SNAP25 internalization is dynamin independent and F actin dependent. (A) PC12 cells expressing GFP-SNAP25 (top row) or GFP-SNAP25 and ARF6^Q67L (bottom row) were incubated with Texas-Red 10 kDa dextran for 5 min. Arrowheads indicate the colocalization of GFP-SNAP25 (green) with dextran (red) in peripheral endosomes (top) or the accumulation of dextran in the SNAP25-containing vacuole induced by ARF6^Q67L expression (bottom). (B) PC12 cells expressing dynamin wild type (top row) or dynamin K44A mutant (bottom row) and GFP-SNAP25 (green) were incubated with Alexa 568-conjugated Tf (red) for 30 min. Tf uptake and perinuclear SNAP25 were quantitated in images of 50 randomly selected cells. Expression of dynamin K44A inhibited Tf uptake without affecting perinuclear SNAP25. (C) PC12 cells expressing GFP-SNAP25 (green) were incubated with CTxB-Alexa 648 (red) for 5 or 15 min. There was little colocalization of CTxB with SNAP25 in peripheral endosomes in 5-min incubations (top row), whereas CTxB colocalized extensively with GFP-SNAP25 in the perinuclear region in 15-min incubations (bottom row, arrowheads). (D) PC12 cells expressing GFP-SNAP25 (green) and ARF6^Q67L were fixed for staining with Alexa 568-phalloidin (red). Projected images indicated that the SNAP25-containing vacuole is coated with F-actin (arrowheads). (E) Top row, PC12 cells expressing ARF6^WT-GFP (green) were treated with 1 μM latrunculin A for 30 min, fixed, and stained with monoclonal SNAP25 antibody (red). SNAP25 accumulated on ARF6-positive tubular invaginations from the plasma membrane (arrowheads). Bottom row, PC12 cells expressing GFP-SNAP25 (green) were treated with latrunculin A for 10 min and incubated with Alexa 568-conjugated Tf for 30 min (red). Latrunculin A did not affect Tf uptake but blocked internalization of SNAP25. (F) Intracellular SNAP25 was quantitated in 50 randomly selected cells after treatment with 1 μM latrunculin A for indicated times. The ratio of intracellular to total immunofluorescence × 100% was plotted as mean values (•). Solid line, which corresponds to a theoretical curve based on the rate constant for SNAP25 recycling back to the plasma membrane (k₁ = 2.86% min^-1; see Figure 6) and the assumption that SNAP25 internalization was blocked (k₂ = 0), fit well to experimental data points (r = 0.975).

SNAP25-containing endosomes merge with Tf-positive endosomal compartments. (A) PC12 cells expressing GFP-SNAP25 (green) and human Tf receptor were incubated with Alexa 568-conjugated Tf (red) for indicated times. Arrowheads indicate the colocalization of GFP-SNAP25 and Tf. Bar, 10 μm. (B) PC12 cells expressing Rab5^Q79L-GFP (green) were fixed and immunostained for endogenous SNAP25 (red). Arrowheads indicate the accumulation of endogenous SNAP25 on Rab5^Q79L-positive endosomes. PC12 cells expressing myc-syntaxin 13 (red) and ARF6^Q67L (C–E) or ARF6^T27N (F) were fixed and stained for endogenous SNAP25 (green). (C) There was significant colocalization of SNAP25 and syntaxin 13 in the perinuclear region. (D) Near the ventral surface of the cells, some of SNAP25 endosomes contained syntaxin 13 (arrowheads), whereas other SNAP25 endosomes did not contain syntaxin 13 (circles). (E) In cells expressing higher levels of ARF6^Q67L, SNAP25 was trapped in an endosomal vacuole where it segregated from syntaxin-13-positive endosomes. (F) ARF6^T27N expression interfered with the internalization of SNAP25 and little colocalization of syntaxin 13 with SNAP25 was seen in the perinuclear region. (G) Expression of ARF6^Q67L and ARF6^T27N mutants alter the coimmunoprecipitation of syntaxin 13 with SNAP25. SNAP25 antibody immunoprecipitates were prepared from detergent lysates of cells expressing myc-syntaxin 13 and ARF6^WT, ARF6^Q67L, or ARF6^T27N. Immunoprecipitates were analyzed by Western blotting with c-myc antibody to quantitate myc-syntaxin 13. Mean values with SEM are shown for three experiments. Coimmunoprecipitated myc-syntaxin 13 was significantly (p < 0.05) reduced in ARF6^Q67L- and ARF6^T27N-expressing cells.

