<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

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

<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

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