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Key Points Mechanisms such as differential reliance on the secretory pathway contribute to the differentiation of axons and dendrites. A number of transcription factors confer neurons with distinctive dendrite morphologies. Down syndrome cell adhesion molecule (DSCAM) has an evolutionarily conserved role in mediating self-avoidance of dendrites such that the dendrites of each neuron can spread out. Certain types of neurons exhibit homotypic repulsion of their dendrites to allow tiling of their dendritic fields for maximal coverage without ambiguity. Tiling and dendrite maintenance can be differentially regulated by the NDR (nuclear DBF2-related) kinase family members Tricornered and Warts, respectively. They in turn are regulated by the tumour suppressor Hippo. In Drosophila melanogaster , dendrite expansion in sensory neurons is scaled to precisely match the growing epidermis. This is controlled by the microRNA bantam in epithelial cells, which signals to adjacent neurons. Defects in dendrite morphogenesis might contribute to mental disorders such as schizophrenia and autism. Jan and Jan discuss various mechanisms by which neurons acquire their type-specific dendrite morphology. These have largely emerged from studies in Drosophila melanogaster , but are broadly applicable to vertebrates. The possible contribution of defects in dendrite morphogenesis to mental disorders such as autism is also considered. Type-specific dendrite morphology is a hallmark of the neuron and has important functional implications in determining what signals a neuron receives and how these signals are integrated. During the past two decades, studies on dendritic arborization neurons in Drosophila melanogaster have started to identify mechanisms of dendrite morphogenesis that may have broad applicability to vertebrate species. Transcription factors, receptor–ligand interactions, various signalling pathways, local translational machinery, cytoskeletal elements, Golgi outposts and endosomes have been identified as contributors to the organization of dendrites of individual neurons and the placement of these dendrites in the neuronal circuitry. Further insight into these mechanisms will improve our understanding of how the nervous system functions and might help to identify the underlying causes of some neurological and neurodevelopmental disorders.

Electron cryo-microscopy density maps of mouse TMEM16A reconstituted in nanodiscs or solubilized in detergent reveal two functional states of calcium-activated chloride channels. TMEM16A structure solved The diverse TMEM16 membrane protein family contains Ca( II )-activated chloride channels, lipid scramblases and cation channels. TMEM16A mediates chloride-ion permeation, which controls neuronal signalling, muscle contraction and numerous other physiological functions. In this issue of Nature , two groups have solved the structure of TMEM16A by using cryo-electron microscopy, providing insights into the function of this channel. Unlike other ligand-gated ion channels, the Ca( II ) ion interacts with the pore directly, where a glycine residue acts as a flexible hinge to adjust calcium sensitivity. Raimund Dutzler and colleagues report the structure of the protein in both Ca( II )-free and Ca( II )-bound states, which shows how calcium binding facilitates the structural rearrangements involved in channel activation. In the second Letter, Lily Jan and colleagues present two functional states of TMEM16A in the glycolipid LMNG and in nanodiscs, with one and two Ca( II ) ions bound, respectively. The closed conformation observed in nanodiscs is proposed to show channel rundown after prolonged Ca( II ) activation. Calcium-activated chloride channels (CaCCs) encoded by TMEM16A 1 , 2 , 3 control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system 4 , 5 , 6 , 7 . To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores 8 , 9 . Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase 10 , 11 , 12 , as well as subnanometre-resolution electron cryo-microscopy 12 . Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca 2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca 2+ occupancy requires membrane depolarization in order to open (C.J.P. et al ., manuscript submitted). The structure in nanodiscs has two Ca 2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca 2+ . Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting. The endolysosomal pathway plays an important role in regulating protein and lipid sorting and degradation. Here, the authors show that TMEM16K, an endoplasmic reticulum lipid scramblase, forms ER-endosome contact sites where it regulates endosomal sorting.

Genetic and biochemical evidence details that the tumour supressor Hippo is an upstream regulator of the tumour suppressors Warts/Lats, and that loss of Warts/Lats leads to proper dendritic tiling. Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields 1 , 2 , 3 , is established through a ‘like-repels-like’ behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila 4 , 5 , 6 , 7 . Here we report that the other NDR family kinase, the tumour suppressor Warts/Lats (Wts) 8 , 9 , 10 , regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. We further provide biochemical and genetic evidence for the tumour suppressor kinase Hippo (Hpo) 11 , 15 as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis.

TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. Every cell in the body is surrounded by a barrier called the cell membrane. There are, however, a number of ways that molecules can pass through this membrane to either enter or leave the cell. Calcium-activated channels are a group of proteins that are embedded within the cell membrane and that allow different ions to pass through the membrane. These proteins are involved in a number of processes in a variety of tissues, for example in the gut, lungs and nervous system. A family of proteins called TMEM16 includes a number of calcium-activated channels that have been recently identified. However, it is not clear how these TMEM16 channel proteins detect the calcium ions that cause them to open. Two ideas have been suggested: the calcium ions might be detected by a protein called calmodulin, which then forces the channel to open; alternatively, the calcium ions might be detected by the channel protein itself. Tien, Peters et al. have now tested both of these ideas by focusing on a calcium-activated channel protein called TMEM16A, which allows chloride ions to pass through membranes. The possible role of calmodulin was tested in several ways, such as by preventing it from binding to the TMEM16A protein or from binding to calcium. However, none of these changes affected the opening of the channel; so Tien, Peters et al. concluded that calmodulin is not involved in these channels being activated by calcium ions. Next, Tien, Peters et al. tested specific parts of the TMEM16A channel protein itself to see if they were involved in calcium detection instead. Proteins are made from smaller building blocks called amino acids, and it is known that some amino acids are more likely to bind to calcium ions than others. There are 38 of these amino acids in the TMEM16A channel that are also found in other members of the TMEM16 family in both fruit flies and mammals. Tien, Peters et al. found that replacing five of these with other amino acids made the channel less sensitive to calcium. Further experiments suggested that four of these five amino acids are clustered at the site where a calcium ion might bind to the TMEM16A channel protein, which suggests that the protein itself can detect calcium directly. The next challenge will be to understand how calcium ions binding to the site on the TMEM16A channel protein can cause the channel to open to allow the chloride ions to pass through.

