Mechanical forces are fundamental regulators of biology. Individual cells can detect environmental forces and transform them into intracellular biochemical actions, which impact gene expression, metabolism, and differentiation. In turn, this phenomenon of “mechanotransduction” at the cellular level affects tissue- and organ-level function and can shape disease progressions. Tools that enable researchers to genetically harness mechanotransduction would therefore be powerful for developing of novel tissue engineering and cell therapy technologies. However, synthetically engineering mechanotransduction in cells has remained difficult. In this thesis, we control how cells respond to molecular forces by engineering modular mechanosensitive receptors. Using a structured-guided approach, we engineered force-sensitive protein domains that, when inserted into synthetic Notch receptors, vary the input-output relationship between mechanical force and cellular action. We demonstrate that the mechanical strength of these domains can be systematically tuned through mutagenesis. We show that our synthetic mechanoreceptors enable the design of signaling networks where tensile forces in the environment are recorded as measurable and specifiable biochemical responses, such as myogenic differentiation in mouse embryonic fibroblasts. We then present additional technologies for modulating the Notch mechanoreceptor’s endogenous mechanical strength, ligand-mediated activation, and protease-regulated activation. Taken together, this dissertation introduces a mechanogenetic framework for synthetically controlling mechanotransduction in mammalian cells, informs the design of future synthetic force-sensitive pathways, and provides valuable tools for the study of Notch signaling in development and disease. / 2022-09-30T00:00:00Z

No description available.

<p>Pain is defined by the international association of pain as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Most people have experienced one form of pain or another and although such experiences can be unsavory, pain serves the basic need for the detection of dangerous stimuli that can cause bodily harm. Because pain serves such an essential need, it is important to understand how the nervous system processes and encodes noxious or potentially tissue damaging stimuli. This neural processing is called nociception. </p><p>In this study, I use Drosophila larvae as a genetic model organism to study nociception. In response to noxious thermal and mechanical stimuli, Drosophila larvae perform a nociceptive defensive behavior (termed nocifensive) where larvae rotate in a corkscrew like fashion along the long axis causing them to move in a lateral direction. Using this behavior and genetic tools which can manipulate neuronal output, we have identified the sensory neurons which serve as larval nociceptors as class IV multidendritic sensory neurons. Further characterization of these larval nociceptors, has also shown that they are both cholinergic and peptidergic.</p><p>After the identifying the larval nociceptors, I next identified several molecular components which are required for larval mechanical nociception. I have found that the degenerin epithelial sodium channel (DEG/ENaC) called pickpocket is required for larval mechanical nociception by using genetic mutants and RNAi knockdwon. In addition, after performing a screen using RNAi to knockdown ion channel transcripts in larval nociceptors, I have identified two other DEG/ENaC channels which are required for larval mechanical nociception. DEG/ENaCs are particularly interesting because they have been identified as candidate mechanotransducers in C. elegans for the gentle touch behavior. I propose that DEG/ENaCs may serve as candidate mechanotransducers in larval mechanical nociception because they are not generally required for neuronal excitability. However, future research will be required to establish their true role in mechanical nociceptive signaling.</p><p>In addition to DEG/ENaCs, transient receptor potential (TRP) channels also play a role in nociception. painless, a channel that was first identified in a thermal nociception screen on Drosophila larvae, is required for both thermal and mechanical nociception. The last section shows that multiple isoforms of painless exist and that these different isoforms may play different roles in thermal and mechanical nociception. </p><p>Taken together, these results have begun to establish Drosophila larva as a model for studying nociception. I have identified the sensory neurons used as larval nociceptors and shown that DEG/ENaC channels play an important role in larval mechanical nociception.</p> / Dissertation

