Presynaptic UNC-31 (CAPS) is required to activate the G alpha(s) 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 G alpha(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 G alpha(s) are different parts of the same pathway and that the UNC-31/G alpha(s) pathway is functionally distinct from the presynaptic G alpha(q) pathway with which it interacts. UNC-31 acts upstream of G alpha(s) because mutations that activate the G alpha(s) pathway confer similar levels of strongly hyperactive, coordinated locomotion in both unc-31 null and (+) backgrounds. Using cell-specific promoters, we show that both UNC-31 and the G alpha(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 G alpha(s) pathway near synaptic active zones.

Figure 1.

Model of the synaptic signaling network. Solid lines indicate that direct interactions are known or likely, while dashed lines and/or large gaps between line endpoints and downstream effectors indicate predicted interactions or predicted missing components. Proteins that promote locomotion and/or neurotransmitter release are shown in green, while proteins that inhibit locomotion and/or neurotransmitter release are shown in red. This study addresses the relationship between the dense-core vesicle priming protein UNC-31 (CAPS) and the synaptic signaling network. See the text and the following references for details: Maruyama and Brenner (1991); Mendel et al. (1995); Segalat et al. (1995); Brundage et al. (1996); Koelle and Horvitz (1996); Hajdu-Cronin et al. (1999); Lackner et al. (1999); Miller et al. (1999, 2000); Nurrish et al. (1999); Richmond et al. (1999, 2001); Robatzek and Thomas (2000); Chase et al. (2001); van der Linden et al. (2001); Robatzek et al. (2001); Tall et al. (2003); Reynolds et al. (2005); and Schade et al. (2005).

Figure 2.

Conserved UNC-31 (CAPS) domains and a null mutation. Shown is a scale drawing of C. elegans UNC-31 depicting the known domains of CAPS (its vertebrate ortholog), as defined by Grishanin et al. (2002). The pleckstrin homology (PH) domain (489–596 of UNC-31) mediates specific interactions with the plasma membrane, while a C-terminal domain (1078–1260) mediates association with dense-core vesicles (Grishanin et al. 2002). A C2 domain (367–461) identified by Grishanin et al. (2002) is of unknown function. Percentages indicate the percent identity to rat CAPS (GenBank no. U16802). Note that the region of highest homology is the UNC-13 homology domain (domain DUF1041, using the NCBI conserved domain search tool; 696–892). A core subregion (734–815) of the UNC-13 homology domain is 84% identical to rat CAPS-1. The overall identity between UNC-31 and rat CAPS-1 is 49%. The region deleted by the unc-31(e928) mutation, as indicated, includes the entire C-terminal half of UNC-31 (553–1260 of 1260 aa total). This analysis is based on UNC-31 protein sequence in Wormbase, freeze version 130. All amino acid positions refer to C. elegans UNC-31.

Figure 3.

