A post-docking role for active zone protein Rim

Abstract

Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.

Fig. 1

Organization of the C. elegans unc-10 gene. (a) Genomic region of the unc-10 gene including selected restriction sites. Exons are shown as colored boxes that define domains using the same color code as in (b). Zinc finger, PDZ and C2 domains were identified using the SMART program. The glutamine- and asparagine-rich domain (Q/N) was delineated by visual inspection. The position of unc-10 point mutations (arrows) and deletions (|\#x02014;|) are depicted below the genomic map. Genomic regions included in plasmids used in this work are also illustrated. Open boxes in the plasmids indicate deletions of the corresponding unc-10 genomic regions. Green fluorescent protein (GFP) sequences inserted in plasmids are denoted as labeled boxes. (b) Comparison of the domain organization of the C. elegans UNC-10 and rat Rim1 proteins. The percentage sequence identity between shared domains and the positions of alternative splicing are depicted. The alanine and proline rich domain (A/P) and alternative splice forms of Rim1 were identified previously. (c) Sequence alignments of selected domains of Rim homologs. Alignments were created and formatted using the clustalW and SeqVu programs.

Fig. 2

Rim protein localizes to a subdomain of the synapse. Whole mounts of wild type (a–e) and unc-10(md1117) (f, g) C. elegans fixed and prepared for immunohistochemical detection of Rim (green) and RAB-3 (red) proteins. (a) A lateral view of the head region of an adult demonstrates that Rim protein is abundant in the ventral and dorsal nerve cords and in the nerve ring. Scale bar, 20 μm. (b, c) Closer inspection of the nerve ring (b) and ventral nerve cord (c) reveals that Rim is restricted to discrete puncta. (d) A view of the dorsal nerve cord illustrates that Rim is more discretely localized compared to the synaptic vesicle-associated protein RAB-3. (e) A view of an SAB neuron innervating head muscle demonstrates that one or two Rim punctum are localized within each RAB-3 staining varicosity. Scale bar (b–e), 2 μm. (f) Rim (green) but not RAB-3 (red) immunoreactivity is absent in unc-10(md1117) animals as visualized in the SAB neurons. (g) Isolated view of the Rim channel (green) of (f) confirms that Rim immunoreactivity is absent in the unc-10(md1117) mutant. Scale bar (f), (g) 20 μm.

Fig. 3

Behavioral defects of Rim mutants. (a) Bright field micrographs of young adult wild type and unc-10 mutant animals. (b) Behavior of wild type, unc-10 mutant and unc-10 transgenic animals after 12 h in the presence of varying concentrations of aldicarb. pRIM6 was used as the source of Rim (+), pRIM19 at the source of RimΔZn, pRIM9 as the source of RimΔC2B and pRIM10 as the source of RimΔZnQ. RimΔZnQ 1 and 2 are two independent lines expressing this construct.

Fig. 4

Localization of Rim, RAB-3 and UNC-13 in different mutant backgrounds. (a–e) Whole-mount mutant C. elegans animals fixed and prepared for immunohistochemical detection of Rim (green) and RAB-3 (red) proteins. (a) Rim protein is localized indistinguishably from the wild type despite the absence of RAB-3 in rab-3(js49) animals. Compare with Fig. 2a–c. (b) Rim protein localizes normally in a transgenic unc-10(md1117) Rim null animal expressing the RimΔZn construct pRIM19. (c) Both Rim and RAB-3 are normally localized in the severe unc-13(s69) mutant. (d, e) Images at two focal planes showing that Rim is localized in discrete puncta at the nerve ring despite the mislocalization and accumulation of RAB-3 in neuronal cell bodies (ganglia) in an unc-104(e1265) kinesin mutant. (f, g) Whole-mount mutant C. elegans animals fixed and prepared for immunohistochemical detection of long form of UNC-13 (red) in wild type (f) and unc-10(md1117) Rim null (g) mutant animals. UNC-13 localization in a Rim mutants is indistinguishable from wild type. Scale bars, 2 μm.

Fig. 5

Structure of neuromuscu-lar junctions in Rim mutants. Electron micrographs of neuro-muscular junctions from young adult wild type and Rim mutants. The presynaptic specialization (blue arrowhead), and representative synaptic vesicles (red arrowhead) and docked synaptic vesicles (green arrowhead) are labeled. Scale bar, 200 nm.

Fig. 6

Defects in evoked and spontaneous release at the neuromuscular junction. (a) Representative evoked synaptic responses of voltage-clamped muscle (holding potential, −60 mV) in the wild type and in Rim mutants in response to a 2-ms stimulus to motor neurons in the ventral nerve cord in the presence of 3 mM calcium. (b) Mean amplitudes of evoked responses from the wild type (n = 22) and unc-10(md1117) Rim mutants (n = 11). Errors bars, s.e.m. (c) Representative traces of endogenous currents in muscles from the wild type and unc-10(md1117) mutants in 3 mM calcium. (d) Average frequencies of endogenous synaptic currents in the wild type (n = 27) and unc-10(md1117) (n = 14) muscles. (e) Representative evoked synaptic responses of voltage-clamped muscle (holding potential, −60 mV) in the wild type and in Rim mutants in response to consecutive 2-ms stimuli to motor neurons in the ventral nerve cord in 0.4 mM and 5.0 mM calcium. (f) Ratio of the amplitude of evoked responses recorded in muscle in 0.4 versus 5.0 mM calcium in the wild type (n = 14) and unc-10(md1117) (n = 7). (g) Representative trace of spontaneous calcium-free miniature postsynaptic currents in the wild-type and unc-10(md1117) muscles. (h) Average frequencies of spontaneous calcium-free miniature post-synaptic events in wild type (n = 6) and unc-10(md1117) (n = 7) muscles.

Fig. 7

Open syntaxin suppresses Rim-evoked release defect. (a) Representative evoked synaptic responses of voltage-clamped muscle (holding potential, −60 mV) in the wild type, Rim mutant with and without syntaxin constructs in response to a 2-ms stimulus to motor neurons in the ventral nerve cord in the presence of 5.0 mM calcium. The complete genotype of the Rim mutant syntaxin(+) strain is unc-10(md1117) oxIs33; unc-64(js115) and Rim syntaxin(open) strain is unc-10(md1117); unc-64(js115) oxIs34. (b) Mean amplitude of responses ± s.e.m. (wild type, n = 14; unc-10(md1117), n = 7; unc-10(md1117); syx(wt), n = 4; unc-10(md1117); syx(open), n = 10).