Recovery after photobleaching defines the kinetics of SNAP25 internalization and recycling. (A) PC12 cells expressing GFP-SNAP25 (top) or ARF6-GFP (bottom) were treated with 10 μg ml^-1 CHX for 120 min and immunostained for TGN-38 (red) or SNAP25 (red) respectively. (B) Quantitation of relative fluorescence intensity of intracellular compartment of endogenous SNAP25 after treatment with 10 μg ml^-1 CHX for indicated times. Fifty randomly selected cells were quantitated for each time point. (C) Photobleaching of intracellular GFP-SNAP25. Cells expressing GFP-SNAP25 were incubated with CHX for 30 min, and the intracellular compartment (red box) was bleached by the 488-nm laser. Z-section images were collected every 5 min for the next 60 min. (D) Time course of recovery of intracellular GFP-SNAP25 after photobleaching. Plot shows mean relative fluorescence intensity for perinuclear region (•) on three confocal slices near the middle of cells from a typical experiment. Solid line is drawn from model using indicated values of k₁ and k₂. (E) Photobleaching of intracellular pool of GFP-SNAP25 (red box) in CHX-treated cells expressing ARF6^Q67L. (F) Time course of recovery of intracellular GFP-SNAP25 after photobleaching in ARF6^Q67L-expressing cells is shown as increase in mean relative fluorescence intensity (•) on three confocal slices near the middle of cells from a typical experiment. Solid line is drawn from model using indicated values of k₁ and k₂. (G) Two compartment model for SNAP25 trafficking.

SNAP25 is constitutively internalized from the plasma membrane into ARF6-positive tubular vesicles. (A) PC12 cells expressing ARF6^WT-GFP (green) were fixed for immunostaining with a SNAP25 mAb (red). Confocal images shown are projected sections (top row) or merges of optical sections for bottom, middle, and top portions of the cell (middle). Vertical (X/Z) sections from separate fields are shown (bottom). Arrowheads indicate the colocalization of endogenous SNAP25 and ARF6^WT-GFP. (B) Images obtained in a real-time series of live PC12 cells (CHX-treated) expressing GFP-SNAP25 recorded every 30 s. Boxed areas are enlarged to show SNAP25-containing vesicles pinching off (circled). Bars, 5 μm. Cells expressing normal (C) or high (D) levels of ARF6^Q67L-GFP or expressing ARF6^T27N-GFP (E) were immunostained with a monoclonal SNAP25 antibody (red). Projected images and vertical sections (X/Z) from confocal microscopy are shown. Arrowheads indicate invaginations in which ARF6^Q67L and SNAP25 colocalization was apparent. Asterisk in E (X/Z) indicates absence of ARF6^T27N and SNAP25 colocalization in tubular invaginations. Bars, 10 μm. (F) Quantitative analysis of intracellular SNAP25 in randomly selected cells expressing ARF6 and its mutants. The ratio of intracellular to total SNAP25 immunofluorescence × 100% was plotted as mean values (with SEM; asterisks indicate p < 0.001).

