Journal: Journal of cell science

Article Title: Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

doi: 10.1242/jcs.259576

Figure Lengend Snippet: Fig. 5. STX7 interacts with multiple SNARE partners. (A) GBP pulldown was performed with lysates of HeLa cells expressing GFP-vector, and GFP-tagged SNAP23, VAMP2 and VAMP3, pre-treated with 1 mM NEM. Purified GST–GBP (25 µg) was incubated with glutathione–Sepharose beads and allowed to bind with the respective lysates (300 µg) for 5 h at 4°C. Beads were washed, boiled and advanced to immunoblotting using anti-STX7 and anti-GFP antibodies. The number represents the normalized value of prey protein (STX7) with respect to the precipitated protein (GFP-tagged protein) for the blot shown. WCL, whole-cell lysates. (B,C) GBP pulldown was performed with lysates from HEK-293 cells expressing GFP-vector, GFP-tagged VAMP2, STX4 and STX7, pre-treated with 1mM NEM. Purified GST–GBP (25 µg) was incubated with glutathione–Sepharose beads and allowed to bind with the respective lysates from cells expressing GFP-vector (300 µg), GFP–VAMP2 (300 µg), GFP–STX4 (500 µg) or GFP–STX7 (300 µg) for 5 h at 4°C. Beads were washed, boiled and advanced to immunoblotting using anti-STX7, anti-STX2 and anti-GFP antibody. The number represents the normalized value of prey protein with respect to the precipitated protein (GFP-tagged protein) for the blot shown. Results in A–C are representative of three experimental repeats. (D) MDA-MB-231 cells with independent transfection of GFP-tagged VAMP2, VAMP3, VAMP7, SNAP23 or STX4 were immunostained for STX7. Arrowhead indicates colocalized puncta (N=3). Scale bars: 10 μm. (D′) Percentage colocalization was quantified and plotted. N=3, n=140 (GFP–VAMP2), n=92 (GFP–VAMP3), n=154 (GFP–VAMP7), n=100 (GFP–STX4), n=115 (GFP–SNAP23). The data are displayed using SuperPlots; each biological replicate is distinctly color-coded and each dot represents identified percentage colocalization in a field of view (frame), as described in the Materials and Methods, with mean±s.d. N, number of experimental repeats; n, number of cells analyzed.

Article Snippet: Jo u rn al o f Ce ll Sc ie n ce (Millipore, MAB3328), 1:1000 [immunoblotting (IB)], 1:500 (IF); mouse anti-MT1-MMP (R& D Systems, MAB9181-SP), 1:200 (IF); mouse antivinculin (Sigma, V9131), 1:1000 (IB); mouse anti-cortactin (Millipore, 05- 180), 1:1000 (IB), 1:300 (IF); mouse anti-transferrin receptor (Invitrogen, 136800), 1:500 (IF); rabbit anti-actin (Sigma, A2066), 1:2000 (IB); mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB); mouse anti-CD63 (DSHB, H5C6), 1:500 (IF); rabbit anti- STX2 (Proteintech, 55033-1-AP), 1:1000 (IB), mouse anti- STX7 (Santa Cruz Biotechnology, sc-514017), 1:1000 (IB), 1:300 (IF); mouse anti-β1-integrin (P5D2, DSHB), 1:1000 (IB), mouse antiphospho-tyrosine (Millipore, 05-1050), 1:1000 (IB); and rabbit anti-Tks5 (Santa Cruz Biotechnology, sc-30122), 1:500 (IF).

Techniques: Expressing, Plasmid Preparation, Purification, Incubation, Western Blot, Transfection

Journal: Journal of cell science

Article Title: Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

doi: 10.1242/jcs.259576

Figure Lengend Snippet: Fig. 7. The proposed model showing that STX7 interacts with multiple SNAREs and forms multiple distinct SNARE complexes. These complexes assist in the fusion of vesicles carrying MT1-MMP to invadopodia, thus, facilitating ECM degradation. However, upon depletion of STX7, trafficking of MT1-MMP is diverted towards the PM rather than invadopodia. Also, silencing of STX7 abrogates the formation of invadopodia, possibly due to hampered trafficking of signaling molecules or growth factors or an unknown cargo carried by STX7 to promote invadopodia formation. Alternatively, when STX4, VAMP2, VAMP3 or STX7–STX4 is depleted, there is reduced MT1-MMP trafficking to cell surface as well as reduced invadopodia formation.

