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First published online 19 August 2008
doi: 10.1242/jcs.032029

Journal of Cell Science 121, 2983-2991 (2008)
Published by The Company of Biologists 2008
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The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

Emanuele Cocucci1,*, Gabriella Racchetti2, Marjan Rupnik3 and Jacopo Meldolesi1,2,4,5,{ddagger}

1 Vita-Salute San Raffaele University, Center of Excellence in Cell Development, via Olgettina 58, 20132 Milan, Italy
2 Scientific Institute San Raffaele, via Olgettina 60, 20132 Milan, Italy
3 Institute of Physiology, Faculty of Medicine, University of Maribor, Slomskov, trg 15, 2000 Maribor, Slovenia
4 National Institute of Neurosciences, Italy
5 IIT Network, Research Unit of Molecular Neuroscience, via Olgettina 58, 20132 Milan, Italy


Figure 1
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  		Fig. 1. Expression of SNAREs and annexin-2 in cells. For SDS-PAGE, homogenate protein (40 µg) from PC12-27 cells was loaded in all slots. The proteins shown, exhibiting different sizes, were aligned for the sake of space. Notice the almost complete absence of the classical SNAREs of neurotransmitter release (VAMP2, Stx1a and SNAP25) and the abundance of other SNAREs, in particular of VAMP3 and VAMP4, of the three syntaxins and of SNAP23. Annexin-2, which is abundant in PC12-27 cells compared with many other cells, is shown to the extreme right (A2).

 

Figure 2
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  		Fig. 2. Distribution of VAMPs and annexin-2 in PC12-27 cells, relative to the enlargeosome marker desmoyokin. The four series of deconvolved confocal images, obtained from PC12-27 cells that were permeabilized before immunolabelling, illustrate: (left) the distribution of VAMP4, VAMP7 and VAMP8 (A-C, respectively), and of annexin-2 (D); (middle) that of desmoyokin; (right) the merge of the two corresponding images. The VAMP4 results, obtained with a monoclonal antibody raised in our laboratory, are representative also of those obtained with another monoclonal antibody and with two polyclonal antibodies. All the VAMP proteins and annexin-2 appear spread in the cytoplasm, with VAMP4 concentrated preferentially in the perinuclear area (TGN) and annexin-2 showing a positivity at the periphery of the cell. The merge images reveal a significant colocalization of desmoyokin in the cytoplasmic punctae that were positive for VAMP4 and annexin-2 (A,D). No significant level of colocalization is visible for VAMP7 and VAMP8 (B,C). Scale bar: 5 µm.

 

Figure 3
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  		Fig. 3. Distribution of syntaxins and SNAP23 in PC12-27 cells, relative to the enlargeosome marker desmoyokin. The four series of deconvolved confocal images, obtained from PC12-27 cells permeabilized before immunolabeling, illustrate: (left) the distribution of Stx4, Stx6 and Stx13 (A-C, respectively) and of SNAP23 (D); (middle) that of desmoyokin; (right) the merge of the two corresponding images. All the syntaxins and SNAP23 decorate punctae that are spread in the cytoplasm, with different patterns. In all cases the co-labelling of these SNAREs with desmoyokin is very low and below significance (<10%), except for SNAP23 in the area near the plasma membrane. In addition, Stx6 and also Stx13 show intense labelling in the perinuclear area. Notice the intense signalling of Stx13 and SNAP23 at the periphery of the cells. Scale bar: 5 µm.

 

Figure 4
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  		Fig. 4. Enlargeosome exocytosis in PC12-27 cells that were microinjected with antibodies. (A-D) [Ca2+]i and surface-capacitance traces in patched-clamped cells. Photolysis of the NP-EGTA caged Ca2+ compound induced similar [Ca2+]i rises in non-microinjected (A) and microinjected (B-D) cells (bottom traces). The capacitance surface-expansion responses (upper traces) were similar in non-microinjected cells (A) and in cells microinjected with monoclonal anti-ChgB antibody; ChgB is not expressed by PC12-27 cells. (B) By contrast, these responses were greatly reduced in cells that were microinjected with antibodies against SNAP23 (C) and annexin-2 (D). The time and % capacitance increase bars in A are valid also for B-D. (E) Averages ± s.d. of the capacitance-increase data in groups of 10-15 cells. (F-M) The enlargeosome exocytic responses, revealed by surface immunolabeling by desmoyokin (red), that were induced by ionomycin treatment (3 µM, 5 minutes) in cells microinjected with anti-ChgB, anti-SNARE or anti-annexin-2 antibodies (labelled in green) are shown. The cells that exhibit no green labelling were non-microinjected. Enlargeosome exocytoses were approximately the same in non-microinjected cells and in those microinjected with antibodies against ChgB (F), VAMP7 (J), VAMP8 (K) and Stx4 (L); they were greatly reduced in the cells microinjected with antibodies against SNAP23 (G), annexin-2 (H), VAMP4 (I) and Stx6 (M). Scale bar: 5 µm. (N) Averages ± s.d. of the data obtained for the immunolabeling of surface desmoyokin in groups of 18-22 cells stimulated with ionomycin. The indications specified in the bars refer to the microinjected antibodies. (O) Averages ± s.d. of the data obtained for the immunolabeling of surface desmoyokin (d/A) in groups of 18-22 cells, resting (NT) or stimulated with ionomycin (iono) without or with pre-treatment with botulinum toxin E (BtxE).

