-making.

The process of decision-making is quite complex involving different aspects of logic, emotion, and intuition. The process of decision-making can be summarized as choosing the best alternative among a given plethora of options in order to achieve the desired outcome. This requires establishing numerous neural networks between various factors associated with the decision and creation of possible combinations and speculating their possible outcomes. In a nutshell, it is a highly coordinated process consuming the majority of the brain's energy. It has been found that the heart comprises an intrinsic neural system that contributes not only to the decision-making process but also the short-term and long-term memory. There are approximately 40,000 cells present in the heart known as sensory neurites which play a vital role in memory transfer. The heart is quite a mysterious organ, which functions as a blood-pumping machine and an endocrine gland, as well as possesses a nervous system. There are multiple factors that affect this heart ecosystem, and they directly affect our decision-making capabilities. These interlinked relationships hint toward the sensory neurites which modulate cognition and mood regulation. This review article aims to provide deeper insights into the various roles played by sensory neurites in decision-making and other cognitive functions. The article highlights the pivotal role of sensory neurites in the numerous brain functions, and it also meticulously discusses the mechanisms through which they modulate their effects.

Rethinking Opsins.

Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.

Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains.

PAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture. IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

Sensory transduction is required for normal development and maturation of cochlear inner hair cell synapses.

Acoustic overexposure and aging can damage auditory synapses in the inner ear by a process known as synaptopathy. These insults may also damage hair bundles and the sensory transduction apparatus in auditory hair cells. However, a connection between sensory transduction and synaptopathy has not been established. To evaluate potential contributions of sensory transduction to synapse formation and development, we assessed inner hair cell synapses in several genetic models of dysfunctional sensory transduction, including mice lacking transmembrane channel-like (Tmc) 1, Tmc2, or both, in Beethoven mice which carry a dominant Tmc1 mutation and in Spinner mice which carry a recessive mutation in transmembrane inner ear (Tmie). Our analyses reveal loss of synapses in the absence of sensory transduction and preservation of synapses in Tmc1-null mice following restoration of sensory transduction via Tmc1 gene therapy. These results provide insight into the requirement of sensory transduction for hair cell synapse development and maturation.

Putting the Pieces Together: the Hair Cell Transduction Complex.

Identification of the components of the mechanosensory transduction complex in hair cells has been a major research interest for many auditory and vestibular scientists and has attracted attention from outside the field. The past two decades have witnessed a number of significant advances with emergence of compelling evidence implicating at least a dozen distinct molecular components of the transduction machinery. Yet, how the pieces of this ensemble fit together and function in harmony to enable the senses of hearing and balance has not been clarified. The goal of this review is to summarize a 2021 symposium presented at the annual mid-winter meeting of the Association for Research in Otolaryngology. The symposium brought together the latest insights from within and beyond the field to examine individual components of the transduction complex and how these elements interact at molecular, structural, and biophysical levels to gate mechanosensitive channels and initiate sensory transduction in the inner ear. The review includes a brief historical background to set the stage for topics to follow that focus on structure, properties, and interactions of proteins such as CDH23, PCDH15, LHFPL5, TMIE, TMC1/2, and CIB2/3. We aim to present the diversity of ideas in this field and highlight emerging theories and concepts. This review will not only provide readers with a deeper appreciation of the components of the transduction apparatus and how they function together, but also bring to light areas of broad agreement, areas of scientific controversy, and opportunities for future scientific discovery.

Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

A Bacterial Inflammation Sensor Regulates c-di-GMP Signaling, Adhesion, and Biofilm Formation.

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn2+ occupancy, which in turn regulates the catalysis of c-di-GMP by the associated GGDEF domain. We find DgcZ-dependent biofilm formation and HOCl sensing to be regulated in vivo by the conserved zinc-coordinating cysteine. Additionally, point mutants that mimic oxidized CZB states increase total biofilm. A survey of bacterial genomes reveals that many pathogenic bacteria that manipulate host inflammation as part of their colonization strategy possess CZB-regulated diguanylate cyclases and chemoreceptors. Our findings suggest that CZB domains are zinc-sensitive regulators that allow host-associated bacteria to perceive host inflammation through reactivity with HOCl. IMPORTANCE Immune cells are well equipped to eliminate invading bacteria, and one of their primary tools is the synthesis of bleach, hypochlorous acid (HOCl), the same chemical used as a household disinfectant. In this work, we present findings showing that many host-associated bacteria possess a bleach-sensing protein that allows them to adapt to the presence of this chemical in their environment. We find that the bacterium Escherichia coli responds to bleach by hunkering down and producing a sticky matrix known as biofilm, which helps it aggregate and adhere to surfaces. This behavior may play an important role in pathogenicity for E. coli and other bacteria, as it allows the bacteria to detect and adapt to the weapons of the host immune system.

Mechanotransduction Ion Channels in Hearing and Touch.