UNC-31 (CAPS) null mutants and mutants lacking a neuronal Gα_s pathway share similar phenotypes. Note that for purposes of clarity, Gα_q, Gα_s, and Gα_o are at times referred to as Gq, Gs, and Go in this and subsequent figures. (A) unc-31 null mutants look similar to mutants that lack a neuronal Gα_s pathway when viewed on culture plates through a stereomicroscope. Images compare a typical N2 (wild-type) animal with a typical unc-31(e928) null mutant and a mutant that lacks a neuronal Gα_s pathway. The latter's genotype is acy-1(pk1279); ceEx108 [myo-3∷acy-1(+) cDNA], which expresses acy-1 (adenylyl cyclase) only in muscle cells and thus is null for acy-1 in the nervous system (Reynolds et al. 2005). All three animals are feeding in a lawn of bacteria. Note the similar straight posture associated with the paralysis of the unc-31 and neuronal Gα_s pathway null mutants. The image “Mutant lacking a neuronal Gα_s pathway” is reprinted with permission from Reynolds et al. (2005) for comparison. (B) unc-31 and neuronal Gα_s pathway null mutants exhibit a similar degree of paralysis. Shown are the mean locomotion rates, expressed as body bends per minute, of the indicated strains. An egl-30 (Gα_q) reduction-of-function mutant (ad805) (Brundage et al. 1996) that is paralyzed to a similar extent as mutants lacking a neuronal Gα_s pathway or UNC-31 is shown for comparison. unc-31 null mutants are only slightly less paralyzed than animals that lack a neuronal Gα_s pathway. The bar on the right demonstrates that a transgenic array containing the unc-31 (+) cDNA expressed in the nervous system fully rescues the locomotion defect of the unc-31(e928) null. Error bars represent the standard error of the mean for populations of 10 animals. Transgenic arrays are ceEx108 [myo-3∷acy-1(+) cDNA (expressed only in muscle)] and ceEx117 [rab-3∷unc-31(+) cDNA (expressed only in the nervous system)]. The value for acy-1(pk1279); ceEx108 is reprinted with permission from Reynolds et al. (2005) for comparison. (C) unc-31 and neuronal Gα_s pathway null mutants respond normally to the cholinergic agonist levamisole. The graph depicts the contraction of individual animals after a 20-min exposure to various concentrations of levamisole. Error bars represent the standard errors for six individual animals at each concentration. acy-1(pk1279); ceEx108 (a transgenic muscle-rescued acy-1 null mutant) was used as the neuronal Gα_s pathway null. Note that the levamisole sensitivity of unc-31(e928) is indistinguishable from wild type, while the neuronal Gα_s pathway null appears only slightly hypersensitive to levamisole (although this is not statistically significant at any concentration). We did not investigate the extent to which overexpression of the acy-1(+) transgene cDNA in muscle may have contributed to the slight levamisole hypersensitivity, as has been previously shown to occur with muscle-specific expression of acy-1(gf) (Schade et al. 2005). Statistical significance tests used the unpaired t-test with Welch correction, with the level of statistical significance set at P = 0.05. The data for N2 and the neuronal Gs pathway null are reprinted with permission from Reynolds et al. (2005) for comparison and were generated as part of the same experiment with the unc-31 null. (D) unc-31 and neuronal Gα_s pathway null mutants exhibit near-normal aldicarb sensitivity. Shown are the population growth rates of strains on various concentrations of the acetylcholinesterase inhibitor aldicarb. One hundred percent represents the number of progeny produced from a starting population of L1 larvae over a 96-hr period in the absence of aldicarb. Curves are representative of duplicate experiments. Complete genotypes are as follows: unc-31 null, unc-31(e928); Gα_q reduction-of-function mutant, egl-30(ad805) (Brundage et al. 1996); and neuronal Gs pathway null, acy-1(pk1279); ceEx108 (a transgenic muscle-rescued acy-1 null mutant) (Reynolds et al. 2005). The data for N2, egl-30(ad805), and the neuronal Gs pathway null are reprinted with permission from Reynolds et al. (2005) for comparison and were generated as part of the same experiment with the unc-31 null.

Figure 4.

In the synaptic signaling network, UNC-31 (CAPS) and Gα_s function together in a pathway that is functionally distinct from the Gα_q pathway. (A) Circuit model of the synaptic signaling network, depicting the components and mutations used in this experiment. The X's through the Gα_s pathway and UNC-31 indicate null acy-1 (adenylyl cyclase) and unc-31 (CAPS) mutations, while the dashed X through EGL-30 represents the reduction-of-function egl-30 (Gα_q) mutations that were combined with the unc-31 null in this experiment. (B) UNC-31 (CAPS) and neuronal Gα_s are part of the same pathway. Shown are the mean locomotion rates, expressed as body bends per minute, of various strains labeled with arrows. The first group of bars on the left represents wild type and the unc-31 null single mutant. Subsequent groups of bars show strains homozygous for acy-1(pk1279) or two different egl-30 (Gα_q) reduction-of-function mutants as indicated. The first bar in each of these latter groups represents the single mutant that is indicated on the x-axis, while subsequent bars in each group represent double mutants in which the second mutation is the unc-31 null mutation. The final bar on the right shows an egl-30 (Gα_q) null single mutant. Error bars represent the standard error of the mean for 10 animals. Note that most of the wild-type bar is out of range of the graph. The values for acy-1(pk1279) and egl-30(ad810) are reprinted with permission from Reynolds et al. (2005) for comparison.

