Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system.

Sensitive and rapid point-of-care assays have been crucial in the global response to SARS-CoV-2. Loop-mediated isothermal amplification (LAMP) has emerged as an important diagnostic tool given its simplicity and minimal equipment requirements, although limitations exist regarding sensitivity and the methods used to detect reaction products. We describe the development of Vivid COVID-19 LAMP, which leverages a metallochromic detection system utilizing zinc ions and a zinc sensor, 5-Br-PAPS, to circumvent the limitations of classic detection systems dependent on pH indicators or magnesium chelators. We make important strides in improving RT-LAMP sensitivity by establishing principles for using LNA-modified LAMP primers, multiplexing, and conducting extensive optimizations of reaction parameters. To enable point-of-care testing, we introduce a rapid sample inactivation procedure without RNA extraction that is compatible with self-collected, non-invasive gargle samples. Our quadruplexed assay (targeting E, N, ORF1a, and RdRP) reliably detects 1 RNA copy/µl of sample (=8 copies/reaction) from extracted RNA and 2 RNA copies/µl of sample (=16 copies/reaction) directly from gargle samples, making it one of the most sensitive RT-LAMP tests and even comparable to RT-qPCR. Additionally, we demonstrate a self-contained, mobile version of our assay in a variety of high-throughput field testing scenarios on nearly 9,000 crude gargle samples. Vivid COVID-19 LAMP can be an important asset for the endemic phase of COVID-19 as well as preparing for future pandemics.

Molecular Basis of Chemotactile Sensation in Octopus.

Animals display wide-ranging evolutionary adaptations based on their ecological niche. Octopuses explore the seafloor with their flexible arms using a specialized "taste by touch" system to locally sense and respond to prey-derived chemicals and movement. How the peripherally distributed octopus nervous system mediates relatively autonomous arm behavior is unknown. Here, we report that octopus arms use a family of cephalopod-specific chemotactile receptors (CRs) to detect poorly soluble natural products, thereby defining a form of contact-dependent, aquatic chemosensation. CRs form discrete ion channel complexes that mediate the detection of diverse stimuli and transduction of specific ionic signals. Furthermore, distinct chemo- and mechanosensory cells exhibit specific receptor expression and electrical activities to support peripheral information coding and complex chemotactile behaviors. These findings demonstrate that the peripherally distributed octopus nervous system is a key site for signal processing and highlight how molecular and anatomical features synergistically evolve to suit an animal's environmental context.

Angiotensin II type 2 receptor stimulation increases the rate of NG108-15 cell migration via actin depolymerization.

Angiotensin II (Ang II) has been reported to induce migration in neuronal cell types. Using time-lapse microscopy, we show here that Ang II induces acceleration in NG108-15 cell migration. This effect was antagonized by PD123319, a selective AT2 receptor antagonist, but not by DUP753, a selective AT1 receptor antagonist, and was mimicked by the specific AT2 receptor agonist CGP42112. This Ang II-induced acceleration was not sensitive to the inhibition of previously described signaling pathways of the AT2 receptor, guanylyl cyclase/cyclic GMP or p42/p44 mapk cascades, but was abolished by pertussis toxin treatment and involved PP2A activation. Immunofluorescence studies indicate that Ang II or CGP42112 decreased the amount of filamentous actin at the leading edge of the cells. This decrease was accompanied by a concomitant increase in globular actin levels. Regulation of actin turnover in actin-based motile systems is known to be mainly under the control of the actin depolymerizing factor and cofilin. Basal migration speed decreased by 77.2% in cofilin-1 small interfering RNA-transfected NG108-15 cells, along with suppression of the effect of Ang II. In addition, the Ang II-induced increase in cell velocity was abrogated in serum-free medium as well as by genistein or okadaic acid treatment in a serum-containing medium. Such results indicate that the AT2 receptor increases the migration speed of NG108-15 cells and involves a tyrosine kinase activity, followed by phosphatase activation, which may be of the PP2A type. Therefore, the present study identifies actin depolymerization and cofilin as new targets of AT2 receptor action, in the context of cellular migration.

Human melanocortin receptor 2 expression and functionality: effects of protein kinase A and protein kinase C on desensitization and internalization.

The aim of this study was to investigate the short-term regulation of the ACTH receptor human (h) melanocortin receptor 2 (MC2R) by transfection of a c-Myc-tagged hMC2R in the M3 cell line and assess its membrane expression by indirect immunofluorescence. Stimulation with ACTH induced production of cAMP with EC(50) values ranging from 7.6-11.9 nM in transient and stable transfectants, respectively. Pretreatment with ACTH induced a dose-dependent loss of cAMP production, from 1 pm up to 10 nM. Desensitization was also time dependent, with 70% loss of maximal responsiveness occurring after 15-min pretreatment with 10 nM ACTH, followed by a plateau up to 60 min. The decrease in hMC2R responsiveness was abrogated by individual treatment with protein kinase A (PKA) or protein kinase C inhibitors, H-89 and GF109203X. However, when added simultaneously, receptor responsiveness was raised over the maximal hMC2R activity observed in control cells. ACTH-induced loss of cAMP production was accompanied by receptor sequestration into intracellular vesicles (maximum after 30-min exposure). Cotransfection of M3 cells with the c-Myc-tagged hMC2R and beta-arrestin-2-green fluorescence protein along with sucrose treatment revealed that beta-arrestin-2-green fluorescence protein and c-Myc-hMC2R were redistributed in similar intracellular vesicles through a clathrin-dependent, but caveolae-independent, process. Sucrose pretreatment blocked receptor desensitization, indicating that hMC2R desensitization and internalization are interrelated. Moreover, preincubation with H-89 abrogated hMC2R internalization, whereas GF109203X had no effect. In conclusion, the present results indicate that PKA and protein kinase C act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, highlighting the complex nature of the short-term regulatory pattern of this receptor.

Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells.

The binding and signaling properties of neuronal NTS2 neurotensin (NT) receptors were examined in cultured rat cerebellar granule cells. As shown by reverse transcription-PCR, receptor autoradiography, and confocal microscopic localization of fluorescent NT, these cells selectively express the NTS2 receptor subtype. Accordingly, a single apparent class of (125)I-NT-binding sites, with an affinity of 3.1 nm, was detected in cerebellar granule cell cultures. This binding was competed for with high affinity (IC(50) = 5.7 nm) by the NTS2 ligand levocabastine and with low affinity (IC(50) = 203 nm) by the NTS1 antagonist SR48692. Hypertonic acid stripping of surface-bound ligand and hyperosmolar sucrose treatment revealed that 64% of specifically bound (125)I-NT was internalized at equilibrium via a clathrin-dependent pathway. In cells loaded with the Ca(2+)-sensitive fluorescent dye Fluo4, SR48692, but neither NT nor levocabastine, triggered a marked increase in cytosolic [Ca(2+)](i). By contrast, both NT and levocabastine, but not SR48692, induced a sustained (>60 min) activation of the mitogen-activated protein kinases, p42/p44, indicating functional coupling of NTS2 receptors. Complementary experiments carried out on synaptosomes from adult rat cerebellum demonstrated the presence of presynaptic NTS2 receptors. However, in contrast to perikaryal NTS2 sites, these presynaptic receptors did not internalize in response to NT stimulation. Taken together, the present results demonstrate that NTS2 receptors are present both presynaptically and postsynaptically in central neurons and that NT and levocabastine act as agonists on these receptors.