UNC-41/Stonin Functions with AP2 to Recycle Synaptic Vesicles in <em>Caenorhabditis elegans</em>

<div><p>The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (<em>Drosophila</em> Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the <em>unc-41</em> gene, which encodes the stonin ortholog in the nematode <em>Caenorhabditis elegans</em>. Transgenic expression of <em>Drosophila stonedB</em> rescues <em>unc-41</em> mutant phenotypes, demonstrating that UNC-41 is a <em>bona fide</em> member of the stonin family. In <em>unc-41</em> mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of <em>snt-1 unc-41</em> double mutants is stronger than <em>snt-1</em> mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that <em>unc-41</em> mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in <em>apm-2</em> mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in <em>unc-41 apm-2</em> double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.</p> </div

The <i>unc-41</i> gene products are differentially expressed in the <i>C. elegans</i> nervous system.

<p>Animals expressing the <i>Punc-41A::</i>NLS-CFP (green) and <i>Punc-41B::</i>NLS-YFP (red) transgenes were imaged on a confocal microscope. An L1 larva is shown in the left column (panels A, C, and E). One of the six GABA motor neurons (DD) is indicated. The animal’s head is in the upper left of the image, and the animal is positioned with its ventral surface on the inside of the arc (scale bar is 10 µm). Shown in the right column (panels B, D, and F) is the head region of an adult hermaphrodite. The nerve ring (nr), dorsal nerve cord (dnc), and ventral nerve cord (vnc) are indicated for reference. Anterior is to the left, ventral is down, and the bar is 10 µm.</p

Phenotypic rescue of <i>unc-41</i> mutants by transgenic expression of <i>Drosophila</i> stoned B (STNB).

<p>(A) A YFP::STNB fusion protein is correctly trafficked and localized to synaptic regions. Shown is the head of a young adult hermaphrodite; anterior is to the left, and the scale bar is 10 µm. STNB is specifically associated with synaptic regions, including the nerve ring (nr) and dorsal (dnc) and ventral (vnc) nerve cords. (B) Behavioral analyses of young adult hermaphrodites expressing STNB or YFP::STNB transgenes. The strains analyzed were (from left to right) RM2086, RM2655, RM2683, and RM3644; genotypes are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095.s006" target="_blank">List S1</a>. Details of swimming and expulsion assays are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#s4" target="_blank">Materials and Methods</a>. For swimming assays, each bar represents the mean number of body bends/min for 20 individuals of each strain. For expulsion assays, each bar represents the percentage of defecation cycles ending with an enteric muscle contraction (EMC, %) for 10 individuals of each strain. Error bars indicate SEM. Asterisks denote significant difference from <i>unc-41</i> (P<0.0001).</p

Overexpression of synaptotagmin does not rescue <i>unc-41</i> mutants.

<p>(A) Images of synaptotagmin (SNT-1::GFP) under different overexpression conditions. All images were taken at identical settings. High levels of tagged synaptotagmin were observed in the nerve rings of <i>snt-1(md290)</i> or <i>unc-41(e268)</i> mutants when injected at 5 ng/µl. Scale bar is 2 µm. (B) Swimming assays. Details are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#s4" target="_blank">Materials and Methods</a>. Error bars indicate SEM. Rescue of <i>unc-41</i> mutant phenotypes was not observed with the <i>snt-1::GFP</i> construct injected at 1 or 5 ng/µl.</p

Analysis of <i>snt-1 unc-41</i> double mutants.

<p>(A) Swimming assays of wild type, <i>snt-1(md290)</i>, and <i>unc-41(e268)</i> single mutants, and <i>snt-1(md290); unc-41(e268)</i> double mutants (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#s4" target="_blank">Materials and Methods</a>). Each bar represents the mean number of body bends/min for 20 individuals of each strain; error bars indicate SEM. Comparable results were observed with <i>snt-1(md184); unc-41(e268)</i> and <i>snt-1(md290); unc-41(md152)</i> double mutants (data not shown). (B) Immunohistochemical staining for the synaptic vesicle protein UNC-17/VAChT in <i>snt-1(md290); unc-41(e268)</i> double mutants, demonstrating proper subcellular localization of this protein. Shown is a confocal image of cholinergic synapses in a body-sublateral nerve cord of a young adult hermaphrodite; anterior is to the left, and the scale bar is 10 µm.</p

The <i>unc-41</i> gene and UNC-41 proteins.

<p>(A) The <i>unc-41</i> gene consists of 12 exons spanning ∼9 kb of genomic DNA. The two promoter regions are shown in green, and several <i>unc-41</i> mutations are indicated. (B) The <i>unc-41</i> gene products are members of the stonin family. Shown are features of the UNC-41A (∼188 kDa) and B (∼160 kDa) proteins, as well as the <i>Drosophila</i> stoned B and human stonin 2 proteins. Each of these proteins possesses a central stonin-homology domain (SHD) and a C-terminal µ-homology domain (µHD); significant sequence similarity among the proteins is limited to these domains. The brown rectangles indicate proline-rich domains (defined as a sequence of ≥21 amino acids containing ≥33% prolines). Blue circles indicate NPF motifs. Three motifs, shown as triangles, interact with the α-ear domains of the AP2 complex: red triangles indicate WxxF motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Jha1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Ritter1" target="_blank">[30]</a>, orange triangles indicate DPF motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Brett1" target="_blank">[28]</a>, and the green triangle indicates an FxDxF motif <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Brett1" target="_blank">[28]</a>. The pink diamonds indicate C-terminal PDZ domain-binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040095#pone.0040095-Sheng1" target="_blank">[31]</a>. Asterisks indicate that the marked site is not conserved even among closely related species.</p

(A) Localization of UNC-41 is independent of synaptotagmin.

<p>Wild-type and <i>snt-1(md290)</i> young adult hermaphrodites were stained with antibodies to UNC-41. In both wild-type and mutant animals, UNC-41 is specifically associated with synaptic regions, including the nerve ring (nr) and dorsal (dnc) and ventral (vnc) nerve cords. (B) Synaptotagmin (SNT-1) but not UNC-41 requires the synaptic vesicle kinesin UNC-104 for transport to synapses. Wild-type and <i>unc-104(e1265)</i> animals were stained with antibodies to UNC-41 (green) and SNT-1 (red). Synaptotagmin is mislocalized to neuronal cell bodies (cb) and is no longer observed at synapses in the dorsal and ventral nerve cords in <i>unc-104(e1265),</i> whereas UNC-41 is still trafficked to synaptic regions. Slight accumulation of UNC-41 in neuronal cell bodies is also observed in <i>unc-104</i> mutants. All images are of head regions, anterior is to the left, ventral is down, and the scale bar is 10 µm.</p

<i>unc-41</i> mutants have fewer synaptic vesicles at synapses.

<p>(A) Representative images of neuromuscular junctions in ventral nerve cords of the wild type, <i>unc-41(e268)</i>, and <i>unc-41(e268); apm-2(e840)</i> strains. Scale bar is 200 nm. SV, synaptic vesicle. (B) The total number of synaptic vesicles is reduced in <i>unc-41</i> and <i>unc-41; apm-2</i> mutants. (C) The number of docked synaptic vesicles is reduced in <i>unc-41</i> and <i>unc-41; apm-2</i> mutants. (D) Vesicle diameters are slightly increased in <i>unc-41</i> and <i>unc-41; apm-2</i> mutants. All data were obtained exclusively from profiles containing a dense projection. Values in panels B, C, and D represent means; error bars indicate SEM. The number of synapses scored in Panels B and C for wild type, <i>unc-41</i>, and <i>unc-41; apm-2</i> were 38, 40, and 30, respectively for ACh synapses, and 36, 45, and 39, respectively for GABA synapses. The number of synaptic vesicles measured for the data in panel D for wild type, <i>unc-41</i>, and <i>unc-41; apm-2</i> were 733, 380, and 265, respectively for ACh synapses, and 904, 652, and 453, respectively for GABA synapses. Triple asterisks denote significant difference from wild type (P<0.001).</p

The UNC-41 proteins are localized to synaptic regions and colocalize with synaptic vesicle proteins.

<p>(A) Wild-type and <i>unc-41(e268)</i> animals were stained with antibodies to UNC-41 (green) and UNC-10/RIM (red). UNC-41 is specifically associated with synaptic regions: heavy punctate staining is observed in the nerve ring (nr), and the dorsal (dnc) and ventral (vnc) nerve cords. The UNC-41 proteins are absent in <i>unc-41(e268)</i> animals. Head region, anterior is to the left, ventral is down, and the bar is 10 µm. (B) Magnified view of anterior sublateral (SAB) nerve cords stained with antibodies to UNC-41 (green), synaptotagmin (SNT-1; red), and UNC-10/RIM (blue). UNC-41 staining overlaps with SNT-1, but does not colocalize significantly with UNC-10/RIM. Scale bar is 5 µm.</p