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The ability to sense humidity and wetness is an important sensory attribute for many species across the animal kingdom, including humans. Although this sensory ability plays an important role in many human physiological and behavioural functions, as humans largest sensory organ i.e. the skin seems not to be provided with specific receptors for the sensation of wetness (i.e. hygroreceptors), the neurophysiological mechanisms underlying this complex sensory experience are still poorly understood. The aim of this Thesis was to investigate the neurophysiological mechanisms underpinning humans remarkable ability to sense skin wetness despite the lack of specific skin hygroreceptors. It was hypothesised that humans could learn to perceive the wetness experienced when the skin is in contact with a wet surface or when sweat is produced through a complex multisensory integration of thermal (i.e. heat transfer) and tactile (i.e. mechanical pressure and friction) inputs generated by the interaction between skin, moisture and (if donned) clothing. Hence, as both thermal and tactile skin afferents could contribute significantly to drive the perception of skin wetness, their role in the peripheral and central sensory integration of skin wetness perception was investigated, both under conditions of skin s contact with an external (dry or wet) stimulus as well as during the active production of sweat. A series of experimental studies were performed, aiming to isolate the contribution of each sensory cue (i.e. thermal and tactile) to the perception of skin wetness during rest and exercise, as well as under different environmental conditions. It was found that it is not the contact of the skin with moisture per se, but rather the integration of particular sensory inputs which drives the perception of skin wetness during both the contact with an external (dry or wet) surface, as well as during the active production of sweat. The role of thermal (cold) afferents appears to be of a primary importance in driving the perception of skin wetness during the contact with an external stimulus. However, when thermal cues (e.g. evaporative cooling) are limited, individuals seem to rely more on tactile cues (i.e. stickiness and skin friction) to characterise their perception of skin wetness. The central integration of conscious coldness and mechanosensation, as sub-served by peripheral cutaneous A-nerve fibers, seems therefore the primary neural process underpinning humans ability to sense wetness. Interestingly, these mechanisms (i.e. integration of thermal and tactile sensory cues) appear to be remarkably consistent regardless of the modality for which skin wetness is experienced, i.e. whether due to passive contact with a wet stimulus or due to active production of sweat. The novelty of the findings included in this Thesis is that, for the first time, mechanistic evidence has been provided for the neurophysiological processes which underpin humans ability to sense wetness on their skin. Based on these findings, the first neurophysiological sensory model for human skin wetness perception has been developed. This model helps explain humans remarkable ability to sense warm, neutral and cold skin wetness.

Ion pumps establish homeostasis across the membranes of living cells. Hearing is a mechanotransduction event that takes place in a closed compartment containing a fluid high in K+ concentrations. In Drosophila melanogaster, this closed compartment is formed by a scolopale cell that wraps around the dendrite of sensory neurons. The receptor lymph is maintained by the scolopale cell. The lumenal membrane of the scolopale cell is the wall of the compartment containing the receptor lymph, the scolopale space. The ablumenal membrane of the scolopale cell creates the border of the scolopidium. The Na/K pump is located on the ablumenal membrane of the scolopale cell, bringing K+ into the scolopale cell cytoplasm and extruding K electrogenically (Roy et al, 2013). We explored other primary and secondary ion pumps that are involved in creating a K+-rich lumen in the Malpighian tubule (Day et al, 2008; Rodan et al, 2012). We used RNAi technology to knockdown one gene at a time and electrophysiology to measure a sound evoked potential (SEP) that reflects the fly’s ability to hear. We found that knocking down V-ATPase, a proton pump, subunits involved in proton extrusion significantly reduces the SEP of knockdown flies. The involvement of cation chloride cotransporters (CCCs) and cation proton antiporter (CPAs), both secondary ion pumps that use the gradients created by the Na/K pump and V-ATPase respectively to pump other ions up their gradient, is less clear. We found that knocking down Nhe3, a CPA, significantly reduced the SEP when knocked down in the scolopale cell, suggesting it as a partner to the V-ATPase. Knocking down CG31547, a CCC, statistically increased the SEP, possibly a type1 statistical error.

