Active zone protein SYD-2/Liprin- α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Abstract

Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin-α to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin-α acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.

Figure 1:. Synaptic vesicle proteins travel with lysosomal proteins in heterogenous carriers

(A) Quantitation of co-transport of different combinations of synaptic vesicle proteins and lysosomal proteins from kymograph analysis of dual color imaging. The number of animals per genotype (N) ≥ 10. Number of vesicles analyzed (n) > 600. (B) Quantitation of fraction of different lysosomal proteins co-transporting different synaptic vesicle proteins from kymograph analysis of dual color imaging. N ≥ 10; n > 100. (C) Quantitation of fraction of CTNS-1-labelled compartments moving in the anterograde and retrograde direction in different mutants. N ≥ 9 per genotype; the number of CTNS-1-labelled compartments ≥ 20. (D) Quantitation of co-transport of CTNS-1::mCherry and mNeonGreen::RAB-7 in WT animals from sequential dual color imaging at 1.3 fps. CTNS-1 with RAB-7 indicates the fraction of CTNS-1-labelled compartments co-transporting RAB-7. RAB-7 with CTNS-1 indicates the fraction of RAB-7-labelled compartments co-transporting CTNS-1. N ≥ 15 animals; n > 450. (E) Schematic showing the PLM neuron. Red boxes highlight the regions of imaging. The arrow shows the anterograde direction of vesicle motion. (F) GFP::RAB-3, SNG-1::GFP, CTNS-1::mCherry, and RAB-7::mScarlet in the cell body, process and synapses of wildtype PLM neurons. Scale bar: 10 μm.

Figure 2:. LRK-1 and AP-3 act in parallel and through SYD-2 to regulate lysosomal protein trafficking.

(A) Quantitation of co-transport of SNG-1::eGFP and mCherry::RAB-3 in WT, lrk-1(km17), and apb-3(ok429) from kymograph analysis of sequential dual color imaging at 1.3 frames per second (fps). # P-value ≤ 0.05 (One-Way ANOVA with Tukey’s post-hoc test, all comparisons to WT); ns: not significant; Number of animals (N) ≥ 15 per genotype; Number of vesicles analyzed per genotype (n) > 800. (B) Quantitation of fraction of SNG-1-carrying vesicles co-transporting CTNS-1 in WT, lrk-1(km17), lrk-1(km41), apb-3(ok429) and lrk-1(km17) apb-3(ok429), from kymograph analysis of sequential dual color imaging at 1.3 fps. # P-value ≤ 0.05 (Mann–Whitney Test); N ≥ 15 per genotype; n > 500. (C) Quantitation of fraction of SNG-1-carrying vesicles co-transporting RAB-7 from WT, lrk-1(km17), apb-3(ok429) and lrk-1(km17) apb-3(ok429), kymograph analysis of dual color imaging. # P-value ≤ 0.05 (One-Way ANOVA with Tukey’s post-hoc test, all comparisons to WT); ns: not significant; N ≥ 20 per genotype; n > 800. (D) CTNS-1::mCherry in the cell body, process, and synapses of PLM neurons of WT, lrk-1(km17), apb-3(ok429), and lrk-1(km17) apb-3(ok429). Scale bar: 10 μm. Red arrows point to some CTNS-1-labelled compartments. (E) mScarlet::RAB-7 in the cell body, process, and synapses of PLM neurons of WT, lrk-1(km17), apb-3(ok429), and lrk-1(km17) apb-3(ok429). Scale bar: 10 μm. Red arrows point to some RAB-7-labelled compartments. (F) LMP-1::mNeonGreen in the cell body, process, and synapses of PLM neurons of WT, lrk-1(km17), apb-3(ok429), and lrk-1(km17) apb-3(ok429). Scale bar: 10 μm. Red arrows point to some RAB-7-labelled compartments. (G) Images showing APB-3::GFP puncta in the head ganglion cell bodies of WT, lrk-1(km17), and syd-2(ok217). Scale bar: 10 μm. Red boxes highlight the regions of insets with cell bodies from images showing APB-3::GFP in (i) WT, (ii) lrk-1, and (iii) syd-2. (H) Quantitation of the number of APB-3::GFP puncta per cell body in WT, lrk-1(km17), and syd-2(ok217). # P-value ≤ 0.05 (Mann–Whitney Test); ns: not significant; N > 10 animals; n > 75 cell bodies. (I) Quantitation of intensity of APB-3::GFP puncta in cell bodies of WT, lrk-1(km17), and syd-2(ok217). The ratio of the intensity of APB-3::GFP puncta to cytosolic intensity in the cell body is plotted. # P-value ≤ 0.05 (Mann–Whitney Test); ns: not significant; N > 10 animals; n > 75 cell bodies. (J) Quantitation of average size of APB-3::GFP puncta per cell body in WT, lrk-1(km17), and syd-2(ok217). # P-value ≤ 0.05 (Mann–Whitney Test); ns: not significant; N > 10 animals; n > 75 cell bodies.

Figure 3:. SV-lysosomes in lrk-1 and apb-3 mutants are dependent on UNC-104

(A) Schematic showing the PLM neuron. Red boxes highlight the regions of imaging. (B) SNG-1::GFP in the cell body, process, and synapses of PLM neurons showing dependence on UNC-104 in lrk-1(km17) and apb-3(ok429) mutants and their doubles with unc-104(e1265tb120). Scale bar: 10 μm. (C) CTNS-1::mCherry in the cell body, process, and synapses of PLM neurons showing dependence on UNC-104 in lrk-1(km17) and apb-3(ok429) mutants and their doubles with unc-104(e1265tb120). Red arrows highlight fainter CTNS-1 puncta. Scale bar: 10 μm. (D) Quantitation of co-transport of SNG-1 and CTNS-1 in unc-104(e1265tb120), lrk-1(km17); unc-104, and apb-3(ok429); unc-104 from kymograph analysis of sequential dual color imaging done at 1.3 fps. #P-value ≤ 0.05 (Mann–Whitney Test, all comparisons to WT); ns: not significant; Number of animals (N) ≥ 18 per genotype; Number of vesicles (n) > 1200.

Figure 4:. Distribution of SV-lysosomal compartments depends on UNC-104

(A) Quantitation of co-transport of SNG-1 and CTNS-1 in syd-2 mutants and their doubles with lrk-1(km17) and apb-3(ok429), from kymograph analysis of dual color imaging. ok217 refers to the null allele of syd-2, syd-2(ok217); while ju37 refers to the syd-2(ju37) allele. #P-value ≤ 0.05 (Mann–Whitney Test, all comparisons to WT); ns: not significant; Number of animals (N) ≥ 18 per genotype; Number of vesicles (n) > 750. Values for lrk-1 and apb-3 single mutants are the same as those in Fig. 2B. (B) Quantitation of co-transport of SNG-1 and RAB-7 in syd-2(ok217) and its doubles with lrk-1(km17) and apb-3(ok429), from kymograph analysis of dual color sequential imaging at 1.3 fps. P-value > 0.05 (One-Way ANOVA with Tukey’s post-hoc test); ns: not significant; N ≥ 19 per genotype; n > 700. Values for lrk-1 and apb-3 single mutants are the same as those in Fig. 2C. (C) CTNS-1::mCherry in the cell body, process, and synapses of PLM neurons of syd-2(ok217) mutant and its doubles with lrk-1(km17) and apb-3(ok429). Red arrows highlight some CTNS-1-carrying compartments, some fainter. Scale bar: 10 μm. (D) mScarlet::RAB-7 in the cell body, process, and synapses of PLM neurons of syd-2(ok217) mutant and its doubles with lrk-1(km17) and apb-3(ok429). Red arrows highlight some RAB-7-carrying compartments, some fainter. Scale bar: 10 μm. (E) CTNS-1::mCherry in the cell body, process, and synapses of PLM neurons of syd-2(ju37) mutant and its doubles with lrk-1(km17) and apb-3(ok429). Red arrows highlight some CTNS-1-carrying compartments, some fainter. Scale bar: 10 μm. Imaged at 100×. (F) LMP-1::mNeonGreen in the cell body, process, and synapse of PLM neurons of syd-2(ok217) mutant and its doubles with lrk-1(km17) and apb-3(ok429). Red arrows indicate LMP-1-carrying compartments. Scale bar: 10 μm. (G) Quantitation of co-transport of SNB-1 and RAB-3, in syd-2(ok217), from simultaneous dual color imaging at 3 frames per second (fps). # P-value ≤ 0.05 (One-Way ANOVA with Tukey’s post-hoc test); N > 20. (H) Quantitation of co-transport of SNG-1 and RAB-3, in syd-2(ok217), from sequential dual color imaging at 1.3 fps. P-value > 0.05 (One-Way ANOVA with Tukey’s post-hoc test); ns: not significant; N > 15.

