scholarly journals UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans

2021 ◽  
Author(s):  
Kelly H. Oh ◽  
Mia Krout ◽  
Janet E. Richmond ◽  
Hongkyun Kim
Keyword(s):  
Active Zone ◽  
Calcium Influx ◽  
Genetic Screen ◽  
Scaffolding Protein ◽  
Tight Coupling ◽  
Forward Genetic Screen ◽  
Precise Control ◽  
C Elegans ◽  
Vesicle Exocytosis ◽  
Active Zones

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

scholarly journals Aczonin, a 550-Kd Putative Scaffolding Protein of Presynaptic Active Zones, Shares Homology Regions with Rim and Bassoon and Binds Profilin

1999 ◽  
Vol 147 (1) ◽  
pp. 151-162 ◽  
Author(s):  
Xiaolu Wang ◽  
Mark Kibschull ◽  
Michael M. Laue ◽  
Beate Lichte ◽  
Elisabeth Petrasch-Parwez ◽  
...  
Keyword(s):  
Active Zone ◽  
Pdz Domain ◽  
Amino Acid Sequences ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Molecular Architecture ◽  
C2 Domains ◽  
Cytoskeletal Dynamics ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

Neurotransmitter exocytosis is restricted to the active zone, a specialized area of the presynaptic plasma membrane. We report the identification and initial characterization of aczonin, a neuron-specific 550-kD protein concentrated at the presynaptic active zone and associated with a detergent-resistant cytoskeletal subcellular fraction. Analysis of the amino acid sequences of chicken and mouse aczonin indicates an organization into multiple domains, including two pairs of Cys4 zinc fingers, a polyproline tract, and a PDZ domain and two C2 domains near the COOH terminus. The second C2 domain is subject to differential splicing. Aczonin binds profilin, an actin-binding protein implicated in actin cytoskeletal dynamics. Large parts of aczonin, including the zinc finger, PDZ, and C2 domains, are homologous to Rim or to Bassoon, two other proteins concentrated in presynaptic active zones. We propose that aczonin is a scaffolding protein involved in the organization of the molecular architecture of synaptic active zones and in the orchestration of neurotransmitter vesicle trafficking.

scholarly journals MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

2017 ◽  
Author(s):  
Peishan Yi ◽  
Chao Xie ◽  
Guangshuo Ou
Keyword(s):  
Cell Cycle ◽  
Sensory Neurons ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Genetic Screen ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Neuronal Cilia ◽  
Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

scholarly journals Impairing one sensory modality enhances another by reprogramming peptidergic circuits inCaenorhabditis elegans

2021 ◽  
Author(s):  
Giulio Valperga ◽  
Mario de Bono
Keyword(s):  
Synaptic Input ◽  
Sensory Modality ◽  
Genetic Screen ◽  
Sensory Loss ◽  
Forward Genetic Screen ◽  
Sensory Inputs ◽  
C Elegans ◽  
Transcriptional Reprogramming ◽  
Sensory Responses ◽  
Necessary And Sufficient

AbstractAnimals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. To probe these mechanisms, we perform a forward genetic screen for mutants with enhanced O2perception inC. elegans. Multiple mutants exhibiting increased responsiveness to O2concomitantly show defects in other sensory responses. One mutant,qui-1, defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca2+responses in the ADL chemosensory neurons. We find that ADL’s responsiveness to pre-synaptic input from O2- sensing neurons is heightened inqui-1and other sensory defective mutants resulting in an enhanced neurosecretion. Expressingqui-1selectively in ADL rescues both thequi-1ADL neurosecretory phenotype and enhanced escape from 21% O2. Profiling of ADL neurons indicates its acquired O2-evoked neurosecretion is the result of a transcriptional reprogramming that up-regulates neuropeptide signalling. We show that the conserved neuropeptide receptor NPR-22 is necessary and sufficient in ADL to enhance its neurosecretion levels. Sensory loss can thus confer cross-modal plasticity by re-wiring peptidergic circuits.

scholarly journals Caenorhabditis elegans RIG-I Homolog Mediates Antiviral RNA Interference Downstream of Dicer-Dependent Biogenesis of Viral Small Interfering RNAs

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Stephanie R. Coffman ◽  
Jinfeng Lu ◽  
Xunyang Guo ◽  
Jing Zhong ◽  
Hongshan Jiang ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Virus Infection ◽  
Rna Virus ◽  
Genetic Screen ◽  
Small Interfering Rnas ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Terminal Domains

ABSTRACT Dicer enzymes process virus-specific double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans . Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans . Using a mapping-by-sequencing strategy, we uncovered four loss-of-function alleles of drh-1 , three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsiRNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral dsRNA replicative intermediates. Interestingly, vsiRNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsiRNA hot spots when DRH-1 is functional. As RIG-I translocates on long dsRNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsiRNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long dsRNA precursors. IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans , suggesting an analogous role for DRH-1 as an intracellular dsRNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans . Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral siRNAs in C. elegans . As mammals produce Dicer-dependent viral siRNAs to target RNA viruses, our findings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs.

scholarly journals Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans

2018 ◽  
Author(s):  
Kirthi C. Reddy ◽  
Tal Dror ◽  
Ryan S. Underwood ◽  
Guled A. Osman ◽  
Christopher A. Desjardins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pathogen Resistance ◽  
Genetic Screen ◽  
Specific Gene ◽  
Defense Gene Expression ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Evolutionary Features ◽  
Species Specific ◽  
Mutant Phenotypes

AbstractImmune genes are under intense pressure from pathogens, which cause these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. These phenotypic and evolutionary features of pals-22 and pals-25 are strikingly similar to species-specific R gene pairs in plants that control immunity against co-evolved pathogens.

