NOMPC-dependent mechanotransduction shapes the dendrite of proprioceptive neurons.

Animal locomotion depends on proprioceptive feedback which is generated by mechanosensory neurons. We recently demonstrated that the evolutionarily conserved stumble (stum) gene is essential for mechanical transduction in a group of proprioceptive neurons in Drosophila leg joints. A specialized dendritic ending of the stum-expressing neurons is stretched by changes in joint position, suggesting that the dendritic site is specifically tuned for joint proprioception. Here, we show that the stum-expressing neurons express the mechanosensory channel NOMPC. In nompC mutants responses to joint position were abolished, indicating that NOMPC is the mechanosensitive channel in stum-expressing neurons. The NOMPC protein had a similar subcellular distribution as STUM, being located specifically at the dendritic site that is stretched by joint motions, thus validating that this site is a specialized sensory compartment. In the absence of NOMPC the sensory portion of the dendrite was misshapen, generating membrane protrusions. Thus, we have shown that mechanical responsiveness at a specialized dendritic site is essential for determination of the fine dendritic structure. The abnormal morphology at the sensory compartment of non-active neurons suggests that the dendrite samples for a responsive anchoring site and stabilizes the structure that elicits the effective mechanotransduction.

A migrating ciliary gate compartmentalizes the site of axoneme assembly in Drosophila spermatids.

BACKGROUND: In most cells, the cilium is formed within a compartment separated from the cytoplasm. Entry into the ciliary compartment is regulated by a specialized gate located at the base of the cilium in a region known as the transition zone. The transition zone is closely associated with multiple structures of the ciliary base, including the centriole, axoneme, and ciliary membrane. However, the contribution of these structures to the ciliary gate remains unclear. RESULTS: Here we report that, in Drosophila spermatids, a conserved module of transition zone proteins mutated in Meckel-Gruber syndrome (MKS), including Cep290, Mks1, B9d1, and B9d2, comprise a ciliary gate that continuously migrates away from the centriole to compartmentalize the growing axoneme tip. We show that Cep290 is essential for transition zone composition, compartmentalization of the axoneme tip, and axoneme integrity and find that MKS proteins also delimit a centriole-independent compartment in mouse spermatids. CONCLUSIONS: Our findings demonstrate that the ciliary gate can migrate away from the base of the cilium, thereby functioning independently of the centriole and of a static interaction with the axoneme to compartmentalize the site of axoneme assembly.

The effect of stress on motor function in Drosophila.

Exposure to unpredictable and uncontrollable conditions causes animals to perceive stress and change their behavior. It is unclear how the perception of stress modifies the motor components of behavior and which molecular pathways affect the behavioral change. In order to understand how stress affects motor function, we developed an experimental platform that quantifies walking motions in Drosophila. We found that stress induction using electrical shock results in backwards motions of the forelegs at the end of walking strides. These leg retrogressions persisted during repeated stimulation, although they habituated substantially. The motions also continued for several strides after the end of the shock, indicating that stress induces a behavioral aftereffect. Such aftereffect could also be induced by restricting the motion of the flies via wing suspension. Further, the long-term effects could be amplified by combining either immobilization or electric shock with additional stressors. Thus, retrogression is a lingering form of response to a broad range of stressful conditions, which cause the fly to search for a foothold when it faces extreme and unexpected challenges. Mutants in the cAMP signaling pathway enhanced the stress response, indicating that this pathway regulates the behavioral response to stress. Our findings identify the effect of stress on a specific motor component of behavior and define the role of cAMP signaling in this stress response.

The stum gene is essential for mechanical sensing in proprioceptive neurons.

Animal locomotion depends on proprioceptive feedback, which is generated by mechanosensory neurons. We performed a genetic screen for impaired walking in Drosophila and isolated a gene, stumble (stum). The Stum protein has orthologs in animals ranging from nematodes to mammals and is predicted to contain two transmembrane domains. Expression of the mouse orthologs of stum in mutant flies rescued their phenotype, which demonstrates functional conservation. Dendrites of stum-expressing neurons in legs were stretched by both flexion and extension of corresponding joints. Joint angles that induced dendritic stretching also elicited elevation of cellular Ca(2+) levels-not seen in stum mutants. Thus, we have identified an evolutionarily conserved gene, stum, which is required for transduction of mechanical stimuli in a specific subpopulation of Drosophila proprioceptive neurons that sense joint angles.

Ionic leakage underlies a gain-of-function effect of dominant disease mutations affecting diverse P-type ATPases.

Type II P-type ATPases (PAIIs) constitute a family of conserved proteins that actively generate ionic gradients across membranes. Mutations in genes encoding PAIIs can cause heritable dominant diseases, with suggested etiology of haploinsufficiency. Using a Drosophila melanogaster genetic screen, we identified a dominant mutation altering the PAII member sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA). This mutation conferred temperature-sensitive uncoordination in a gain-of-function manner. We established that this gain-of-function phenotype is linked to dominant disease-causing mutations affecting various human PAIIs. We further found that heterologous expression of mutant PAIIs elicited ion leakage that was exacerbated at elevated temperatures. Therefore, these dominant mutations result in ionic leakage and render PAIIs susceptible to deleterious effects from elevated temperatures. Accordingly, it was recently reported that missense mutations affecting the Na(+)/K(+) ATPase can elicit ionic leakage. We propose that ionic leakage is a pervasive gain-of-function mechanism that can underlie a variety of dominant PAII-related diseases.

Dissection of gain control mechanisms in Drosophila mechanotransduction.

Mechanoreceptor cells respond to a vast span of stimulus intensities, which they transduce into a limited response-range using a dynamic regulation of transduction gain. Weak stimuli are detected by enhancing the gain of responses through the process of active mechanical amplification. To preserve responsiveness, the gain of responses to prolonged activation is rapidly reduced through the process of adaptation. We investigated long-term processes of mechanotransduction gain control by studying responses from single mechanoreceptor neurons in Drosophila. We found that mechanical stimuli elicited a sustained reduction of gain that we termed long-term adaptation. Long-term adaptation and the adaptive decay of responses during stimuli had distinct kinetics and they were independently affected by manipulations of mechanotransduction. Therefore, long-term adaptation is not associated with the reduction of response gain during stimulation. Instead, the long-term adaptation suppressed canonical features of active amplification which were the high gain of weak stimuli and the spontaneous emission of noise. In addition, depressing amplification using energy deprivation recapitulated the effects of long-term adaptation. These data suggest that long-term adaptation is mediated by suppression of active amplification. Finally, the extent of long-term adaptation matched with cytoplasmic Ca(2+) levels and dTrpA1-induced Ca(2+) elevation elicited the effects of long-term adaptation. Our data suggest that mechanotransduction employs parallel adaptive mechanisms: while a rapid process exerts immediate gain reduction, long-term adjustments are achieved by attenuating active amplification. The slow adjustment of gain, manifest as diminished sensitivity, is associated with the accumulation of Ca(2+).

The role of Drosophila Piezo in mechanical nociception.

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.

Preserving cell shape under environmental stress.

Maintaining cell shape and tone is crucial for the function and survival of cells and tissues. Mechanotransduction relies on the transformation of minuscule mechanical forces into high-fidelity electrical responses. When mechanoreceptors are stimulated, mechanically sensitive cation channels open and produce an inward transduction current that depolarizes the cell. For this process to operate effectively, the transduction machinery has to retain integrity and remain unfailingly independent of environmental changes. This is particularly challenging for poikilothermic organisms, where changes in temperature in the environment may impact the function of mechanoreceptor neurons. Thus, we wondered how insects whose habitat might quickly vary over several tens of degrees of temperature manage to maintain highly effective mechanical senses. We screened for Drosophila mutants with defective mechanical responses at elevated ambient temperatures, and identified a gene, spam, whose role is to protect the mechanosensory organ from massive cellular deformation caused by heat-induced osmotic imbalance. Here we show that Spam protein forms an extracellular shield that guards mechanosensory neurons from environmental insult. Remarkably, heterologously expressed Spam protein also endowed other cells with superb defence against physically and chemically induced deformation. We studied the mechanical impact of Spam coating and show that spam-coated cells are up to ten times stiffer than uncoated controls. Together, these results help explain how poikilothermic organisms preserve the architecture of critical cells during environmental stress, and illustrate an elegant and simple solution to such challenge.

Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways.

Mammals can taste a wide repertoire of chemosensory stimuli. Two unrelated families of receptors (T1Rs and T2Rs) mediate responses to sweet, amino acids, and bitter compounds. Here, we demonstrate that knockouts of TRPM5, a taste TRP ion channel, or PLCbeta2, a phospholipase C selectively expressed in taste tissue, abolish sweet, amino acid, and bitter taste reception, but do not impact sour or salty tastes. Therefore, despite relying on different receptors, sweet, amino acid, and bitter transduction converge on common signaling molecules. Using PLCbeta2 taste-blind animals, we then examined a fundamental question in taste perception: how taste modalities are encoded at the cellular level. Mice engineered to rescue PLCbeta2 function exclusively in bitter-receptor expressing cells respond normally to bitter tastants but do not taste sweet or amino acid stimuli. Thus, bitter is encoded independently of sweet and amino acids, and taste receptor cells are not broadly tuned across these modalities.

TRP channel proteins and signal transduction.

TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.