Microtubule-based localization of a synaptic calcium-signaling complex is required for left-right neuronal asymmetry in C. elegans

Abstract

The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. Here, we show that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. We identified kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity.

Fig. 1.

Nocodazole treatment disrupts left-right AWC asymmetry. (A-C) Expression of the reporter gene str-2p::GFP in wild-type (A), nocodazole-treated (B) and tir-1(tm3036lf) (C) animals. In some of nocodazole-treated animals, the AWC axons are distorted, as indicated by arrowheads (B). Scale bar: 10 μm. Anterior is leftwards and ventral is towards the bottom. Arrows indicate the AWC cell bodies. (D) The AWC signaling pathway.

Fig. 2.

Nocodazole disrupts synaptic localization of TIR-1, UNC-43 and NSY-1 in the AWC axons. (A,B) Images of TIR-1::DsRed in AWC neurons of nocodazole-treated animals exhibiting 1AWC^OFF/1AWC^ON (A) and 2AWC^ON (B) phenotypes. Arrowheads indicate TIR-1::DsRed puncta in the AWC axons. Asterisks indicate cell bodies. (C) The average intensity of TIR-1::DsRed in the AWC axons. (D,E) Images of UNC-43::GFP in AWC neurons of nocodazole-treated animals. (F) The average intensity of UNC-43::GFP in the AWC axons. (G,H) Images of NSY-1::GFP in AWC neurons of nocodazole-treated animals. (I) The average intensity of NSY-1::GFP in the AWC axons. Images were taken from L1 animals. Scale bar: 10 μm. A.U., arbitrary unit. Error bars indicate the s.e.m.; t-test; n>15 for each transgene in 1AWC^OFF/1AWC^ON or 2AWC^ON animals.

Fig. 3.

UNC-104 is localized adjacent to TIR-1 in the AWC axons. (A-C) Images of unc-104p::GFP (A) and odr-1p::DsRed (B). Co-expression of GFP and DsRed in AWC appears yellow (C). Arrows indicate the cell body of AWC. (D-F) Images of TIR-1::DsRed (D) and UNC-104::GFP (E). UNC-104::GFP is localized adjacent to TIR-1::DsRed in AWC axons (F). Inset shows higher magnification of the area outlined. Scale bar: 10 μm.

Fig. 4.

unc-104/kif1a acts non-cell autonomously in AWC^ON to specify AWC^OFF fate. (A,B) AWC phenotypes of unc-104(e1265) mutants expressing the transgene unc-104p::unc-104; odr-1p::DsRed in both AWC neurons (A) or in one of the two AWC neurons (B).

Fig. 5.

The kinesin motor unc-104/kif1a is required for the localization of TIR-1, UNC-43 and NSY-1 in the AWC axons. (A,B) Localization of TIR-1::GFP in AWC neurons of wild-type (A) and unc-104(e1265) (B). (C) The average intensity of TIR-1::GFP in the AWC axons. (D,E) Localization of UNC-43::GFP in AWC neurons of wild-type (D) and unc-104(e1265) (E). (F) The average intensity of UNC-43::GFP in AWC axons. (G,H) Localization of NSY-1::GFP in AWC neurons of wild-type (G) and unc-104(e1265) (H). (I) The average intensity of NSY-1::GFP in AWC axons. Images were taken from L1 animals. Asterisks indicate cell bodies. Scale bar: 10 μm. A.U., arbitrary unit. Error bars indicate the s.e.m.; t-test; n=20 for each transgene per genotype in C,F,I.

Fig. 6.

Dynamic trafficking of TIR-1 in the AWC axons is dependent on unc-104/kif1a. (A) Representative images of moving TIR-1::GFP puncta in wild-type AWC axons. Red arrowheads indicate retrogradely moving puncta and blue arrowheads indicate anterogradely moving puncta. (B) Kymograph of the movie shown in A. The speed of the retrograde movement within the two red arrowheads is 1.2 μm/second; the speed of the anterograde movement between the two blue arrowheads is 1.9 μm/second. (C) The percentage of anterograde and retrograde trafficking events. n=314 and n=101 trafficking events in wild type and unc-104(e1265), respectively. Error bars indicate the s.e.m. Z-test for two proportions.