Figure 5.

Epistasis analysis suggests that the neuronal Gα_s pathway is downstream of UNC-31 and functionally inactive in unc-31 nulls. (A) Circuit model of the Gα_q and Gα_s branches of the synaptic signaling network depicting the components and mutations used in this experiment. “Blinker marks” next to Gα_q and Gα_s signify the strong gain-of-function mutations used in this experiment, while the “X” through UNC-31 indicates the unc-31 null background into which we transferred each gain-of-function mutation. (B) The paralysis of unc-31 nulls is converted to hyperactive locomotion by a mutation that activates the Gα_s pathway, and activating the Gα_q pathway in the unc-31 null confers a phenotype similar to activating the Gα_q pathway in a neuronal Gα_s pathway null. Shown are the mean locomotion rates, expressed as body bends per minute, of various strains. The first group of three bars on the left represents wild-type and single-mutant control strains, which are color coded in the inset. In the right two sets of bars, double- and single-mutant strains homozygous for unc-31(e928) or acy-1(pk1279); ceEx108 (a strain lacking a neuronal Gα_s pathway) are grouped together as indicated. Note that activating the Gα_s pathway in the unc-31 null results in a locomotion rate nearly as hyperactive as the gsa-1 gain-of-function single mutant, whereas activating the Gα_q pathway in either an unc-31 null background or a background lacking a neuronal Gα_s pathway results in an approximately wild-type locomotion rate along with a tendency to knot or coil (photos). Error bars represent the standard error of the mean for 8–10 animals. A portion of the data in this graph is reprinted with permission from Reynolds et al. (2005) for comparison. See also supplemental QuickTime movies (http://www.genetics.org/supplemental/). (C and D) Activating either the Gα_s pathway or the Gα_q pathway in the unc-31 null mutant causes hypersensitivity to aldicarb. Shown are the population growth rates of strains on various concentrations of the acetylcholinesterase inhibitor aldicarb. One hundred percent represents the number of progeny produced from a starting population of L1 larvae over a 96-hr period in the absence of aldicarb. Each strain's response, including the wild-type control, is indicated. gsa-1(gf), gsa-1(ce81); egl-30(gf), egl-30(tg26); and unc-31(0), unc-31(e928). The data for gsa-1(ce81) are reprinted with permission from Schade et al. (2005) for comparison. (E) Phorbol esters induce identical aldicarb hypersensitivity in wild-type and unc-31 null mutants. Shown is a graph depicting the percentage of animals that are paralyzed, over a time course, on plates containing 1 mm aldicarb and/or 5 μm phorbol myristate acetate. Strains and conditions are indicated with arrows. Note that both wild-type and unc-31 null mutants that have been treated with phorbol ester are equally hypersensitive to the paralytic effects of aldicarb (their lines overlap over most of the time course), which suggests that they are both releasing similar amounts of acetylcholine. Error bars represent the standard error of the mean for three independent populations of 20 animals each.

Figure 6.

