Supplemental Material for Noma and Jin, 2018

<i>knjIs1[Prgef-1-GFP::DLK-1L + Cbr-unc-119 + Pttx-3-RFP]</i> strain shows uncoordination. The movie was taken using a stereomicroscope Stemi 508 (Zeiss) with a digital camera L-835 (Hozan).<br

TTBK-3 is required for synapse maintenance during remodeling.

<p>(A) Schematic of heat shock assay, where animals are maintained at 20^°C, undergo a 2hr heat shock during the developmental stage being tested, after which they are returned to 20^°C and assayed at young adult stage. (B-E) Quantification of % animals with normal behavior (B, D) and synapse remodeling (C, E) following heat shock at L1-L4 stages. Data collected from 3 independent biological replicates, with n>10 animals each, and presented as mean ± SEM. Statistics- 2-Way ANOVA followed by Bonferroni posttest; ***p<0.001. (F) Representative images of DD neuron synapses along the DNC in adult animals imaged using <i>P</i>_{<i>unc-25</i>}-SNB-1-GFP (<i>juIs1</i>). Scale bar: 10 μm.</p

Differential regulation of polarized synaptic vesicle trafficking and synapse stability in neural circuit rewiring in <i>Caenorhabditis elegans</i>

<div><p>Neural circuits are dynamic, with activity-dependent changes in synapse density and connectivity peaking during different phases of animal development. In <i>C</i>. <i>elegans</i>, young larvae form mature motor circuits through a dramatic switch in GABAergic neuron connectivity, by concomitant elimination of existing synapses and formation of new synapses that are maintained throughout adulthood. We have previously shown that an increase in microtubule dynamics during motor circuit rewiring facilitates new synapse formation. Here, we further investigate cellular control of circuit rewiring through the analysis of mutants obtained in a forward genetic screen. Using live imaging, we characterize novel mutations that alter cargo binding in the dynein motor complex and enhance anterograde synaptic vesicle movement during remodeling, providing <i>in vivo</i> evidence for the tug-of-war between kinesin and dynein in fast axonal transport. We also find that a casein kinase homolog, TTBK-3, inhibits stabilization of nascent synapses in their new locations, a previously unexplored facet of structural plasticity of synapses. Our study delineates temporally distinct signaling pathways that are required for effective neural circuit refinement.</p></div

SYD-1 function requires SAX-3.

<p>(A) The <i>syd-1(ju2)</i> null mutation does not enhance guidance defects in <i>sax-3(ky123)</i> null mutants. (B) The <i>syd-1(ju2)</i> null mutation enhances guidance defects in <i>sax-3(ky200)</i> hypomorphic mutants. For experiments with the <i>sax-3(ky123)</i> null mutants, we scored only severe HSN defects (the axon never reaches the ventral nerve cord). For the <i>sax-3(ky200)</i> hypomorphic mutants, we did not observe any severe HSN axon guidance defects, but only mild defects (the axon trajectory is defective but eventually reaches the ventral nerve cord). Therefore, for experiments with the <i>sax-3(ky200)</i> hypomorphic mutants, we scored mild axon guidance defects. For all experiments, n≥200. Bracket indicates statistically significant difference, Z test for proportions (*p<0.01).</p

<i>ju1279</i> and <i>ju993</i> are novel alleles of <i>dhc-1</i> and <i>dnc-4</i>, respectively.

<p>(A) Gene structure of <i>dhc-1</i>, with <i>ju1279</i> and reference alleles <i>or195</i> and <i>js319</i> marked. <i>ju1279</i> alters the N-terminal Heavy chain domain 1, <i>or195</i> alters the coiled coil stalk and <i>js319</i> alters region D6 of the motor. (B) Representative images of DD synapses along the DNC (<i>P</i>_{<i>flp-13</i>}-SNB-1::GFP (<i>juIs137</i>)) in adult animals. Ex-DHC-1 denotes extrachromosomal copies of wild type DHC-1. Scale bar: 10 μm. (C) Quantification of synaptic puncta in the DNC of adult animals. Data are mean ± SEM; n>10 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. (D) Gene structure of <i>dnc-4</i>, with <i>ju933</i> and the reference allele <i>or633</i> also marked. (E) Quantification of synaptic puncta in the DNC (<i>P</i>_{<i>unc-25</i>}-SNB-1::GFP (<i>juIs1</i>)) of adult animals. Ex-DNC-4(+) denotes extrachromosomal copies of wild type DNC-4. Data are mean ± SEM; n>8 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. (F) Quantification of synaptic puncta in the DNC (<i>P</i>_{<i>flp-13</i>}-SNB-1::GFP (<i>juIs137</i>)) of adult animals. Animals were cultured at two different temperatures, the permissive (20^°C) and restrictive (25^°C) temperatures for <i>or633</i>, starting from late L1. Data are mean ± SEM; n>8 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ns- not significant.</p

