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First published online 20 May 2003
doi: 10.1242/jcs.00467

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Ectopic expression of syntaxin 1 in the ER redirects TI-VAMP- and cellubrevin-containing vesicles

¹ Membrane Traffic and Neuronal Plasticity, INSERM U536, Institut du Fer-à-Moulin, 75005 Paris, France
² Morphogenesis and Cell Signaling, CNRS UMR 144, Institut Curie, 75005 Paris, France

View larger version (72K): [in a new window]   Fig. 1. Cognate v-SNAREs were mistargeted in HeLa cells expressing syntaxin 1. (A) HeLa cells transfected with syntaxin 1 were fixed and double stained for syntaxin 1 and for the endogenous ER marker calreticulin. Insets show confocal images acquired at high magnification of a syntaxin-1-expressing cell. Note the colocalization between syntaxin 1 and calreticulin. (B,C) HeLa cells transfected with syntaxin 1 or co-transfected with syntaxin 1 and nSec1 as indicated were fixed and double stained for syntaxin 1 and endogenous TI-VAMP, cellubrevin (Cb) or endobrevin (Eb). Transfected cells are marked with an asterisk. A non-transfected cell in C (low panel) is indicated with a triangle. Note that TI-VAMP vesicular staining was lost in cells expressing syntaxin 1 alone but not in those co-expressing syntaxin 1 and nSec1. In syntaxin-1-expressing cells the perinuclear enrichment characteristic of cellubrevin (arrows) was also lost. By contrast, endobrevin vesicular staining was unaffected. Bar, 6 µm. Bar in the inset, 2.5 µm.

View larger version (133K): [in a new window]   Fig. 4. SNAP-23 was redistributed to the ER in cells expressing syntaxin 1. HeLa cells transfected with syntaxin 1 or co-transfected with syntaxin 1 and nSec1 as indicated were fixed and double stained for syntaxin 1 and endogenous SNAP-23 (A) or the plasma membrane marker Na+/K+ ATPase (B). Note the colocalization of SNAP-23 and ectopic syntaxin 1 (arrowhead) and the absence of SNAP-23 from the plasma membrane in cells expressing syntaxin 1 alone and the plasma membrane staining of SNAP-23 in non-transfected cells or in cells co-transfected with syntaxin 1 plus nSec1 (arrows). By contrast, endogenous Na+/K+ ATPase is not relocalized from the plasma membrane in syntaxin-1-expressing cells. Bar, 7 µm.

View larger version (37K): [in a new window]   Fig. 6. Ectopic expression of syntaxin 1 did not affect transferrin recycling. HeLa cells were transfected with syntaxin 1 (upper panels) or co-transfected with syntaxin 1 and nSec1 (lower panels). 24 hours later cells were allowed to internalize transferrin for 1 hour at 37°C, washed and either fixed immediately (uptake) or incubated for a further 1 hour at 37°C before fixation (release). Notice that both the uptake and the release of transferrin were comparable in both kinds of transfected cells and indistinguishable from non-transfected cells, independently of the intracellular distribution of syntaxin 1. Bar, 5 µm.

View larger version (71K): [in a new window]   Fig. 8. Ectopic expression of syntaxin 1 altered the distribution and trafficking of TI-VAMP vesicles' cargo CD63. (A) (Upper panels) Non-transfected HeLa cells double stained for endogenous TI-VAMP and CD63. (Lower panels) HeLa cells transfected with syntaxin 1 were fixed and stained for syntaxin 1 and CD63 24 hours after transfection. Note the almost complete colocalization between TI-VAMP and CD63 in non-transfected cells. In cells transfected with syntaxin 1 (asterisk) the CD63 typical punctate staining was lost and instead the nuclear envelope (arrow) was labeled. Insets show confocal images acquired at high magnification of a syntaxin-1-expressing cell. (B) HeLa cells transfected with syntaxin 1 or with syntaxin 1 plus nSec1 were incubated for 1 hour at 4°C with anti-CD63 antibodies prior to fixation and processing for immunofluorescence with anti-syntaxin 1 antibodies. Note the plasma membrane binding of the anti-CD63 antibody in cells co-transfected with syntaxin 1 plus nSec1, which is indistinguishable from non-transfected cells. By contrast, cells expressing and retaining syntaxin 1 in the ER did not bind to anti-CD63 antibodies. Bar, 6 µm. Bar in the inset, 2 µm.