Article Snippet: Jo u rn al o f Ce ll Sc ie n ce (Millipore, MAB3328), 1:1000 [immunoblotting (IB)], 1:500 (IF); mouse anti-MT1-MMP (R& D Systems, MAB9181-SP), 1:200 (IF); mouse antivinculin (Sigma, V9131), 1:1000 (IB); mouse anti-cortactin (Millipore, 05- 180), 1:1000 (IB), 1:300 (IF); mouse anti-transferrin receptor (Invitrogen, 136800), 1:500 (IF); rabbit anti-actin (Sigma, A2066), 1:2000 (IB); mouse anti-GFP (Roche, 11814460001), 1:2000 (IB); mouse anti-γ-tubulin (Sigma, T6557), 1:3000 (IB); mouse anti-CD63 (DSHB, H5C6), 1:500 (IF); rabbit anti- STX2 (Proteintech, 55033-1-AP), 1:1000 (IB), mouse anti- STX7 (Santa Cruz Biotechnology, sc-514017), 1:1000 (IB), 1:300 (IF); mouse anti-β1-integrin (P5D2, DSHB), 1:1000 (IB), mouse antiphospho-tyrosine (Millipore, 05-1050), 1:1000 (IB); and rabbit anti-Tks5 (Santa Cruz Biotechnology, sc-30122), 1:500 (IF).

Techniques:

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Stimulus-frequency-dependent responses of Stx7-SEP in comparison to SypHy. Neurons expressing respective SEP fusion constructs were subjected to sequential stimulation ranging from 5 to 40 Hz (200 APs) at 5 min intervals. Left images are representative images of SypHy (top), Stx7-SEP (bottom) at rest, at the end of stimulation at 5, 10, 20, and 40 Hz, and upon application of NH 4 Cl at the end of recordings. Right traces show representative traces of each SEP-fluorescence change in boutons, indicated by arrows. Data were normalized to fluorescence signals upon application of NH 4 Cl at the end of recordings. b Experimental scheme to estimate the kinetics of exocytosis, as well as sizes of total recycling SV pools. Neurons were pretreated with 2 µM bafilomycin A1 for 60 s and then stimulated with 600 APs at different stimulation frequencies. After cessation of stimulation, 50 mM NH 4 Cl was applied and fluorescence during NH 4 Cl application was used to normalized fluorescence signals at individual boutons. c Recycling pool of SypHy and Stx7-SEP. Cells expressing SypHy (left) and Stx7-SEP (right) were stimulated with 600 APs at different frequencies (5, 10, 20, and 40 Hz). d Replots of SypHy (black) and Stx7-SEP (red) responses of the results in ( c ) as a function of stimulus numbers (No. of AP).

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Comparison, Expressing, Construct, Fluorescence