Sensory neurons enable animals to detect environmental changes and avoid harm. An intriguing open question concerns how the various attributes of sensory neurons arise in development. Drosophila melanogaster larvae undergo a behavioral transition by robustly activating a thermal nociceptive escape behavior during the second half of larval development (third instar). The Class IV dendritic arborization (C4da) neurons are multimodal sensors which tile the body wall of Drosophila larvae and detect nociceptive temperature, light, and mechanical force. In contrast to the increase in nociceptive behavior in the third instar, we find that ultraviolet light-induced Ca 2+ activity in C4da neurons decreases during the same period of larval development. Loss of ecdysone receptor has previously been shown to reduce nociception in third instar larvae. We find that ligand-dependent activation of ecdysone signaling is sufficient to promote nociceptive responses in second instar larvae and suppress expression of subdued (encoding a TMEM16 channel). Reduction of subdued expression in second instar C4da neurons not only increases thermal nociception but also decreases the response to ultraviolet light. Thus, steroid hormone signaling suppresses subdued expression to facilitate the sensory switch of C4da neurons. This regulation of a developmental sensory switch through steroid hormone regulation of channel expression raises the possibility that ion channel homeostasis is a key target for tuning the development of sensory modalities. During their lives, animals encounter a broad range of stimuli from their surroundings including heat, light and touch. The ability to appropriately respond to such stimuli is crucial for survival as it allows the animals to avoid predators and other dangers, locate food and shelter, and find mates. Fruit fly larvae are a useful model for studying how animals respond to unpleasant (known as painful) heat stimuli. When something hot touches a larva, the larva rolls away to avoid the stimulus. The heat stimulates electrical activity in a type of neuron known as C4da neurons on the surface of the larva. Ultraviolet light and several other stimuli are also able to activate electrical activity in C4da neurons, resulting in the larvae changing the direction they move to avoid the stimuli. Only older fly larvae respond to painful heat stimuli and previous studies found that a hormone receptor protein is required for this response. However, it remains unclear how this response develops as the larvae age. Jaszczak et al. studied the behavior of fly larvae and electrical activities of C4da neurons in response to painful heat and ultraviolet light. The experiments found that painful heat triggered more rolling behavior from older larvae than those of younger larvae. In contrast, ultraviolet light triggered lower levels of electrical activity in the C4da neurons of older larvae than those of younger larvae. The team raised the levels of a hormone known as ecdysone and found that this increased the rolling behavior in younger larvae. They then increased the amount of receptor protein for this hormone in the neurons and found that it decreased the levels of another protein called Subdued in the C4da neurons. This in turn increased the neurons’ response to painful heat and decreased their response to ultraviolet light. Jaszczak et al. propose that as the larva develops, ecdysone reduces the levels of Subdued, which promotes C4da neurons to switch their sensitivity from detecting ultraviolet light to painful heat. In the future, better understanding of what causes pain sensations in developing animals will help us search for factors that cause long-term pain conditions in humans.

From breathing to walking, rhythmic movements encompass physiological processes important across the entire animal kingdom. It is thought by many that the generation of rhythmic behavior is operated by a central pattern generator (CPG) and does not require peripheral sensory input. Sensory feedback is, however, required to modify or coordinate the motor activity in response to the circumstances of actual movement. In contrast to this notion, we report here that sensory input is necessary for the generation of Drosophila larval locomotion, a form of rhythmic behavior. Blockage of all peripheral sensory inputs resulted in cessation of larval crawling. By conditionally silencing various subsets of larval peripheral sensory neurons, we identified the multiple dendritic (MD) neurons as the neurons essential for the generation of rhythmic peristaltic locomotion. By recording the locomotive motor activities, we further demonstrate that removal of MD neuron input disrupted rhythmic motor firing pattern in a way that prolonged the stereotyped segmental motor firing duration and prevented the propagation of posterior to anterior segmental motor firing. These findings reveal that MD sensory neuron input is a necessary component in the neural circuitry that generates larval locomotion.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

TMEM16B (ANO2) is the Ca2+-activated chloride channel expressed in multiple brain regions, including the amygdala. Here we report that Ano2 knockout mice exhibit impaired anxiety-related behaviors and context-independent fear memory, thus implicating TMEM16B in anxiety modulation. We found that TMEM16B is expressed in somatostatin-positive (SOM+) GABAergic neurons of the central lateral amygdala (CeL), and its activity modulates action potential duration and inhibitory postsynaptic current (IPSC). We further provide evidence for TMEM16B actions not only in the soma but also in the presynaptic nerve terminals of GABAergic neurons. Our study reveals an intriguing role for TMEM16B in context-independent but not context-dependent fear memory, and supports the notion that dysfunction of the amygdala contributes to anxiety-related behaviors.