G protein-coupled receptor kinase-2 (GRK-2) controls exploration through neuropeptide signaling in Caenorhabditis elegans.

Animals alter their behavior in manners that depend on environmental conditions as well as their developmental and metabolic states. For example, C. elegans is quiescent during larval molts or during conditions of satiety. By contrast, worms enter an exploration state when removed from food. Sensory perception influences movement quiescence (defined as a lack of body movement), as well as the expression of additional locomotor states in C. elegans that are associated with increased or reduced locomotion activity, such as roaming (exploration behavior) and dwelling (local search). Here we find that movement quiescence is enhanced, and exploration behavior is reduced in G protein-coupled receptor kinase grk-2 mutant animals. grk-2 was previously shown to act in chemosensation, locomotion, and egg-laying behaviors. Using neuron-specific rescuing experiments, we show that GRK-2 acts in multiple ciliated chemosensory neurons to control exploration behavior. grk-2 acts in opposite ways from the cGMP-dependent protein kinase gene egl-4 to control movement quiescence and exploration behavior. Analysis of mutants with defects in ciliated sensory neurons indicates that grk-2 and the cilium-structure mutants act in the same pathway to control exploration behavior. We find that GRK-2 controls exploration behavior in an opposite manner from the neuropeptide receptor NPR-1 and the neuropeptides FLP-1 and FLP-18. Finally, we show that secretion of the FLP-1 neuropeptide is negatively regulated by GRK-2 and that overexpression of FLP-1 reduces exploration behavior. These results define neurons and molecular pathways that modulate movement quiescence and exploration behavior.

Dimensionality of locomotor behaviors in developing C. elegans.

Adult animals display robust locomotion, yet the timeline and mechanisms of how juvenile animals acquire coordinated movements and how these movements evolve during development are not well understood. Recent advances in quantitative behavioral analyses have paved the way for investigating complex natural behaviors like locomotion. In this study, we tracked the swimming and crawling behaviors of the nematode Caenorhabditis elegans from postembryonic development through to adulthood. Our principal component analyses revealed that adult C. elegans swimming is low dimensional, suggesting that a small number of distinct postures, or eigenworms, account for most of the variance in the body shapes that constitute swimming behavior. Additionally, we found that crawling behavior in adult C. elegans is similarly low dimensional, corroborating previous studies. Further, our analysis revealed that swimming and crawling are distinguishable within the eigenworm space. Remarkably, young L1 larvae are capable of producing the postural shapes for swimming and crawling seen in adults, despite frequent instances of uncoordinated body movements. In contrast, late L1 larvae exhibit robust coordination of locomotion, while many neurons crucial for adult locomotion are still under development. In conclusion, this study establishes a comprehensive quantitative behavioral framework for understanding the neural basis of locomotor development, including distinct gaits such as swimming and crawling in C. elegans.

Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis.

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilin's SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.

INX-18 and INX-19 play distinct roles in electrical synapses that modulate aversive behavior in Caenorhabditis elegans.

In order to respond to changing environments and fluctuations in internal states, animals adjust their behavior through diverse neuromodulatory mechanisms. In this study we show that electrical synapses between the ASH primary quinine-detecting sensory neurons and the neighboring ASK neurons are required for modulating the aversive response to the bitter tastant quinine in C. elegans. Mutant worms that lack the electrical synapse proteins INX-18 and INX-19 become hypersensitive to dilute quinine. Cell-specific rescue experiments indicate that inx-18 operates in ASK while inx-19 is required in both ASK and ASH for proper quinine sensitivity. Imaging analyses find that INX-19 in ASK and ASH localizes to the same regions in the nerve ring, suggesting that both sides of ASK-ASH electrical synapses contain INX-19. While inx-18 and inx-19 mutant animals have a similar behavioral phenotype, several lines of evidence suggest the proteins encoded by these genes play different roles in modulating the aversive quinine response. First, INX-18 and INX-19 localize to different regions of the nerve ring, indicating that they are not present in the same synapses. Second, removing inx-18 disrupts the distribution of INX-19, while removing inx-19 does not alter INX-18 localization. Finally, by using a fluorescent cGMP reporter, we find that INX-18 and INX-19 have distinct roles in establishing cGMP levels in ASK and ASH. Together, these results demonstrate that electrical synapses containing INX-18 and INX-19 facilitate modulation of ASH nociceptive signaling. Our findings support the idea that a network of electrical synapses mediates cGMP exchange between neurons, enabling modulation of sensory responses and behavior.

The C-terminal of CASY-1/Calsyntenin regulates GABAergic synaptic transmission at the Caenorhabditis elegans neuromuscular junction.

The C. elegans ortholog of mammalian calsyntenins, CASY-1, is an evolutionarily conserved type-I transmembrane protein that is highly enriched in the nervous system. Mammalian calsyntenins are strongly expressed at inhibitory synapses, but their role in synapse development and function is still elusive. Here, we report a crucial role for CASY-1 in regulating GABAergic synaptic transmission at the C. elegans neuromuscular junction (NMJ). The shorter isoforms of CASY-1; CASY-1B and CASY-1C, express and function in GABA motor neurons where they regulate GABA neurotransmission. Using pharmacological, behavioral, electrophysiological, optogenetic and imaging approaches we establish that GABA release is compromised at the NMJ in casy-1 mutants. Further, we demonstrate that CASY-1 is required to modulate the transport of GABAergic synaptic vesicle (SV) precursors through a possible interaction with the SV motor protein, UNC-104/KIF1A. This study proposes a possible evolutionarily conserved model for the regulation of GABA synaptic functioning by calsyntenins.