Acid-sensing ion channels (ASICs) are proton-gated cation channels expressed throughout the nervous system. These channels are activated by acidic pH conditions within an attainable physiologic range. The specific function of these channels has proven to be elusive, but it is clear that they are involved in various neuronal processes, both in the central nervous system as well as in the periphery.In order to further study the functions of these channels in an animal model system, transgenic animals were generated that overexpress individual ASIC subunits: ASIC2a and ASIC3. Transgenic proteins were detectable in brain and peripheral nervous tissue, and each had differential effects on acid-gated current properties in cultured neurons.Transgenes included N-terminal epitope tags to distinguish from endogenous ASICs, and expression was driven by a pan-neuronal promoter. Mechanical thermal sensory behaviors were tested in the transgenic mice. However, no effect was observed in these behaviors. The most interesting effect of overexpressing ASIC3 was the resulting impairment of conditioned fear behaviors in the transgenic animals without effect on unconditioned fear. ASIC3 transgenic behave like ASIC1a knockout mice in conditioned fear behaviors. Transgenic ASIC3 interacts with endogenous ASIC1, and is likely altering subunit composition of ASIC channels in the brain without abolishing proton-gated currenst like in the ASIC1a knockout. Overexpressing these two ASIC subunits in transgenic animals has produced tools that may be used to further study the functions of these channels. While this still is an artificial setting for studying ASIC functions, it nonetheless provides an in vivo method to study the effects of altering subunit composition in a whole animal and its behavioral effects, as well as in vivo expression of transgenes that can be studies biochemically. It is hopeful that studying localization in the transgenic mice will afford a better understanding of the localization and function of endogenous channels without the limitations of generating antibodies against endogenous mouse ASIC proteins, which is still in progress.

Irritable Bowel Syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. Visceral hypersensitivity is the most commonly reported symptom of IBS, yet is the least adequately treated. Mechanosensitive information from the colon is relayed to the CNS by extrinsic colonic primary afferent nerves which have their cell bodies within dorsal root ganglia (DRG). This thesis aims to identify the contribution of several putatively mechanosensitive ion channels (ASIC1, 2 and 3, TRPV4 and TRPA1) toward detection of mechanical stimuli in the colon. This involvement is assessed by both molecular and functional means. The abundance of each of these channels was assessed by comparing expression within whole DRG against that in specifically colonic DRG neurons using an in situ hybridization methodology developed as part of this PhD. The functional role TRPV4 and TRPA1 impart toward colonic mechanosensation was investigated by recording responses to mechanical stimuli from colonic primary afferent fibres and comparing the results from mice genetically modified to lack either TRPV4 or TRPA1 with those of their intact littermates. The results from these studies indicate expression patterns within whole DRG do not provide accurate representation of the organ of interest, with abundances of each of the channels investigated differing between colonic DRG cells and the whole DRG. In particular ASIC3 and TRPV4 are preferentially expressed in colonic DRG neurons, unlike ASIC2 and TRPA1. Further, TRPV4 is functionally restricted to detection of noxious mechanical stimuli in the colon, while expression of TRPA1 is more widespread and functionally less restricted. Each of these channels are each potential targets for the treatment of IBS as each affects specific aspects of colonic mechanotransduction. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1347202 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Sciences, 2008