Plasma membrane SNAP25 colocalizes with PIP₂ and ARF6. (A) SNAP25 (red) and PIP₂ (green) colocalize in permeable PC12 cells. Permeabilized cells were incubated with Mg-ATP and rat brain cytosol for 30 min, fixed and stained with monoclonal PIP₂ antibody (green) and polyclonal SNAP25 antibody (red). Middle and bottom confocal sections of cells are shown. Arrowheads indicate examples where SNAP25 and PIP₂ puncta colocalize. (B) Immunoreactive PIP₂ was detected in permeable PC12 cells expressing ARF6^WT-GFP (top row) or ARF6^Q67L-GFP (bottom row). Arrowheads indicate colocalization of immunoreactive SNAP25 (red), PIP₂ (blue), and ARF6-GFP proteins (green). (C) Cells expressing a PLCδ₁-PH-GFP fusion protein (green) were treated with 1 μM latrunculin A for 30 min, fixed, and stained with a monoclonal SNAP25 antibody (red). Arrowheads indicate the colocalization of SNAP25 and the PLCδ₁-PH-domain-GFP protein. All bars, 10 μm.

BFA inhibits SNAP25 recycling to the plasma membrane. PC12 cells were treated with 5 μg ml^-1 BFA for 30 min or with 3 μg ml^-1 nocodazole (NOC) for 60 min where indicated. Immunocytochemistry was conducted to localize SNAP25 (A–D), Golgi mannosidase II (A), TGN-38 (B), Rab11 (C, top row), or cathepsin D (C, bottom row). Perinuclear SNAP25 colocalized with TGN-38 and Rab11 but not with mannosidase II or cathepsin D. (D) BFA treatment for 60 min promoted an accumulation of perinuclear SNAP25 detected by immunocytochemistry. (E) The accumulation of perinuclear SNAP25 was quantitated in 50 randomly selected cells for indicated time points after BFA addition. The ratio of intracellular to total SNAP25 immunofluorescence × 100% was plotted as mean values (•). A theoretical curve (solid line) based on the rate constant for SNAP25 internalization (k₂ = 0.44% min^-1) derived from FRAP analysis (see Figure 6) and the assumption that SNAP25 recycling back to the plasma membrane was blocked (k₁ = 0) fit well to experimental data points (r = 0.953). (F) BFA treatment converted some membrane-associated SNAP25 to cytosolic SNAP25. PC12 cells were treated with 5 μg ml^-1 BFA for 60 min where indicated. Membrane (10 μg of protein corresponding to 0.3% of total) and cytosol (5 μg protein corresponding to 0.65% of total) fractions were analyzed for SNAP25 by Western blotting and densitometry. Correcting for total recovered membrane and cytosol protein, we calculated that 97% of the total SNAP25 was membrane-associated in untreated cells, whereas only 50% was membrane-associated in BFA-treated cells. (G) Immunoreactive SNAP25 (green) colocalized with expressed ARF6-HA (red) in an expanded recycling endosome compartment after 60 min of BFA treatment (arrowheads).

SNAP25 is not cointernalized with syntaxin 1A nor with vesicle constituents after exocytosis. (A) PC12 cells expressing ARF6^WT-, ARF6^Q67L-, or Rab5^Q79L-GFP (green) were immunostained for syntaxin-1A (red). Asterisks indicate enlarged endosomal vacuoles that did not contain syntaxin 1A. (B) PC12 cells expressing wild-type dynamin-HA or K44A dynamin-HA were incubated with synaptotagmin I antibody in 59 mM K⁺ buffer for 20 min and fixed for immunostaining with HA antibody (green) and anti-rabbit IgG (red). Cells expressing K44A dynamin exhibited reduced synaptotagmin I antibody uptake (arrowheads). (C) Cells expressing HA-ARF6^WT (blue) and GFP-SNAP25 (green) were incubated for indicated times in 59 mM K⁺ buffer with synaptotagmin I antibody (red). Arrows indicate sites of vesicle fusion that colocalize GFP-SNAP25 and synaptotagmin I antibody on the plasma membrane. Arrowheads indicate the colocalization of internalized synaptotagmin I antibody with ARF6^WT.