scholarly journals Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+channels

2017 ◽  
Vol 114 (38) ◽  
pp. E8081-E8090 ◽  
Author(s):  
Fujun Luo ◽  
Xinran Liu ◽  
Thomas C. Südhof ◽  
Claudio Acuna
Keyword(s):  
Active Zone ◽  
Neurotransmitter Release ◽  
Information Transfer ◽  
Bipolar Cells ◽  
Mouse Retina ◽  
Tight Coupling ◽  
Ribbon Synapses ◽  
Vesicle Exocytosis ◽  
Stimulus Secretion Coupling

Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+buffer EGTA. These findings suggest that RBPs tether L-type Ca2+channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

scholarly journals Shank promotes action potential repolarization by recruiting BK channels to calcium nanodomains

2021 ◽  
Author(s):  
Luna Gao ◽  
Jian Zhao ◽  
Evan L. Ardiel ◽  
Qi Hall ◽  
Stephen Nurrish ◽  
...  
Keyword(s):  
Potassium Channels ◽  
Action Potential ◽  
Gene Copy Number ◽  
Bk Channels ◽  
Scaffolding Protein ◽  
Gene Copy ◽  
Tight Coupling ◽  
C Elegans ◽  
Calcium Dependent ◽  
Action Potential Repolarization

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here we show that the C. elegans SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1’s effects on AP duration are mediated by changes in BK channels. In shn-1 mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased shn-1 gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote nanodomain coupling of BK with CaV1.

scholarly journals Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

2020 ◽  
Author(s):  
Kade M. Power ◽  
Jyothi S. Akella ◽  
Amanda Gu ◽  
Jonathon D. Walsh ◽  
Sebastian Bellotti ◽  
...  
Keyword(s):  
Genetic Screen ◽  
Mucociliary Transport ◽  
Neuronal Function ◽  
Post Translational Modifications ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Neuronal Cilia ◽  
Microtubule Stability ◽  
Progressive Degeneration ◽  
Downstream Effects

AbstractCiliary microtubules are subject to post-translational modifications that act as a “Tubulin Code” to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in the tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of microtubules and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 does not localize to cilia yet plays a role in regulating axonemal microtubule stability. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into human ciliopathies and neurodegenerative diseases.

scholarly journals ELKS active zone proteins as multitasking scaffolds for secretion

Open Biology ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 170258 ◽  
Author(s):  
Richard G. Held ◽  
Pascal S. Kaeser
Keyword(s):  
Plasma Membrane ◽  
Active Zone ◽  
Molecular Mechanisms ◽  
Scaffolding Protein ◽  
Functional Roles ◽  
Vesicle Traffic ◽  
Synaptic Vesicle Exocytosis ◽  
Variable Extent ◽  
Vesicle Exocytosis ◽  
Presynaptic Plasma Membrane

Synaptic vesicle exocytosis relies on the tethering of release ready vesicles close to voltage-gated Ca 2+ channels and specific lipids at the future site of fusion. This enables rapid and efficient neurotransmitter secretion during presynaptic depolarization by an action potential. Extensive research has revealed that this tethering is mediated by an active zone, a protein dense structure that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Although roles of individual active zone proteins in exocytosis are in part understood, the molecular mechanisms that hold the protein scaffold at the active zone together and link it to the presynaptic plasma membrane have remained unknown. This is largely due to redundancy within and across scaffolding protein families at the active zone. Recent studies, however, have uncovered that ELKS proteins, also called ERC, Rab6IP2 or CAST, act as active zone scaffolds redundant with RIMs. This redundancy has led to diverse synaptic phenotypes in studies of ELKS knockout mice, perhaps because different synapses rely to a variable extent on scaffolding redundancy. In this review, we first evaluate the need for presynaptic scaffolding, and we then discuss how the diverse synaptic and non-synaptic functional roles of ELKS support the hypothesis that ELKS provides molecular scaffolding for organizing vesicle traffic at the presynaptic active zone and in other cellular compartments.

scholarly journals Liprin-α/SYD-2 determines the size of dense projections in presynaptic active zones in C. elegans

2013 ◽  
Vol 203 (5) ◽  
pp. 849-863 ◽  
Author(s):  
Maike Kittelmann ◽  
Jan Hegermann ◽  
Alexandr Goncharov ◽  
Hidenori Taru ◽  
Mark H. Ellisman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
High Resolution ◽  
Active Zone ◽  
Neuromuscular Junctions ◽  
Main Role ◽  
C Elegans ◽  
Active Zones ◽  
Dense Projections

Synaptic vesicle (SV) release is spatially and temporally regulated by a network of proteins that form the presynaptic active zone (AZ). The hallmark of most AZs is an electron-dense projection (DP) surrounded by SVs. Despite their importance for our understanding of triggered SV release, high-resolution analyses of DP structures are limited. Using electron microscopy, we show that DPs at Caenorhabditis elegans neuromuscular junctions (NMJs) were highly structured, composed of building units forming bays in which SVs are docked to the AZ membrane. Furthermore, larger ribbonlike DPs that were multimers of the NMJ building unit are found at synapses between inter- and motoneurons. We also demonstrate that DP size is determined by the activity of the AZ protein SYD-2/Liprin-α. Whereas loss of syd-2 function led to smaller DPs, syd-2 gain-of-function mutants displayed larger ribbonlike DPs through increased recruitment of ELKS-1/ELKS. Therefore, our data suggest that a main role of SYD-2/Liprin-α in synaptogenesis is to regulate the polymerization of DPs.

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