Knocking out the inhibitory Gα_o pathway affects the unc-31 null in a manner similar to activating the Gα_q pathway or to phorbol ester treatment. (A) Circuit model of the synaptic signaling network depicting the components and mutations used in this experiment. The X's through GOA-1 and UNC-31 indicate the null goa-1 and unc-31 mutations used in this experiment, which were goa-1(sa734) and unc-31(e928). (B) Knocking out the inhibitory Gα_o pathway only partially suppresses the paralysis of the unc-31 null. Shown are the mean locomotion rates, expressed as body bends per minute. The bars on the left represent wild-type and the goa-1 null single-mutant control strains, while the bars on the right represent strains containing the unc-31 null mutation. Note that knocking out the Gα_o pathway improves the locomotion rate of the unc-31 null only to a level that is ∼18% of the goa-1 null single mutant and ∼40% of wild type. Error bars represent the standard error of the mean for 8–10 animals. The value for goa-1(sa734) is reprinted with permission from Reynolds et al. (2005) for comparison. (C and D) Knocking out unc-31 does not affect the strong aldicarb hypersensitivity caused by knocking out the Gα_o pathway. (C) The population growth rates of strains on various concentrations of aldicarb. One hundred percent represents the number of progeny produced from a starting population of L1 larvae over a 96-hr period in the absence of aldicarb. Note that the strong aldicarb hypersensitivity of the goa-1(0) single mutant is closely matches that of a double mutant that is null for both unc-31 and goa-1, despite the rather sluggish locomotion rate of this double mutant (B). (D) The acute response of these strains to a fixed concentration of 2 mm aldicarb over an 80-min time course (using paralysis as the endpoint). Error bars represent the standard errors of populations of 25 animals at each time point.

Figure 7.

The activated Gα_s pathway suppresses the paralysis of unc-31 null mutants via a highly specific, short timescale mechanism. (A) Activating the Gα_s pathway does not strongly rescue the paralysis of Gα_q reduction-of-function mutants that are paralyzed to a similar degree as unc-31 nulls. Shown are the mean locomotion rates, expressed as body bends per minute. The first group of bars on the left represents wild-type and single-mutant control strains, which are color coded in the inset. Subsequent groups of bars show strains homozygous for unc-31(e928) or two different egl-30 (Gα_q) reduction-of-function mutants as indicated on the x-axis. The first bar in each of these latter groups represents the single mutant that is indicated on the x-axis, while subsequent bars in each group represent double mutants in which the second mutation activates a component of the Gα_s pathway as ordered in the inset. The mutations in the inset activate ACY-1 (adenylyl cyclase) or protein kinase A [the latter is an indirect activation via strongly reducing the function of the KIN-2 regulatory subunit, as described (Reynolds et al. 2005)]. Error bars represent the standard error of the mean for 8–10 animals. Gaps between bars in the egl-30 groups represent double mutants that have not been constructed. The values for the single mutants acy-1(md1756), acy-1(ce2), and kin-2(ce179) are reprinted with permission from Schade et al. (2005) for comparison. (B) Activating the Gα_s pathway suppresses the paralysis of unc-31 null mutants via a short timescale that is not dependent on development. Shown are the mean locomotion rates, expressed as body bends per minute. Dark and light blue bars indicate locomotion rates without or with heat-shock treatment, respectively. The pkIs296 transgene used in this experiment was produced in a previous study (Korswagen et al. 1997). Error bars represent the standard error of the mean for populations of 8 animals. The values for the pkIs296 single mutant (plus or minus heat shock) are reprinted with permission from Schade et al. (2005) for comparison.

Figure 8.

Transgenic site-of-action studies using cell-specific promoters. (A) The neuronal, and not the muscle, Gα_s pathway contributes most significantly to rescuing the paralysis of the unc-31 null. Shown are the mean locomotion rates, expressed as body bends per minute, of various strains. The first group of four bars on the left represents wild-type and single-mutant control strains, which are color coded in the inset. In the right set of bars, double- and single-mutant strains homozygous for unc-31(e928) are grouped together as indicated and labeled. Gα_s pathway activation transgenes consisted of an acy-1 cDNA containing the P260S gain-of-function mutation (Reynolds et al. 2005) driven by either the rab-3 or the myo-3 promoter (for neuronal or muscle-specific expression, respectively). acy-1(ce2) is the native genomic mutation containing the P260S gain-of-function mutation. Error bars represent the standard error of the mean for 10 animals. The values for the acy-1(ce2), ceIs11, and ceIs6 single mutants are reprinted with permission from Schade et al. (2005) for comparison. (B) UNC-31 and the neuronal Gα_s pathway can function together in the same neurons. Shown are the mean locomotion rates, expressed as body bends per minute, of various strains. Note that the unc-17β promoter used here does not drive expression in the VC neurons in the ventral cord, which could also contribute to locomotion rate. Error bars represent the standard error of the mean for 10 animals. Transgenic array descriptions are ceEx117 [rab-3∷unc-31(+) cDNA], ceIs19 [unc-17β∷unc-31(+) cDNA], and ceIs28 [unc-17β∷acy-1(P260S gain-of-function) cDNA].