Caveolae are small plasma membrane invaginations present in many different cell types, which have been linked to diverse cellular functions, including cell signalling, membrane rearrangements and lipid regulation. The caveolae markers, members of the caveolin family of proteins, are essential for caveola formation and function. Recently, however, a protein named PTRF (Polymerase I and Transcript Release Factor) or cavin, originally identified as a nuclear factor that regulates transcription in vitro, was shown to be associated with caveolae in adipocytes. In the first chapter of this thesis, I have used the zebrafish Danio rerio to investigate the relation of PTRF/cavin to caveolae as well as caveola function in vivo. During zebrafish development, PTRF/cavin was highly expressed in the notochord in 18 h, 24 h and 35 h post-fertilization embryos, as detected by in situ hybrydization. Analysis of later development stages showed that PTRF/cavin is also present in the otic vesicle, brachial arches, and periderm. Disruption of PTRF/cavin expression, via morpholino-mediated inhibition, caused severely defective development of the notochord as well as heart edema, in a dose-dependent manner. PTRF/cavin knockdown embryos had curved notochords and were shorter than the controls. Examination of the notochord by electron microscopy showed that the number of caveolae was greatly reduced in PTRF/cavin-morpholino-injected embryos. Similar effects were observed when caveolin-1, the major protein of caveolae in non-muscle cells, was down-regulated. Altogether, these results indicated a role for PTRF/cavin during formation and/or stabilization of caveolae as well as an essential role for caveolae during zebrafish embryo development. Combined with results obtained in mammalian cells, these findings identify PTRF/cavin as the first component of a caveolar coat, required for caveola formation and function (Hill et al., 2008). We subsequently identified a family of PTRF/cavin-related proteins, the cavins, that all associate with caveolae. Using biochemistry, light microscopy, and FRET-based approaches we characterised PTRF/cavin and the new members of this family of proteins SDR/cavin-2, SRBC/cavin-3 and MURC/cavin-4. We have shown that the four members of the cavin family form a multi-protein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and then associate with caveolin at the plasma membrane caveolae; interestingly, caveolin is essential for the plasma membrane translocation of the cavin complex, and in caveolin-1 knockout cells the four cavin proteins are restricted to the cytosol. PTRF/cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The four cavin proteins present distinct patterns of tissue expression, which suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. MURC/cavin-4 is expressed predominantly in muscle and its distribution is perturbed in human muscle disease associated with caveolin-3 dysfunction, identifying MURC/cavin-4 as a novel muscle disease candidate caveolar protein. To functionally investigate the relation of cavins and caveolae, we explored a caveolar function in mechanosensation. Through the use of hypo-osmotic media, we induced membrane-stretch and showed that the increased membrane tension leads to dissociation of the caveolin-cavin module and caveola disassembly as observed by immunofluorescence and FLIM/FRET techniques. Once released from caveolae, caveolin was seen internalized in late endosomes and lysosomes. Cavin-1, on the other hand, was found to be diffused in the cytosol and from there it was translocated to the nuclear compartment. The nuclear translocation was observed in several different cell types, which suggests a universal role for nuclear cavin-1, and was independent of caveolin expression. Analysis of live cells using real-time FLIM/FRET showed that cells quickly respond to variations in membrane tension by dissociation/re-association of caveolin and cavin-1. Altogether, in the course of this project, I was able to show that cavin-1 is an essential regulator of caveola biogenesis in cultured cells and in vivo. Cavin-1 and the other members of the PTRF/Cavin family form a multiprotein complex that is recruited to caveolae by caveolin and coats plasma membrane caveolae. The association between cavin-1 and caveolin is crucial for caveolae assembly and this interaction has a role in the cellular sensation of plasma membrane tension. Under high membrane tensions, caveolin and cavin-1 dissociate with the consequent flattening of caveolae. Under these circumstances, caveolin is internalized into enlarged endosomes and lysosomes while cavin-1 is translocated to the nucleus, identifying for the first time a caveola- to nucleus signalling pathway. The exact role of nuclear cavin-1 under plasma membrane stretch is now amenable to analysis.

