The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons

Stx3 is polarized to axons and axon tips, and Stx4 to dendrites in hippocampal neurons.

<p>(A) Localization of Stx3 and Stx4 expressed in mature neurons. Neurons were coinfected with adenoviruses expressing either myc-Stx3 (left) or myc-Stx4 (right), and GFP. Total Stx expression was determined by immunolabeling after membrane permeabilization using anti-myc antibody, and cell morphology was highlighted with anti-GFP antibody. Neurons are representative of several hundred cells. Bar, 20 μm. Inset shows magnified view of a region of dendrite (same bar for inset, 8 μm). (B) Surface and internal labeling of Stx3 and Stx4 was determined with rat anti-myc antibody, followed by membrane permeabilization, and labeling for internal Stx3 (mouse anti-myc) and GFP. Surface Stx3 was prominent in axon tips, whereas Stx4 was localized to dendritic spines, shaft and cell soma. Images are montages of 2–4 images taken at identical exposure settings. Arrowheads mark the axon, which was identified in the GFP image; arrows denote axon tips. Bars, 20 μm.</p

Mistargeting of Stx3 does not alter polarized localization of cytoskeletal proteins MAP2 (somatodendritic) or Tau (axonal).

<p>(A) Endogenous MAP2 was labeled in neurons (DIV 4) 2 days after cotransfection with Stx3, Stx3Δ38 or Stx4 together with GFP. (B) Quantification of the axon:dendrite polarity index for endogenous MAP2 and Stx constructs. MAP2 was polarized to the somatodendritic region, and was not significantly different between groups, whereas Stx3 was primarily axonal, Stx3Δ38 was throughout the neurons, and Stx4 was primarily somatodendritic. (C) Endogenous Tau was labeled in neurons (DIV 5) 2 days after cotransfection with Stx3, Stx3Δ38 or Stx4 together with GFP. (D) Quantification of the axon:dendrite polarity index for endogenous Tau. Tau was strongly polarized to the axon, and was not significantly different between groups. Bar, 20 μm.</p

Stx3 overexpression results in increased axonal length and branching.

<p>(A) DIV4 neurons coexpressing GFP in the absence (left) or presence of Stx3 (middle) or Stx4 (right). Stx3-expressing neurons had longer axons, with no difference in dendrites or in axonal width observed between conditions. To highlight morphology, images show GFP labeling of representative neurons. Scale bar, 50 μm. (B-D) Quantification of neuronal morphology showed that Stx3-expressing cells had increased total axonal length, increased number of long axonal branches, and increased length of the longest primary axon compared to control. Error bars, SEM; N = 33–35 in B, C and D; N = 183–315 in E; * P≤ 0.01. This experiment was repeated independently three times.</p

The N-terminal FMDE motif of Stx3 is required for axonal targeting.

<p>(A) Stx3 wild-type and mutant constructs are schematically depicted. Tandem myc epitope tags are present at the C-terminus (extracellular domain) to facilitate surface labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163671#pone.0163671.ref023" target="_blank">23</a>]. Stx3Δ38 contains a 38 amino acid N-terminal truncation. In Stx3_AAA, three residues of the <u>F</u>M<u>DE</u> motif of Stx3 were changed to alanine as indicated by ‘AAA’. (B) Mutant Stx3 constructs lacking the FMDE motif are mistargeted to the somatodendritic, as well as axonal plasma membrane. Neurons were coinfected with adenoviruses expressing Stx3Δ38 or Stx3_AAA and GFP, and were live surface labeled with anti-myc antibody (red). Arrowheads denote axon. Bar, 20 μm. (C) Quantification of axon:dendrite polarity index for surface (red bars) and internal (black bars) Stx3, Stx3Δ38 and Stx4 in mature hippocampal neurons. PI ratios for Stx3Δ38 and Stx4 were significantly reduced compared to Stx3. Error bars, SEM; N = 14–18; * P < 0.0001.</p

Mistargeting of Stx3 disrupts axonal polarization of NgCAM and neurexin 1 but not dendritic polarization of transferrin receptor.

<p>(A) NgCAM is targeted to the axon surface when expressed alone (with tdTomato) or in the presence of wild type Stx3, but when cotransfected with Stx3Δ38 or Stx3_AAA, NgCAM is mislocalized to the somatodendritic region together with mutant syntaxin. Neurons were cotransfected with NgCAM-GFP in the absence or presence of Stx3, Stx3Δ38, Stx3_AAA, or Stx4. After overnight expression, the cells were surface labeled for plasma membrane NgCAM (8D9 antibody), and then were permeabilized and internally labeled with anti-myc to detect total syntaxin, and anti-GFP to label total NgCAM. Arrows indicate dendrite, arrowheads denote axon; axon segments are at or near the axon tip; bar, 20 μm. (B) Quantification of axon:dendrite polarity index for surface NgCAM or TfR coexpressed with Stx constructs. NgCAM polarity index was decreased in neurons coexpressing Stx3Δ38 or Stx3_AAA, whereas TfR, a somatodendritic cargo, was somatodendritic under all conditions tested (see images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163671#pone.0163671.s004" target="_blank">S4 Fig</a>). Error bars, SEM; N = 20–21 for NgCAM, 13–14 for TfR; * P ≤ 0.0001. (C) Nrxn is targeted to the axon when expressed alone (with tdTomato), but is mislocalized to the somatodendritic region when coexpressed with Stx3 mutants. Bar, 20 μm. (D) Axon:dendrite polarity index for surface Nrxn was reduced when Nrxn was coexpressed with Stx3Δ38 or Stx3_AAA compared to control. Error bars, SEM; N = 26–30; * P<0.0001.</p

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons