Role of endosome recycling pathway in NGF-induced neurite extension. (A) PC12 cells expressing GFP (white) or GFP plus BoNT E LC (black) were treated with NGF for 2 d to induce neurite outgrowth. Neurite length in transfected cells was measured and grouped into the indicated categories. (B) PC12 cells expressing ARF6^WT (white), ARF6^Q67L (black), or ARF6^T27N (gray) were treated with NGF for 2 d to induce neurite outgrowth. (C) PC12 cells expressing ARF6^WT or ARF6^Q67L were treated with NGF for 2 d to induce neurite outgrowth. SNAP25 (green) and ARF6 (red) were localized by immunofluorescence. Arrowheads indicate examples of ARF6 colocalization with SNAP25. Cells expressing high levels of ARF6^Q67L (bottom row) exhibited vacuoles containing SNAP25 and very short neurites.

BoNT E expression destabilizes an endosomal SNARE complex. (A) PC12 cells were transfected with the indicated plasmids and SNAP25 immunoprecipitates were prepared from detergent lysates for analysis by Western blotting for myc-syntaxin 13. myc-Syntaxin 13 in SNAP25 immunoprecipitates decreased in cells expressing BoNT E LC. (B) PC12 cells were transfected with the indicated plasmids and myc-syntaxin 13 immunoprecipitates were prepared from detergent lysates for analysis by Western blotting for VAMP 2-GFP. VAMP 2-GFP in myc-syntaxin 13 immunoprecipitates was decreased in BoNT E LC-expressing cells but was restored in cells coexpressing GFP-SNAP25 D179K. (C) PC12 cells transfected with plasmids encoding VAMP 2-GFP, myc-syntaxin 13, and BoNT E LC as indicated were fixed for staining with myc and SNAP25 antibodies. VAMP 2 (green) colocalized with syntaxin 13 (red) and SNAP25 (cyan) in the perinuclear RE in control cells (top), whereas syntaxin 13 was dispersed to a peripheral location away from perinuclear VAMP 2 in BoNT E LC-expressing cells (bottom).

Summary of the trafficking itinerary of SNAP25. SNAP25 is internalized into tubulovesicular endosomes from the plasma membrane by a dynamin-independent, ARF6-regulated pathway (right), which is distinct from the clathrin-dependent pathway of endocytosis (left) (Aikawa et al., 2006). Expression of ARF6^T27N blocks SNAP25 internalization, whereas ARF6^Q67L expression enhances SNAP25 internalization but blocks merger of SNAP25 endosomes into SEs. SNAP25 endosomes lack syntaxin 1 but merge with syntaxin 13 in the SE (Aikawa et al., 2006). Tubular elements derived from the SE either coalesce to form the RE compartment or fuse with a preexisting RE compartment. The trafficking of cargo from peripheral SEs to the perinuclear RE requires intact SNAP25 and is inhibited by BoNT E. Syntaxin 13/SNAP25-containing SE elements encounter VAMP 2 in the RE to form SNARE complexes consisting of SNAP25/syntaxin 13/VAMP 2.

BoNT E expression inhibits the transit of syntaxin 13 to the VAMP 2-positive recycling endosome. (A and B) PC12 cells transfected with plasmids encoding GFP-Rab5, GFP-Rab11, or BoNT E LC as indicated were stained with rabbit SNAP25 and mouse TGN38 antibodies. Arrowheads indicate examples of overlap of SNAP25 (red) with Rab5 (green) or Rab11 (green) in cells expressing BoNT E. (C) PC12 cells transfected with plasmids encoding myc-syntaxin 13, GFP-SNAP25, or GFP-SNAP25 D179K and BoNT E LC as indicated were stained with monoclonal myc antibody. Arrowheads indicate examples of colocalization of syntaxin 13 (red) with SNAP25 (green). The perinuclear distribution of syntaxin 13 disrupted by BoNT E LC was restored by expression of SNAP25 D179K. (D) Hippocampal neurons transfected with plasmids encoding syntaxin 13-GFP (green) and BoNT E LC as indicated were stained with monoclonal SNAP25 antibody (red). (E) The immunofluorescence intensity of syntaxin 13 in images similar to those in C was quantitated in 50 randomly selected cells, and the percentage of perinuclear to total fluorescence was plotted as mean values ± SD; *** corresponds to p < 0.001.

BoNT E expression inhibits the transit of SNAP25 to the recycling endosome. (A) Representative Western blot shows the effect of BoNT E LC expression on endogenous SNAP25 (marked End, bottom) and on GFP-SNAP25 or GFP-SNAP25 D179K (top). (B) Representative Western blot shows the effect of BoNT E LC expression on endogenous SNAP25 (bottom) or rat or human GFP-SNAP23 (top) detected with monoclonal GFP (top) or SNAP25 (bottom) antibodies. Asterisks designate the cleaved product SNAP25 (1-180). GFP-D179K SNAP25, rat SNAP-23 and human SNAP-23 were not cleaved by BoNT E. (C) PC12 cells were transfected with plasmids encoding GFP-SNAP25, GFP-SNAP25 D179K, or GFP-SNAP23 with or without BoNT E LC as indicated. The distribution of GFP-SNAP25 in a perinuclear RE pool was altered by coexpression of BoNT E (arrowheads). (D) The fluorescence intensity of GFP-SNAP25 or GFP-SNAP23 was quantitated in 50 randomly selected cells, and the percentage of perinuclear to total fluorescence was plotted as mean values ± SD; *** and * indicate p < 0.001 and p < 0.05, respectively, for comparison with control by t test. The perinuclear distribution of SNAP25 was restored in cells expressing GFP-SNAP25 D179K.