Notre capacité à communiquer et à apprécier la musique repose sur une discrimination de fréquences couvrant une large gamme de fréquences sonores. Cette propriété résulte de cellules mécanosensorielles « ciliées », qui sont réglées pour répondre de façon maximale à une fréquence caractéristique qui varie monotoniquement le long de l’axe de l’organe auditif, la cochlée. Les mécanismes cellulaires et moléculaires qui définissent la fréquence d’une cellule ciliée et régulent sa valeur pour différentes cellules afin de couvrir la gamme auditive demeurent néanmoins inconnus. Notre hypothèse de travail est que cette fréquence est réglée en partie par les propriétés mécaniques passives et actives de la « touffe ciliaire », l’antenne mécanosensorielle de la cellule ciliée. A l’aide d’une préparation excisée de la cochlée du rat, nous avons combiné l’iontophorèse de chélateurs de calcium (BAPTA ou EDTA) pour casser les liens de bout-de-cil qui connectent les stéréocils voisins de la touffe ciliaire, une stimulation grâce à un micro-jet de fluide pour estimer la raideur de la touffe ciliaire et des enregistrements en « patch-clamp » de courants de transduction afin de compter le nombre de liens de bout-de-cil intacts qui contribuent à la réponse. Avec les mouvements évoqués par la rupture des liens de bout-de-cil et avec nos mesures de raideur, nous avons pu estimer la tension dans toute la touffe ciliaire, ainsi que la tension dans un seul lien de bout-de-cil en connaissant le nombre de liens qui contribuent à cette tension. Dans les cellules ciliées externes, qui sont impliquées dans l’amplification du stimulus sonore mais qui n’envoient pas d’information neuronale au cerveau, nous avons observé un gradient de tension et de raideur lorsque la fréquence caractéristique de la cellule ciliée augmente, suggérant que ces paramètres physiques peuvent être impliqués dans le réglage d’une cellule ciliée à sa fréquence caractéristique. Au contraire, pour les cellules ciliées internes, les vraies cellules sensorielles de la cochlée, nos observations ne montrent pas de gradient significatif. De plus, nous avons observé des mouvements de la touffe ciliaire induits par la variation de la concentration en calcium, correspondant à une tension accrue pour des concentrations en calcium plus faibles. Ces mouvements sont similaires à ceux évoqués dans d’autres classes de vertébrés, tels que chez la grenouille ou chez la tortue. Ainsi, nos résultats réconcilient les expériences faites chez les vertébrés inférieurs et chez le mammifère, et montrent l’implication des gradients de la mécanique de la touffe ciliaire pour l’importante sélectivité fréquentielle de la cochlée / Our ability to communicate and appreciate music relies on acute frequency discrimination over a broad range of sound frequencies. This property results from the operation of mechanosensory “hair" cells, which are each tuned to respond maximally to a characteristic frequency that varies monotonically along the axis of the auditory organ, the cochlea. The cellular and molecular mechanisms that set the characteristic frequency of a hair cell and regulate its value among different cells to cover the auditory range have remained elusive. Our working hypothesis is that tuning results in part from passive and active mechanical properties of the “hair" bundle, the mechanosensory antenna of the hair cell.Using an excised preparation from the rat cochlea, we combined iontophoresis of a calcium chelator (BAPTA or EDTA) to break the tip links that interconnect neighbouring stereocilia of the hair-cell bundle, fluid-jet stimulation to estimate hair-bundle stiffness and patch-clamp recordings of transduction currents to count the number of intact transduction channels contributing to the response. From the movements evoked by tip-link breakage and our stiffness measurements, we were able to estimate tension in the whole hair bundle as well as, knowing the number of tip links contributing to this tension, in a single tip link.In outer hair cells, which are involved in sound amplification but do not send neural information to the brain, we observed a gradient of tension and stiffness from the low-frequency to the high-frequency end of the cochlea, suggesting that these physical parameters may help tune the hair cell to its characteristic frequency. Interestingly, with inner hair cells - the true sensors of the cochlea, our observations do not show any significant gradient. Furthermore, we observed calcium-evoked hair-bundle movements corresponding to an increased tension in the tip links at decreased concentrations of calcium. These movements were similar to those evoked in other classes of vertebrates, such as the frog or the turtle. Together, our results reconcile experiments performed in lower vertebrates with those performed in mammals and show the implication of hair-bundle mechanical gradients in the sharp frequency tuning of the cochlea