View larger version (119K): [in a new window]   Fig. 2. Colocalization of GFP-TIVAMP and GFP-cellubrevin but not of GFP-endobrevin with syntaxin 1 in the ER of syntaxin-1-expressing cells. MDCK cells stably expressing GFP-TIVAMP, GFP-cellubrevin (GFP-Cb) or GFP-endobrevin (GFP-Eb) were fixed 24 hours after transfection with syntaxin 1. Cells were stained for syntaxin 1 (red) and calreticulin (blue). Insets show confocal images acquired at high magnification of the same transfected cells shown in the lower magnification panels. GFP-TIVAMP and GFP-Cb lost their characteristic vesicular pattern and instead colocalized with calreticulin in syntaxin-1-expressing cells, as shown by the triple colocalization in the merge panels (light pink). GFP-Eb pattern was not affected by syntaxin 1 expression, as indicated by the colocalization of syntaxin 1 and calreticulin in syntaxin-1-expressing MDCK cells, but not of GFP-Eb, in the merge panel (magenta/purple). Bar in low magnification panels, 7 µm; bar in high magnification insets, 4 µm.

View larger version (32K): [in a new window]   Fig. 3. TI-VAMP and cellubrevin interacted specifically in the ER with ectopic syntaxin 1. (A) HeLa cells were either co-transfected with GFP-Cb and Stx1 (1), or transfected with each cDNA alone (2,3), as indicated. 24 hours after transfection, cells were lysed and the extract from co-transfected cells was immediately immunoprecipitated (1), whereas extracts from cells transfected with only GFP-Cb or Stx1 were mixed and then immunoprecipitated (2+3) with the antibodies indicated. (B) HeLa cells were transfected with syntaxin 1 (1) or co-transfected with syntaxin 1 and nSec1 (2). 24 hours after transfection, cells were lysed and processed for immunoprecipitation with either anti-syntaxin 1 mouse monoclonal antibody (1 and 2) or with control mouse immunoglobulins (1C and 2C). Proteins were resolved by SDS-PAGE, and western blots were probed with the antibodies indicated. Note that cellubrevin, SNAP-23 and TI-VAMP but not VAMP4, endobrevin or Vti1b were recovered in the syntaxin 1 immunoprecipitate. SM, starting material.

View larger version (35K): [in a new window]   Fig. 5. Inhibition of TIVAMP-GFP and Cb-GFP transport to the plasma membrane in cells expressing syntaxin 1. HeLa cells were transfected with either TIVAMP-GFP or Cb-GFP alone (upper panels) or co-transfected with syntaxin 1 (lower panels), as indicated. 24 hours after transfection cells were incubated with anti-GFP antibodies for 1 hour at 37°C and processed for immunofluorescence. Note that when expressed alone, TIVAMP-GFP and Cb-GFP displayed their typical staining patterns and efficiently took up the anti-GFP antibody from the medium. However, when co-expressed together with syntaxin 1, TIVAMP-GFP and Cb-GFP were both retained in the ER and their expression at the plasma membrane and endocytosis was inhibited. Bar, 6 µm.

View larger version (72K): [in a new window]   Fig. 7. TI-VAMP relocalization in syntaxin-1-expressing cells was microtubule dependent. (A) MDCK cells stably expressing GFP-TIVAMP incubated with or without 5 µM nocodazole for 1 hour were fixed and stained for -tubulin. Note that in the presence of nocodazol, microtubules were completely disrupted. (B) MDCK cells stably expressing GFP-TIVAMP incubated with or without 5 µM nocodazole for 1 hour prior to transfection with syntaxin 1 were fixed and stained for syntaxin 1 8 hours after transfection. In control conditions (Cont), the expression of syntaxin 1 and its retention in the ER induced a relocalization of GFP-TIVAMP towards syntaxin-1-positives structures in the ER (arrows). By contrast, in the absence of functional microtubules (+Noc) the localization of GFP-TIVAMP in transfected cells was indistinguishable from non-transfected cells. Bar, 4 µm.

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