Presynaptic UNC-31 (CAPS) Is Required to Activate the Gαs Pathway of the Caenorhabditis elegans Synaptic Signaling Network

Abstract

C. elegans mutants lacking the dense-core vesicle priming protein UNC-31 (CAPS) share highly similar phenotypes with mutants lacking a neuronal Ga s pathway, including strong paralysis despite exhibiting near normal levels of steady-state acetylcholine release as indicated by drug sensitivity assays. Our genetic analysis shows that UNC-31 and neuronal Ga s are different parts of the same pathway and that the UNC-31/Ga s pathway is functionally distinct from the presynaptic Ga q pathway with which it interacts. UNC-31 acts upstream of Ga s because mutations that activate the Ga s pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (1) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the Ga s pathway function in cholinergic motor neurons to regulate locomotion rate. Using immunostaining we show that UNC-31 is often concentrated at or near active zones of cholinergic motor neuron synapses. Our data suggest that presynaptic UNC-31 activity, likely acting via dense-core vesicle exocytosis, is required to locally activate the neuronal Ga s pathway near synaptic active zones.A T least two distinct vesicle systems, synaptic vesicles and dense core vesicles, mediate the presynaptic release of neurotransmitters. The synaptic vesicle system mediates the highly localized, rapid release of classical neurotransmitters from active zones. Presynaptic dense core vesicle release does not occur at morphologically defined active zones and is generally associated with neuropeptide release. Little is known about whether and how these two vesicle systems interact at the synapse during the execution of behaviors. Both vesicle systems release their contents in response to electrically induced calcium influx (Martin 2003), and each system has a mechanism for priming vesicles, which is the process by which vesicles become competent to fuse in response to the calcium signal. Synaptic vesicles use an UNC-13-based system (Aravamudan et al. 1999;Augustin et al. 1999;Richmond et al. 1999Richmond et al. , 2001. Dense core vesicles use a CAPS-based system (Grishanin et al. 2004). UNC-13 and CAPS are distantly related as indicated by a shared UNC-13 homology domain (Grishanin et al. 2002).In either system, if vesicles are not primed, they will not release neurotransmitter in response to stimuli, even if calcium enters the synapse or cell (Hay and Martin 1992;Aravamudan et al. 1999;Augustin et al. 1999;Richmond et al. 1999). Although the pathways regulating dense core vesicle priming are poorly understood, previous studies have shown that diacylglycerol (DAG) and cAMP can regulate the readily releasable (primed) pool of synaptic vesicles (Chen and Regehr 1997;Stevens and Sullivan 1998;Waters and Smith 2000;Kidokoro et al. 2004;Virmani et al. 2005). Genetic studies in the model organism Caenorhabditis elegans have identified a network of pathways that appears to regulate priming in the synaptic vesicle system. These studies have suggested a model, the synaptic signal...

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release fromCaenorhabditis elegansMotor Neurons

“…We prepared freeze-cracked 4% paraformaldehyde fixed worms for immunostaining as described (Charlie et al 2006a;Charlie et al 2006b), with the exception that we fixed worms for anti-FMRFamide staining for 16 h at 6° C . For immunostaining of FMRFamide neuropeptides, we co-immunostained N2 and unc-43(ce685) fixed animals using Rabbit antiFMRFamide antiserum (1/2000) and Goat anti-UNC-47 antiserum (1/12,000) ), using Donkey anti-Rabbit Dylight 550 and Donkey anti-Goat Dylight 488 secondary antibodies (Pierce).…”

“…We dual-labeled the GFP with Donkey anti-Mouse Dylight 488 and Donkey anti-Mouse Dylight 650 secondaries, and we labeled the EGL-21 primary antibody with a Donkey anti-Rabbit Dylight 550 secondary. For immunostaining of UNC-31 (CAPS), we triple-stained wild type and unc-43(ce685) fixed animals using Goat anti-UNC-31 (1/200; KM16C-3.1; Charlie et al, 2006a), Mouse anti-UNC-17 (1/5000 from monoclonal ascites fluid; , and Rabbit anti-UNC-10 (1/240; RIM5431; (Koushika et al 2001). We used previously described methods for immunostaining of fixed animals (Charlie et al 2006a;Charlie et al 2006b), with the exception that we only spread 750 animals per slide at the final mounting step.…”

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

“…The unc-17 promoter (Punc-17) included 3180 bp upstream of the SL1 trans-splice site (Alfonso et al 1994a); it drives expression exclusively in cholinergic neurons (Rand et al 2000). A derivative of this promoter, designated Punc-17b, drives expression in the AS, DA, DB, VA, and VB cholinergic motor neurons of the ventral nerve cord (Charlie et al 2006).…”

“…The unc-17 promoter (Punc-17) included 3180 bp upstream of the SL1 trans-splice site (Alfonso et al 1994a); it drives expression exclusively in cholinergic neurons (Rand et al 2000). A derivative of this promoter, designated Punc-17b, drives expression in the AS, DA, DB, VA, and VB cholinergic motor neurons of the ventral nerve cord (Charlie et al 2006).DNA injections: Transgenic nematodes were generated by microinjection of DNA (plasmids and/or PCR products), essentially as described by Mello and Fire (1995). The final DNA concentration was adjusted to 50 ng/ml by the addition of pBluescript.…”

UNC-73/Trio RhoGEF-2 Activity Modulates Caenorhabditis elegans Motility Through Changes in Neurotransmitter Signaling Upstream of the GSA-1/Gαs Pathway

“…Ga q (C. elegans EGL-30) and Ga s (GSA-1) positively influence neurotransmitter release while Ga o (GOA-1) activity reduces neurotransmitter release through inhibition of the Ga q /EGL-30 pathway (HajduCronin et al 1999;Lackner et al 1999;Miller et al 1999;Reynolds et al 2005;Schade et al 2005;Charlie et al 2006). Our previous genetic analysis placed unc-73 RhoGEF-2 activity downstream of Ga o /GOA-1 and Williams et al (2007) more specifically identified the UNC-73E RhoGEF-2 isoform as a Ga q /EGL-30 effector acting in parallel to phospholipase C b (EGL-8) (Steven et al 2005).…”