Volume 13, Issue 2, Pages 156-160 (January 2003) The t-SNARE Syntaxin 4 Is Regulated during Macrophage Activation to Function in Membrane Traffic and Cytokine Secretion  Julia K Pagan, Fiona G Wylie, Shannon Joseph, Charlotte Widberg, Nia J Bryant, David E James, Jennifer L Stow  Current Biology  Volume 13, Issue 2, Pages 156-160 (January 2003) DOI: 10.1016/S0960-9822(03)00006-X

Figure 1 SNARE Protein Levels Change in Response to LPS (A) Cytosol (C) and total microsomal membranes (M) (2 μg protein/lane), prepared from cultured RAW264 cells after treatment for 2 or 24 hr with LPS (100 ng/ml), were analyzed by SDS-PAGE and immunoblotting with a panel of antibodies to detect Syntaxins (Syn) and the other proteins indicated. The sources of the antibodies are as follows: Syntaxin 13 was provided by Rohan Teasdale (University of Queensland); Syntaxin 2 was kindly provided by Keith Mostov (UCSF); and Syntaxins 6 and 11 (BD Transduction Laboratories, Lexington, NK), synaptotagmin (Transduction Laboratories), and antibodies to other proteins were made in our laboratories. Protein staining of actin is included as a loading control. (B) Average densitometric values from immunoblots from four experiments were used to calculate the relative amount of each protein at different times. The fold changes in some of the cell surface-associated SNARE and SM proteins are shown in the first graph. In the second graph, the expression patterns of Syntaxins in exocytic (solid symbols) and endocytic (open symbols) pathways are compared. (C) Immunofluorescence staining [13] of endogenous Syntaxin 4 on fixed, permeablized RAW264 cells showing brighter staining of Syntaxin 4 on the surface of cells after 2 hr of LPS treatment. (D) Cell homogenates were fractionated [13] to produce fractions that are enriched for high-density membranes (HDM), low-density membranes (LDM), plasma membrane (PM), and cytosol (CYT) and that were then separated on SDS-PAGE. Immunoblotting with specific antibodies detected Munc18c and Syntaxin 4 together in plasma membrane and high-density membranes, while a control, vesicle-associated protein, GAIP, was enriched in low-density membranes. (E) Munc18c was immunoprecipitated from cell extracts, and the proteins in the supernatant (SN) and immunoprecipitate (IP) were analyzed by immunoblotting. Syntaxin 4, but not Syntaxin 2, coimmunoprecipitates with Munc18c in macrophages. Current Biology 2003 13, 156-160DOI: (10.1016/S0960-9822(03)00006-X)

Figure 2 Cell Surface Delivery of TNFα in Activated Macrophages (A) Immunofluorescence staining of TNFα in fixed, permeabilized RAW264 [9] cells shows the accumulation of newly synthesized TNFα in the perinuclear Golgi complex 2 hr after activation with LPS. By 24 hr, TNFα synthesis has ceased. (B) Staining of fixed, unpermeabilized cells after treatment with LPS for 1 hr in the absence or presence of a TACE inhibitor. Newly synthesized TNFα is normally cleaved rapidly from the cell surface, where it is then difficult to detect; the inhibitor blocks this cleavage and results in retention and increased surface staining of TNFα. The single-cell assay for cell surface delivery of TNFα in cells treated with LPS and inhibitor is shown in the color panels; cells are stained sequentially to detect surface TNFα on unpermeabilized cells (red), followed by permeabilization and staining for an intracellular protein, in this case, the Golgi vesicle-associated protein γ-adaptin (green). Both markers are visualized together in the merged image. Current Biology 2003 13, 156-160DOI: (10.1016/S0960-9822(03)00006-X)

Figure 3 The t-SNARE Syntaxin 4 Regulates Cell Surface Delivery of TNFα (A) Cells were microinjected with fusion proteins corresponding to the cytoplasmic tails of Syntaxin 2 and Syntaxin 4 [13] (GST-Syn2-tail, GST-Syn4-tail, and GST alone) diluted in microinjection buffer (10 mM KH2PO4 [pH 7.2] and 75 mM KCl, containing Texas red-conjugated dextran) to a final concentration of ∼1 mg/ml. After 5 hr of recovery, cells were treated with LPS and TACE inhibitor for 1 hr, then fixed and immunostained for surface TNFα. The levels of surface TNFα staining (green) in microinjected cells, depicted by Texas red staining, were compared to surrounding uninjected cells. Images were analyzed to measure pixel intensity within a defined area at the cell surface; 50 injected cells were measured for each condition in three separate experiments, and the results are shown in the graph along with standard errors of the mean. (B) GFP-Munc18c cDNA (10 μg) was introduced into RAW264 cells by electroporation; the next day, cells were treated with LPS to induce synthesis of TNFα and were treated with TACE inhibitor for 1 hr prior to fixation. Unpermeabilized cells were immunostained to detect surface TNFα, which was then assessed on GFP-labeled cells compared to surrounding cells. These images are representative of >50 fields containing transfected cells in each of three separate experiments. (C) A cDNA (10 μg) encoding HA-tagged Syntaxin 4 was electroporated into cells that were then treated with LPS and TACE inhibitor. Unpermeabilized cells were immunostained to detect surface TNFα, then cells were permeabilized and stained with an HA antibody. TNFα surface staining was compared in cells expressing HA-Syntaxin 4 and in untransfected cells in three replicate experiments. Images were analyzed to measure pixel intensity within a defined area at the cell surface; >50 transfected cells were measured in each of three separate experiments, and the results are shown in the graph with standard errors of the mean. Current Biology 2003 13, 156-160DOI: (10.1016/S0960-9822(03)00006-X)