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Double immunostaining of Stx7 and Syp in cultured hippocampal neurons at 14 DIV. An upper panel shows representative axonal localization of Stx7 (green) and Syp (red). Magnified images of numbered areas (1−3) are shown individually below. The specificity of the Stx7 antibody was confirmed in independent experiments where Stx7 expression level was reduced by specific shRNA (Supplementary Fig. ). Scale bars indicate 2 µm. b Triple-immunofluorescence for Stx7 (blue), Syp (red), and an active zone maker Bassoon (BSN, green). An upper panel shows representative images. Magnified images of the numbered areas (1−3) are shown below. Scale bars indicate 1 µm. c Immunoelectron micrographs of SypHy (left) and Stx7-SEP (right) at presynaptic terminals. Immunogold labeling was intensified by silver enhancement. Arrowheads indicate both edges of the active zone deduced from postsynaptic density structures. Scale bars indicate 100 nm. d Number of immunoparticles as a function of the area of presynaptic varicosity. Sixteen varicosities for SypHy (black) and 24 varicosities for Stx7-SEP (red) were analyzed. e A box-whisker plot showing densities of SypHy immunoparticles (black) and Stx7-SEP immunoparticles (red) calculated from ( d ). p < 0.0001 with unpaired t -test with Welch’s correction. f A cumulative plot of distances of immunoparticles to the nearest AZ membrane. Note that vertical lines from the edges of AZs were drawn manually, and only immunoparticles inside the enclosed areas were measured for the analysis (Supplementary Fig. 9). Statistical significance was evaluated with Kolmogorov−Smirnov test ( p = 0.0003). g A representative quantification of Stx7 in native SVs purified from rat brains. Various amounts of recombinant GST-Stx7-N-terminal domain (GST-Stx7-N) and a fixed amount of purified SV fraction (vesicle concentration, 26.7 nM; protein concentration, 99.7 ng/μL) were subjected to quantitative western blot analysis (see also Supplementary Fig. for complete datasets and control experiments for Syb2). h Signal intensities of bands were quantified and plotted as a function of moles of GST-Stx7-N. A red circle indicates the signal intensity measured for 1.0 µg SV shown in ( g ).

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Double Immunostaining, Cell Culture, Expressing, shRNA, Immunofluorescence, Labeling, Whisker Assay, Membrane, Purification, Recombinant, Concentration Assay, Protein Concentration, Western Blot, Control

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Responses of Stx7-SEP (right) in the presence of normal (2 mM, black) and high (8 mM, red) external Ca 2+ . For comparison, responses of SypHy under identical conditions are shown in the left panel. b Effects of CIP (red) on exocytosis of total recycling pool monitored by SypHy. Responses at 10 Hz, 600APs (left) and 20 Hz, 600APs (right) with bafilomycin treatment are shown. Control experiments without CIP in respective conditions are shown in black. Bottom box-whisker plots show quantitative comparisons of total recycling pool sizes and rise kinetics. Normalized fluorescence peaks (left) or the time constants of rise time (τ exo ) (right) during 10 Hz or 20 Hz stimulation are compared. c Effects of CIP (red) on exocytosis of the total recycling pool monitored by Stx7-SEP. Responses at 20 Hz, 600APs (left) with bafilomycin treatment are shown. Control experiments without CIP in the respective conditions are shown in black. Right box-whisker plots show quantitative comparisons of total recycling pool sizes and rise kinetics. Normalized fluorescence peaks (left) or the time constants of rise time (τ exo ) (right) during 20 Hz stimulation are compared. All traces are average traces from >150 boutons.

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Comparison, Control, Whisker Assay, Fluorescence

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Effects of latrunculin A (Lat-A, 5 µM; red) on responses of SypHy (upper traces) and of Stx7-SEP (lower traces) elicited by 10 Hz (left) and 40 Hz (right) stimulation. Control experiments without Lat-A in the respective conditions are shown in black. b Effects of Lat-A (red traces) on recycling of SypHy and Stx7-SEP in the presence of 8 mM external Ca 2+ . Control experiments without Lat-A in the respective conditions are shown in black. c Effects of Lat-A (red) on exocytosis of total recycling pool monitored by SypHy. Responses at 10 Hz, 600APs (left) and 20 Hz, 600APs (right) with bafilomycin treatment are shown. Control experiments without Lat-A in the respective conditions are shown in black. Bottom box-whisker plots show quantitative comparisons of total recycling pool sizes and rise kinetics. Normalized fluorescence peaks (left) or the time constants of rise time (τ exo ) (right) during the 10 Hz or 20 Hz stimulation are compared. All traces are average traces from 50 to 150 boutons.