Neuropeptide-Driven Cross-Modal Plasticity following Sensory Loss in Caenorhabditis elegans.

Sensory loss induces cross-modal plasticity, often resulting in altered performance in remaining sensory modalities. Whereas much is known about the macroscopic mechanisms underlying cross-modal plasticity, only scant information exists about its cellular and molecular underpinnings. We found that Caenorhabditis elegans nematodes deprived of a sense of body touch exhibit various changes in behavior, associated with other unimpaired senses. We focused on one such behavioral alteration, enhanced odor sensation, and sought to reveal the neuronal and molecular mechanisms that translate mechanosensory loss into improved olfactory acuity. To this end, we analyzed in mechanosensory mutants food-dependent locomotion patterns that are associated with olfactory responses and found changes that are consistent with enhanced olfaction. The altered locomotion could be reversed in adults by optogenetic stimulation of the touch receptor (mechanosensory) neurons. Furthermore, we revealed that the enhanced odor response is related to a strengthening of inhibitory AWC→AIY synaptic transmission in the olfactory circuit. Consistently, inserting in this circuit an engineered electrical synapse that diminishes AWC inhibition of AIY counteracted the locomotion changes in touch-deficient mutants. We found that this cross-modal signaling between the mechanosensory and olfactory circuits is mediated by neuropeptides, one of which we identified as FLP-20. Our results indicate that under normal function, ongoing touch receptor neuron activation evokes FLP-20 release, suppressing synaptic communication and thus dampening odor sensation. In contrast, in the absence of mechanosensory input, FLP-20 signaling is reduced, synaptic suppression is released, and this enables enhanced olfactory acuity; these changes are long lasting and do not represent ongoing modulation, as revealed by optogenetic experiments. Our work adds to a growing literature on the roles of neuropeptides in cross-modal signaling, by showing how activity-dependent neuropeptide signaling leads to specific cross-modal plastic changes in neural circuit connectivity, enhancing sensory performance.

MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation.

MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-ß, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration.

A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores.

Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two α-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD.

Synaptic activity regulates the abundance and binding of complexin.

Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.

A Quantitative Analysis of Locomotor Patterns in Developing C. elegans.

Adult animals display robust locomotion, yet the timeline and mechanisms of how juvenile animals acquire coordinated movements and how these movements evolve during development are not well understood. Recent advances in quantitative behavioral analyses have paved the way for investigating complex natural behaviors like locomotion. In this study, we tracked the swimming and crawling behaviors of the nematode Caenorhabditis elegans from postembryonic development through to adulthood. Our principal component analyses revealed that adult C. elegans swimming is low dimensional, suggesting that a small number of distinct postures, or eigenworms, account for most of the variance in the body shapes that constitute swimming behavior. Additionally, we found that crawling behavior in adult C. elegans is similarly low dimensional, corroborating previous studies. However, our analysis revealed that swimming and crawling are distinct gaits in adult animals, clearly distinguishable within the eigenworm space. Remarkably, young L1 larvae are capable of producing the postural shapes for swimming and crawling seen in adults, despite frequent instances of uncoordinated body movements. In contrast, late L1 larvae exhibit robust coordination of locomotion, while many neurons crucial for adult locomotion are still under development. In conclusion, this study establishes a comprehensive quantitative behavioral framework for understanding the neural basis of locomotor development, including distinct gaits such as swimming and crawling in C. elegans.

The endophilin curvature-sensitive motif requires electrostatic guidance to recycle synaptic vesicles in vivo.

Curvature-sensing mechanisms assist proteins in executing particular actions on various membrane organelles. Here, we investigate the functional specificity of curvature-sensing amphipathic motifs in Caenorhabditis elegans through the study of endophilin, an endocytic protein for synaptic vesicle recycling. We generate chimeric endophilin proteins by replacing the endophilin amphipathic motif H0 with other curvature-sensing amphipathic motifs. We find that the role of amphipathic motifs cannot simply be extrapolated from the identity of their parental proteins. For example, the amphipathic motif of the nuclear pore complex protein NUP133 functionally replaces the synaptic role of endophilin H0. Interestingly, non-functional endophilin chimeras have similar defects-producing fewer synaptic vesicles but more endosomes-and this indicates that the curvature-sensing motifs in these chimeras have a common deficiency for reforming synaptic vesicles. Finally, we convert non-functional endophilin chimeras into functional proteins by changing the cationic property of amphipathic motifs, successfully reprogramming the functional specificity of curvature-sensing motifs in vivo.

Presynaptic autophagy is coupled to the synaptic vesicle cycle via ATG-9.