The ability of living organisms to detect mechanical force originates from mechanotransduction ion channels, which convert membrane tension into electrical or chemical signals that are transmitted to the brain. A variety of studies on touch and sound perception in both vertebrates and invertebrates have broadened our understanding of mechanotransduction and identified promising candidates for mechanotransduction ion channels. Here, we discussed the physiological properties of mechanotransduction ion channels in hearing and touch, the identification of their molecular entities, and recent structural studies providing insights to their gating mechanisms in force sensing. We present an updated review of the evidence supporting several candidates, including NOMPC, Brv1, and TMC channels, as mechanotransduction ion channels and highlight their qualifications satisfying the specific criteria proposed for a mechanotransducer.

Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice.

AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.

Neuropod Cells: The Emerging Biology of Gut-Brain Sensory Transduction.

Guided by sight, scent, texture, and taste, animals ingest food. Once ingested, it is up to the gut to make sense of the food's nutritional value. Classic sensory systems rely on neuroepithelial circuits to convert stimuli into signals that guide behavior. However, sensation of the gut milieu was thought to be mediated only by the passive release of hormones until the discovery of synapses in enteroendocrine cells. These are gut sensory epithelial cells, and those that form synapses are referred to as neuropod cells. Neuropod cells provide the foundation for the gut to transduce sensory signals from the intestinal milieu to the brain through fast neurotransmission onto neurons, including those of the vagus nerve. These findings have sparked a new field of exploration in sensory neurobiology-that of gut-brain sensory transduction.

An Adaptive-Threshold Mechanism for Odor Sensation and Animal Navigation.

Identifying the environmental information and computations that drive sensory detection is key for understanding animal behavior. Using experimental and theoretical analysis of AWCON, a well-described olfactory neuron in C. elegans, here we derive a general and broadly useful model that matches stimulus history to odor sensation and behavioral responses. We show that AWCON sensory activity is regulated by an absolute signal threshold that continuously adapts to odor history, allowing animals to compare present and past odor concentrations. The model predicts sensory activity and probabilistic behavior during animal navigation in different odor gradients and across a broad stimulus regime. Genetic studies demonstrate that the cGMP-dependent protein kinase EGL-4 determines the timescale of threshold adaptation, defining a molecular basis for a critical model feature. The adaptive threshold model efficiently filters stimulus noise, allowing reliable sensation in fluctuating environments, and represents a feedforward sensory mechanism with implications for other sensory systems.

The Pivotal Role of TRP Channels in Homeostasis and Diseases throughout the Gastrointestinal Tract.

The transient receptor potential (TRP) channels superfamily are a large group of proteins that play crucial roles in cellular processes. For example, these cation channels act as sensors in the detection and transduction of stimuli of temperature, small molecules, voltage, pH, and mechanical constrains. Over the past decades, different members of the TRP channels have been identified in the human gastrointestinal (GI) tract playing multiple modulatory roles. Noteworthy, TRPs support critical functions related to the taste perception, mechanosensation, and pain. They also participate in the modulation of motility and secretions of the human gut. Last but not least, altered expression or activity and mutations in the TRP genes are often related to a wide range of disorders of the gut epithelium, including inflammatory bowel disease, fibrosis, visceral hyperalgesia, irritable bowel syndrome, and colorectal cancer. TRP channels could therefore be promising drug targets for the treatment of GI malignancies. This review aims at providing a comprehensive picture of the most recent advances highlighting the expression and function of TRP channels in the GI tract, and secondly, the description of the potential roles of TRPs in relevant disorders is discussed reporting our standpoint on GI tract-TRP channels interactions.

Merkel Cells Release Glutamate Following Mechanical Stimulation: Implication of Glutamate in the Merkel Cell-Neurite Complex.

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons through synaptic contact. However, the detailed mechanisms for intercellular communication between MCs and neurons have yet to be clarified. The present study examined the increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by direct mechanical stimulation of MCs. We also measured [Ca2+]i in the trigeminal ganglion neurons (TGs) following direct mechanical stimulation to the MCs in an MC-TGs coculture. The MCs were isolated from hamster buccal mucosa, while TGs were isolated from neonatal Wistar rats. Both cell populations showed depolarization-induced [Ca2+]i. Direct mechanical stimulation to MCs increased [Ca2+]i, showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca2+]i in the TGs. These changes were significantly suppressed by antagonists of glutamate-permeable anion channels (4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid; DIDS), and non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptors (MK801). Apyrase, an ATP-degrading enzyme, and suramin, a non-selective P2 purinergic receptor antagonist, did not exert inhibitory effects on these [Ca2+]i increases in the TGs following MC stimulation. These results indicated that MCs are capable of releasing glutamate, but not ATP, in response to cellular deformation by direct mechanical stimulation. The released glutamate activates the NMDA receptors on TGs. We suggest that MCs act as mechanoelectrical transducers and establish synaptic transmission with neurons, through the MC-neurite complex, to mediate mechanosensory transduction.