Intragenic mutations in <i>tba-1</i> and a novel <i>tbb-2</i> mutation suppress synapse remodeling defects in <i>tba-1(gf) dlk-1(0)</i>.

<p>(A) Schematic of remodeling of DD neuron synapses. In young larvae, pre-synaptic terminals are visualized along the ventral nerve cord (VNC) using GFP- tagged synaptobrevin (SNB-1::GFP). These synapses are completely eliminated in wild type animals, to form new synapses along the dorsal nerve cord (DNC).(B) Schematic of an adult DD neuron, with the red box representing the region of interest. Representative images of DD synapses in the adult DNC of various genotypes, visualized using <i>P</i>_{<i>unc-25</i>}<i>-</i>SNB-1::GFP (<i>juIs1</i>). Scale bar: 10 μm. (C) Bright field images of a <i>tba-1(gf) dlk-1(0)</i> animal and an animal isolated following EMS mutagenesis of <i>tba-1(gf) dlk-1(0)</i>. Suppressors were isolated based on improved behavior, with a total of 8 intragenic <i>tba-1</i> mutants and 8 suppressors with mutations in genes besides <i>tba-1</i> and <i>dlk-1</i>. (D) Representative images of DD synapses in the adult DNC of various genotypes, visualized using <i>P</i>_{<i>unc-25</i>}-SNB-1::GFP (<i>juIs1</i>). Scale bar: 10 μm. (E) Quantification of synaptic puncta in the DNC of adult animals. Data are mean ± SEM; n>10 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. (F) DNC synapses in <i>tba-1(gf) dlk-1(0); tbb-2(ju1535); juIs1</i> animals that either lack or contain a rescuing transgene expressing wild type TBB-2 (Ex-TBB-2(+)).</p

<i>dhc-1(ju1279)</i> enhances anterograde transport during synapse remodeling.

<p>(A) Schematic of imaging region (black box) in the DD neuron. SVs move in both the anterograde (blue solid arrow) and retrograde directions (pink dotted arrow) during remodeling. (B, C) Quantification of: (B) number of mobile vesicles, (C) their direction of movement during remodeling for various genotypes. Data are mean ± SEM; n = no. of animals (shown on (B)). Statistics: One-way ANOVA followed by Tukey’s posttest;*p<0.05, ***p<0.001, **p<0.01, n.s.-not significant (D Model of bidirectional cargo transport during DD neuron synapse remodeling. a) In wild type animals, kinesin (red) and the dynein (blue)-dynactin (yellow) complex transport SVs in both the anterograde and retrograde directions, with more SVs moving towards the dorsal neurite (anterograde). b) <i>unc-116(ju972)</i> (black stars) modifies the MT binding domain of kinesin to enhance both anterograde and retrograde SV transport. c) Almost all SVs move in the anterograde direction in <i>dhc-1(ju1279)</i> (green star) and <i>dnc-4(ju993)</i> (red star) animals, possibly due to a disruption in the interaction between the dynein-dynactin complex and SVs.</p

The function of the <i>syd-1</i> gene in axon guidance is mediated by the SYD-1C isoform.