Autophagy is a cellular degradation pathway essential for neuronal health and function. Autophagosome biogenesis occurs at synapses, is locally regulated, and increases in response to neuronal activity. The mechanisms that couple autophagosome biogenesis to synaptic activity remain unknown. In this study, we determine that trafficking of ATG-9, the only transmembrane protein in the core autophagy pathway, links the synaptic vesicle cycle with autophagy. ATG-9-positive vesicles in C. elegans are generated from the trans-Golgi network via AP-3-dependent budding and delivered to presynaptic sites. At presynaptic sites, ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt endocytosis, including a lesion in synaptojanin 1 associated with Parkinson's disease, result in abnormal ATG-9 accumulation at clathrin-rich synaptic foci and defects in activity-induced presynaptic autophagy. Our findings uncover regulated key steps of ATG-9 trafficking at presynaptic sites and provide evidence that ATG-9 exo-endocytosis couples autophagosome biogenesis at presynaptic sites with the activity-dependent synaptic vesicle cycle.

Circumventing neural damage in a C. elegans chemosensory circuit using genetically engineered synapses.

Neuronal loss can considerably diminish neural circuit function, impairing normal behavior by disrupting information flow in the circuit. Here, we use genetically engineered electrical synapses to reroute the flow of information in a C. elegans damaged chemosensory circuit in order to restore organism behavior. We impaired chemotaxis by removing one pair of interneurons from the circuit then artificially coupled two other adjacent neuron pairs by ectopically expressing the gap junction protein, connexin, in them. This restored chemotaxis in the animals. We expected to observe linear and direct information flow between the connexin-coupled neurons in the recovered circuit but also revealed the formation of new potent left-right lateral electrical connections within the connexin-expressing neuron pairs. Our analysis suggests that these additional electrical synapses help restore circuit function by amplifying weakened neuronal signals in the damaged circuit in addition to emulating the wild-type circuit. A record of this paper's transparent peer review process is included in the Supplemental Information.

Dopamine receptor DOP-1 engages a sleep pathway to modulate swimming in C. elegans.

Animals require robust yet flexible programs to support locomotion. Here we report a pathway that connects the D1-like dopamine receptor DOP-1 with a sleep mechanism to modulate swimming in C. elegans. We show that DOP-1 plays a negative role in sustaining swimming behavior. By contrast, a pathway through the D2-like dopamine receptor DOP-3 negatively regulates the initiation of swimming, but its impact fades quickly over a few minutes. We find that DOP-1 and the GPCR kinase (G-protein-coupled receptor kinase-2) function in the sleep interneuron RIS, where DOP-1 modulates the secretion of a sleep neuropeptide FLP-11. We further show that DOP-1 and FLP-11 act in the same pathway to modulate swimming. Together, these results delineate a functional connection between a dopamine receptor and a sleep program to regulate swimming in C. elegans. The temporal transition between DOP-3 and DOP-1 pathways highlights the dynamic nature of neuromodulation for rhythmic movements that persist over time.

Clinical significance and prognostic value of C-reactive protein/albumin ratio in gastric cancer.

PURPOSE: This study was aimed to evaluate the clinical significance and prognostic value of CRP/albumin ratio (CAR) in patients with gastric cancer. METHODS: The data of 205 gastric cancer patients who underwent surgery was analyzed retrospectively. The association of CAR with the clinical features and prognostic value in gastric cancer was analyzed. The data of this study was combined with previous studies to further determine the prognostic value of CAR in patients with gastric cancer using a meta-analysis method. RESULTS: Cox analysis revealed that preoperative CAR was an independent prognosis indicator in patients with gastric cancer. High expression of CAR indicated a shorter survival time than in those with lower expression. CAR has a higher prognostic value in the 1-, 3-, and 5-year overall survival in patients with gastric cancer. CAR showed significant difference regarding the gastric cancer patients' age, M stage, and clinical stage. The discriminate value of CAR in M stage of gastric cancer was high (area under the curve, 0.809). A meta-analysis combining previous data and our data showed that preoperative CAR demonstrated a significant association with the overall survival of patients with gastric cancer. CONCLUSION: This study demonstrated that preoperative CAR could serve as an important prognostic indicator in patients with gastric cancer.

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release.

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains-C2A and C2B-to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini-mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion.

Exocytosis-the release of the contents of a vesicle--proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells.

The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane.

Synaptotagmin-1 (syt), the putative Ca2+ sensor for exocytosis, is anchored to the membrane of secretory organelles. Its cytoplasmic domain is composed of two Ca2+-sensing modules, C2A and C2B. Syt binds phosphatidylinositol 4,5-bisphosphate (PIP2), a plasma membrane lipid with an essential role in exocytosis and endocytosis. We resolved two modes of PIP2 binding that are mediated by distinct surfaces on the C2B domain of syt. A novel Ca2+-independent mode of binding predisposes syt to penetrate PIP2-harboring target membranes in response to Ca2+ with submillisecond kinetics. Thus, PIP2 increases the speed of response of syt and steers its membrane-penetration activity toward the plasma membrane. We propose that syt-PIP2 interactions are involved in exocytosis by facilitating the close apposition of the vesicle and target membrane on rapid time scales in response to Ca2+.