 

Figure 5
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  		Fig. 5. SNARE downregulation – differential effects on enlargeosome exocytosis. (A) Western blots of VAMP4, Stx6 and VAMP8 in PC12-27 (left) and SH-SY5Y (right) cells, transfected (+) or not (–), with siRNAs. Notice the decrease (by ~50-60%) in the levels of the respective SNARE proteins in the PC12-27 populations transfected with siRNAs for VAMP4, Stx6 and VAMP8; in SH-SY5Y cells, which were all transfected with VAMP4 siRNA, notice the decrease of VAMP4 (by 40%), with no change in VAMP2 levels. The top band (calnexin) is shown to certify that equal protein loads were seeded in the slots. (B,D,F,H) The correlation between surface desmoyokin (d/A), documenting enlargeosome exocytosis, and the total level of individual SNAREs in single PC12-27 (B,D,F) and SH-SY5Y (H) cells non-transfected (blue signs) or transfected (red signs) with the siRNAs specific for the indicated SNAREs and then stimulated with ionomycin (3 µM, 1 minute) is shown. After fixation, the cells were first immunolabelled for surface desmoyokin, then permeabilized and immunolabelled for VAMP4 (circles, B and H), Stx6 (triangles, D) and VAMP8 (squares, F). (B) Notice that, in the population of PC12-27 cells exposed to VAMP4 siRNA (red circles), cells with levels of the SNARE that were distinctly lower than those of the untreated cells (blue circles) (by 90.3±1.7% on average in a group of 20 cells) exhibited a parallel decrease of enlargeosome exocytoses, revealed by the low level of surface appearance of desmoyokin (decreased by 89.9±1.8% compared with non-transfected cells). (H) A similar, although less intense, result was obtained in the SH-SY5Y cells transfected with the human siRNA (–75.2±5.8% for SNARE level and –64.8±7.5% for desmoyokin). (D,F) In PC12-27 cells downregulated for Stx6 (red triangles), the results were closer to those obtained in the same cells with the siRNA for VAMP4 (–79.9±5.6 for SNARE level and –84.6±5.6% for desmoyokin, D), whereas in those downregulated for VAMP8 (red squares, F), the drop in the level of SNARE (–94.7±1.9%) was dissociated from the surface appearance of desmoyokin, which was almost unchanged (–7.8±7.6%). (C,E,G,I) Examples of PC12-27 (C,E,G) and SH-SY5Y (I) cells in the populations of B, D, F and H, i.e. exposed to the siRNAs of VAMP4, Stx6 and VAMP8 and then stimulated with ionomycin, chosen because of their high (C,E,G,I) and low (C',E',G',I') labelling for the three SNAREs. Notice the clear desmoyokin surface labelling of C, E, G and I (yellowish in C and I because of the co-discharge of VAMP4) and its lack in C', E' and I' but not in G'. Scale bar: 5 µm.

 

Figure 6
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  		Fig. 6. Colocalization of VAMP4 and desmoyokin in PC12-27 cells transfected with VAMP4-EGFP. (A-D) Cells transfected with VAMP4-EGFP and immunolabelled in red by anti-GFP and anti-desmoyokin antibodies. Scale bar: 5 µm. (A'-D') The enlargements of the boxes in A-D are shown. The cells in A and A' were fixed while at rest and surface immunolabelled with anti-EGFP without permeabilization. Notice the almost complete lack of red immunolabeling at the surface. The cells in B and C, also non-permeabilized, were fixed after stimulation with ionomycin (3 µM, 5 minutes) and then decorated with anti-GFP and anti-desmoyokin, respectively. The yellowish labelling at the surface documents the incorporation of VAMP4 in the plasma membrane (B') and its colocalization with desmoyokin (C'). (D,D') PC12-27 cells were stimulated, permeabilized and immunolabelled for desmoyokin. Notice that the juxtanuclear EGFP labelling (the TGN) is green, whereas many punctae scattered in the cytoplasm in the proximity of the cell surface are yellowish, documenting the colocalization of VAMP4 and desmoyokin. Only a few green and red punctae are visible in the cytoplasm and also at the surface.

 

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© The Company of Biologists Ltd 2008