SNAP25 is not required for Rab5-dependent homotypic early endosome fusion. (A) Myc antibody immunoprecipitates were prepared from detergent lysates of cells transfected with indicated plasmids and analyzed by Western blotting for VAMP 2-GFP. Coimmunoprecipitation of VAMP 2-GFP with myc-syntaxin 13 was decreased by BoNT E LC expression even in Rab5^Q79L-expressing cells. (B) PC12 cells transfected with plasmids encoding myc-syntaxin 13, GFP-Rab5^Q79L without (top) or with (bottom) BoNT E LC (bottom) were stained with monoclonal myc antibody. Syntaxin 13 (red) localized to Rab5^Q79L-promoted SE vacuoles (green) in control and BoNT E LC-expressing cells (arrowheads). (C) PC12 cells transfected with plasmids encoding ARF6^Q67L and BoNT E LC were stained with SNAP25 antibody. SNAP25 localization (green) to ARF6^Q67L-promoted vacuoles (red) was not prevented in cells expressing BoNT E LC. (D) PC12 cells transfected with plasmids encoding GFP-Rab5^Q79L (green) with either control or ARF6^Q67L- or ARF6^T27N-encoding plasmids were fixed and stained with monoclonal SNAP25 antibody (red). Arrowheads indicate the Rab5^Q79L-induced SE vacuole. (E) PC12 cells transfected with plasmids encoding GFP-SNAP25 (green), human transferrin receptor and ARF6^Q67L or ARF6^T27N as indicated were incubated with Alexa 568-conjugated Tf (red) for 30 min. Alexa 568-Tf accumulated in the perinuclear RE in control cells but not in cells expressing ARF6^Q67L or ARF6^T27N (arrowheads).

Endosome trafficking is inhibited in SNAP25-deficient PC12 cells. (A) PC12 cells transfected with GFP-SNAP25 and control or SNAP25 shRNA plasmids 1 or 2 were subjected to Western blotting with GFP, SNAP25, or Rab 3A antibodies as indicated. SNAP25 shRNA 2 expression extensively down-regulated GFP-SNAP25 and down-regulated endogenous SNAP25 levels in proportion to transfection efficiencies without affecting Rab 3A levels. (B) Densitometry was used to quantitate relative levels of endogenous (end) SNAP25 and GFP-SNAP25 in A. (C) PC12 cells transfected with SNAP25 shRNA 2 plus plasmid encoding syntaxin 13-GFP were stained with SNAP25 antibody. Syntaxin 13-GFP was dispersed into peripheral endosomes in cells lacking SNAP25. (D) PC12 cells were transfected with control or SNAP25 shRNA 2 plasmid and with plasmids encoding syntaxin 13-GFP (green) and transferrin receptor. Cells were imaged after a 30-min incubation with Alexa568-conjugated Tf (red). Bars, 10 μm. (E) The fluorescence intensity of Tf was quantitated in 50 randomly selected cells (similar to D), and the percentage of perinuclear to total fluorescence was plotted as mean values ± SD, with *** denoting p < 0.001 for cells transfected with SNAP25 shRNA 2 versus control plasmid. (F) The fluorescence intensity of syntaxin 13-GFP was quantitated in 50 randomly selected cells (similar to D), and the percentage of perinuclear to total fluorescence was plotted as mean values ± SD, with *** denoting p < 0.001 for cells transfected with SNAP25 shRNA 2 versus control plasmid.

BoNT E inhibits the transit of Tf and CTxB to the recycling endosome. (A) PC12 cells expressing syntaxin 13-GFP, human Tf receptor, and BoNT E LC where indicated were incubated with Alexa 568-conjugated Tf for 5, 15, and 30 min. Extensive colocalization of syntaxin 13-GFP (green) with Tf (red) was observed (arrowheads). In cells expressing BoNT E LC, Tf and syntaxin 13-GFP were retained in peripheral endosomes. (B) The fluorescence intensity of Alexa568-conjugated Tf was quantitated in 50 randomly selected cells, and the percentage in the perinuclear RE to total fluorescence was plotted as mean values ± SD; *** denotes p < 0.001 and *p < 0.05 for comparison with control by t test. (C) PC12 cells expressing GFP-SNAP25, human Tf receptor, and BoNT E LC were incubated with Alexa568-conjugated Tf. Arrowheads indicate examples of colocalization of Tf (red) with GFP-SNAP25 (green). (D and E) PC12 cells expressing GFP-Rab5^Q79L or GFP-SNAP25 or GFP-SNAP25 D179K were incubated with Alexa643-conjugated CTxB on ice for 30 min, washed, and incubated at 37°C for 15 min to internalize the CTxB. CTxB localizes to the vacuolar SE compartment in Rab5^Q79L-expressing cells (D, top). CTxB uptake into a perinuclear RE/TGN compartment is inhibited in BoNT E LC-expressing cells (E, top) and is restored in cells expressing GFP-SNAP25 D179K (E, bottom). Arrowheads indicate examples of colocalization of SNAP25-GFP (green) and CTxB (cyan). (E) The fluorescence intensity of CTxB was quantitated in 50 randomly selected cells, and the percentage of perinuclear to total fluorescence was plotted as mean values ± SD, with *** denoting p < 0.001 for BoNT E-expressing versus control cells.