Figure 5:. SYD-2 is required for UNC-104 dependent of SVp carriers

(A) Schematic of the PLM neuron. The red box highlights the region of imaging. (B) SNG-1::GFP in the cell body, process and synapses of PLM neurons of syd-2(ok217) and unc-104(e1265tb120), and their doubles with lrk-1(km17) and apb-3(ok429). Scale bar: 10 μm. (C) CTNS-1::mCherry in the cell body, process and synapses of PLM neurons of syd-2(ok217) and unc-104(e1265tb120), and their doubles with apb-3(ok429). Scale bar: 10 μm. (D) Quantitation of co-transport of SNG-1 and CTNS-1 in WT, unc-104(e1265tb120), syd-2(ok217), and unc-104; syd-2 from kymograph analysis of sequential dual color imaging at 1.3 fps. P-value > 0.05 (Mann–Whitney Test, all comparisons to WT); ns: not significant; Number of animals (N) > 20 for unc-104; syd-2; Number of vesicles (n) >1000.

Figure 6:. SYD-2 and the AP-1 complex together regulate the polarized distribution of SVps to axons

(A) Schematic of the ASI chemosensory neuron. Red box highlights the region of imaging. (B) SNB-1::GFP in the dendrite of the ASI neuron of WT and two alleles of syd-2 and their doubles with lrk-1(km17) and apb-3(ok429). ok217 represents syd-2(ok217) allele. ju37 represents syd-2(ju37) allele. unc-101(m1) is a substitution mutation in the μ chain of the AP-1 complex causing a premature stop. Red arrows point to the SNB-1::GFP signal at the dendrite tip. Scale bar: 20 μm. Number of animals (N) > 6 per all single mutant genotypes; N > 20 for all double mutant genotypes. (C) CTNS-1::mCherry in the dendrite of the ASI neurons of WT, syd-2(ok217), lrk-1(km17), apb-3(ok429), unc-101(m1), and unc-101(m1); syd-2(ok217). Red arrows point to CTNS-1 compartments in the dendrite. Scale bar: 20 μm. N > 20 per genotype. (D) Density (number of CTNS-1 puncta per 10 μm in the ASI dendrite) of CTNS-1 in the ASI dendrite. # P-values ≤ 0.05 (Mann–Whitney Test, black comparisons against WT and blue comparisons against lrk-1); N > 20 for each genotype. (E) Schematic of C. elegans head showing the pharynx (red) and the head ganglion cell bodies (yellow). (F) Images showing UNC-101::GFP puncta in the head ganglion cell bodies of WT and syd-2(ok217). Scale bar: 10 μm. The red boxes highlight the regions of insets with cell bodies from images showing UNC-101::GFP in (i) WT and (ii) syd-2. (G) Quantitation of intensity of UNC-101::GFP puncta in the head ganglion cell bodies in WT and syd-2(ok217). The ratio of the intensity of UNC-101::GFP puncta to cytosolic intensity in the cell body is plotted. # P-value ≤ 0.05 (One-Way ANOVA with Tukey’s post-hoc test); N > 5 animals; n > 25 cell bodies. (H) Quantitation of average size of UNC-101::GFP puncta per cell body in WT and syd-2(ok217). # P-value ≤ 0.05 (Mann–Whitney Test); N > 5 animals; n > 25 cell bodies. (I) Images showing UNC-101::GFP puncta in the cell bodies of the ventral nerve cord neurons in WT and syd-2(ok217). Scale bar: 10 μm. The red boxes highlight the regions of insets with cell bodies from images showing UNC-101::GFP in (i) WT and (ii) syd-2. (J) Quantitation of intensity of UNC-101::GFP puncta in the cell bodies of the ventral nerve cord in WT and syd-2(ok217). The ratio of the intensity of UNC-101::GFP puncta to cytosolic intensity in the cell body is plotted. # P-value ≤ 0.05 (Mann–Whitney test); N > 5 animals; n > 10 cell bodies.