Figure 9.

Within the ventral nerve cord, UNC-31 is concentrated presynaptically at or near active zones and largely at cholinergic synapses. (A) UNC-31 immunostaining in the ventral nerve cord. Maximum intensity projections of a z-series through the cord are shown. The four panels are different color combinations of the same image. (Top) Red channel showing UNC-17 staining to mark cholinergic vesicle clusters. (Second channel) Merged UNC-17 (red) and UNC-31 (green) channels showing that UNC-31 is concentrated within cholinergic synapses in the ventral cord. (Third channel) Locations of active zones at cholinergic synapses as indicated by an antibody to UNC-10 (blue). Most of the blue spots not associated with cholinergic synapses likely indicate the locations of noncholinergic synapses in the cord. (Bottom) Merged image of all three channels. (B) Enlargements of clusters of synapses in the ventral cord showing that UNC-31 and UNC-10 often localize to the same subsynaptic region (at or near the active zone). Asterisks indicate synaptic sites having strong UNC-31 staining, but weak or little UNC-10. Arrowheads indicate a few examples of synapses with strong UNC-10 and weak UNC-31. Arrows in the bottom right panel indicate noncholinergic active zones that lack concentrated UNC-31.

Figure 10.

UNC-31 immunolocalization in the dorsal and sublateral nerve cords. (A) UNC-31 immunostaining in the dorsal nerve cord. Maximum intensity projections of a z-series through the cord are shown. The three panels are different color combinations of the same image. (Top) Locations of active zones (blue UNC-10) at cholinergic synapses (red UNC-17). Most of the blue spots not associated with cholinergic synapses likely indicate the locations of noncholinergic synapses in the cord (e.g., GABAergic, glutamatergic, and catacholaminergic synapses). (Middle) Merged UNC-17 (red) and UNC-31 (green) channels showing that UNC-31 is largely concentrated within cholinergic synapses in the ventral cord. Note the high correlation of UNC-31 spots with active zones shown in the top channel. (Bottom) Merged image of all three channels. White spots indicate colocalization of all three markers. Arrows at the far left indicate the likely locations of two noncholinergic synapses with significant UNC-31 staining. (B) Enlargement of cholingergic synapses in one of the sublateral nerve cords showing that UNC-31 and UNC-10 often localize to the same subsynaptic region (the active zone). A small synapse containing concentrated UNC-31, but little UNC-10 is indicated with an arrow.

Figure 11.

Alternative models for the function of presynaptic UNC-31 (CAPS). As indicated by our results, both A and B show UNC-31 acting in the same cells as neuronal Gα_s, in the same molecular pathway as Gα_s, upstream of Gα_s, with a site of action at or near the synaptic active zone, and not directly interacting with the Gα_q pathway. Both models depict CAPS acting as a dense-core vesicle priming protein as described in vertebrate studies (Grishanin et al. 2004). (A) UNC-31 exerts its effects on the Gα_s pathway via exocytosis of a dense-core vesicle neurotransmitter/neuropeptide that then locally activates a G protein-coupled receptor that is already present in the presynaptic membrane. (B) UNC-31 exerts its effects on the Gα_s pathway via exocytotic deposition of one or more G protein-coupled receptors in the presynaptic membrane, similar to the phenomena described in vertebrate neuronal cell bodies (Guan et al. 2005).