<p>(A) Schematic of the three isoforms of SYD-1 and location of three mutations. The <i>ju82</i> mutation is a nonsense mutation the affects the SYD-1A and SYD-1B isoforms. The <i>ju2</i> mutation is a nonsense mutation that affects all three isoforms. The <i>tm6234</i> mutation is a deletion that affects all three isoforms. (B) The SYD-1C isoform is necessary and sufficient to mediate the role of SYD-1 in axon guidance. Guidance defects in <i>unc-6</i> null mutants are enhanced by the <i>ju2</i> and <i>tm6234</i> mutations, but not by the <i>ju82</i> mutation. Guidance defects in a <i>syd-1</i>; <i>unc-6</i> double null mutant are rescued by expressing SYD-1C in the HSN neuron with an <i>unc-86</i>::<i>syd-1c</i> transgene. (C) SYD-1C binds to the cytoplasmic domain of UNC-40 fused to GST (GST::UNC-40) and also to the cytoplasmic domain of SAX-3 fused to GST (GST::SAX-3), but not to GST alone. An unrelated protein, luciferase, does not bind to GST, GST::UNC-40, or GST::SAX-3. For reference, an amount of protein (SYD-1C or luciferase) equal to 20% of the amount of protein in the binding assay was run on the gel (20% Input). HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Bracket indicates statistically significant difference, Z test for proportions (**p<0.001).</p

Kinase activity of <i>ttbk-3</i> is required for suppressing <i>tba-1(gf) dlk-1(0)</i>.

<p>(A) Gene structure of <i>ttbk-3</i>, with <i>ju978</i> and the deletion allele <i>tm4006</i> marked. (B) Representative images of DD neuron synapses along the DNC in adult animals imaged using <i>P</i>_{<i>unc-25</i>}-SNB-1-GFP (<i>juIs1</i>). Ex-Muscle-TTBK-3 and Ex-DDneuron-TTBK-3 denotes extrachromosomal copies of wild type TTBK-3 expressed under <i>myo-3</i> (muscle) and <i>flp-13</i> (DD neuron) promoters, respectively. Scale bar: 10 μm. (C) Quantification of synaptic puncta in the DNC (<i>P</i>_{<i>unc-25</i>}-SNB-1::GFP (<i>juIs1</i>)) of adult animals. Ex-TTBK-3 denotes extrachromosomal copies of wild type TTBK-3 expressed under its own promoter. Data are mean ± SEM; n>10 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest; ***p<0.001, ns- not significant. (D) Quantification of synaptic puncta in the DNC (<i>P</i>_{<i>unc-25</i>}-SNB-1::GFP (<i>juIs1</i>)) of adult animals. TTBK-3 contains a kinase domain and a C-terminal coiled-coil domain. Loss of either kinase domain activity (using kinase dead K115A and D209A mutants), or the coiled-coil domain in extrachromosomal copies of TTBK-3 (P_{<i>flp-13</i>}-TTBK-3), result in a failure to rescue <i>tba-1(gf) dlk-1(0); ttbk-3(ju978) juIs1</i> animals. Data are mean ± SEM; n>8 animals per genotype. Statistics: One-Way ANOVA followed by Tukey’s posttest;*p<0.05, ***p<0.001, ns- not significant.</p

SYD-1 promotes axon guidance by negatively regulating the MIG-2 GTPase.

<p>(A) The RhoGAP-like domain of SYD-1 can bind to the active form of MIG-2. Binding between the C-terminal RhoGAP-like domain of SYD-1 (His-SYD-1) and GST-MIG-2 was substantially enhanced after incubation with GTPγS (GST-MIG-2 + GTPγS) relative to incubation with GDP (GST-MIG-2 + GDP). No binding was observed between His-SYD-1 and GST. (B-C) SYD-1 negatively regulates MIG-2 activity. (B) The <i>syd-1(ju2)</i> null mutation enhances HSN guidance defects in partially activated <i>mig-2(gm38)</i> mutants. However, the null mutation in <i>syd-1</i> does not enhance HSN guidance defects in the fully activated <i>mig-2(gm103)</i> mutants. (C) Transgenic expression of SYD-1C suppresses axon guidance defects in the partially activated <i>mig-2(gm38)</i> mutants. HSN axon guidance was scored as defective if the axon failed to reach the ventral nerve cord. For all experiments, n≥200. Brackets indicate statistically significant difference, Z test for proportions (***p<0.0005, **p<0.005).</p