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Control, Whisker Assay, Fluorescence

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Schematic diagram of full length Stx7-SEP, Stx7-SEP lacking the N-terminal domain (Stx7-ΔN-SEP), and Stx7-SEP lacking SNARE motif (Stx7-ΔSNARE-SEP). b Distribution of Stx7-SEP, Stx7-ΔN-SEP, and Stx7-Δ-SNARE-SEP in transfected neurons. Box-whisker plots showing the surface fraction of Stx7-SEP and truncated mutants ( c ) and vesicular pHs of vesicles carrying respective SEPs ( d ). Note that vesicular pH of Stx7-ΔSNARE-SEP could not be calculated, since it was exclusively expressed at the cell surface (N.D. indicates ‘not determined’). e Responses of Stx7-SEP (black traces) and Stx7-ΔN-SEP (red traces) upon 10 Hz (left panels) and 40 Hz stimulation (right panels). f Responses of Stx7-ΔN-SEP upon 10-Hz stimulation in control (black) and after TeNT treatment (red). g Responses of Stx7-ΔN-SEP upon 40 Hz stimulation in control (black) and after Lat-A treatment (red).

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Transfection, Whisker Assay, Control

Journal: Communications Biology

Article Title: The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons

doi: 10.1038/s42003-021-02512-4

Figure Lengend Snippet: a Schematic diagram of SypHy and SypHy co-expressed with Stx7-NTD. Stx7-NTD was placed after a P2A sequence so that all SypHy-positive cells co-expressed Stx7-NTD. b The total recycling pool monitored by SypHy responses in the absence (black) and presence (red) of Stx7-NTD at 10 Hz, 600APs (left) and 20 Hz, 600APs (right) after bafilomycin treatment. Bottom box-whisker plots show quantification of total recycling pool sizes (left) and time constants of rise time (right, τ exo ). c Pretreatment with Lat-A did not further reduce release kinetics upon Stx7-NTD overexpression. Responses of SypHy with Stx7-NTD in the presence of Lat-A (blue) were compared with control (SypHy only, black) and SypHy with Stx7-NTD (red) in response to at 20 Hz, 600APs. A box-whisker plot shows time constants of the rise time (τ exo ). SypHy responses with Stx7-NTD (red) and Stx7-NTD + Lat-A (blue) did not differ significantly ( p > 0.05). d Schematic summary of this study, depicting that Stx7 is preferentially present in the rapidly replenishing SV pool during intense, repetitive stimulation.

Article Snippet: Cells were incubated with rabbit polyclonal anti-GFP antiserum and mouse monoclonal anti-Syb2 antibody (Cl69.1) (kind gifts from Reinhard Jahn) (Figs. b, e, and Supplementary Fig. ), or with rabbit polyclonal anti-Stx7 antibody (Bethyl Laboratories; A304-512A), mouse monoclonal anti-Synaptophysin antibody (Cl7.2), guinea pig polyclonal anti-Synaptophysin antibody (Synaptic Systems; 101 004), and mouse monoclonal anti-Bassoon antibody (Novus Biologicals; SAP7F407)(Fig. ), or chick polyclonal anti-MAP2 antibody (1:1,000; Abcam; ab5392) and anti-Stx7 antibody (Supplementary Figs. , ), for 1 h at RT.

Techniques: Sequencing, Whisker Assay, Over Expression, Control

Journal: Journal of Cell Science

Article Title: Oxidized phagosomal NOX2 complex is replenished from lysosomes

doi: 10.1242/jcs.196931

Figure Lengend Snippet: siRNA knockdown of Stx7, SNAP23 and VAMP8 reduces phagosomal recruitment of cytochrome b 558 and ROS production. (A) Representative western blots and quantification of siRNA knockdown (KD) of stx7 (51% knockdown efficiency), stx12 (56%), SNAP23 (67%) and gp91 phox (92%) relative to levels with a non-targeting siRNA control (NT). GAPDH, loading control ( n =8 donors; two-sided paired Student's t -test). (B) Confocal images of dendritic cells pulsed with Alexa-Fluor-633-conjugated zymosan particles (magenta in merge) and then immunolabeled for gp91 phox (green) with knockdown of VAMP8, stx7, stx12 or SNAP23. Yellow arrowheads, phagosomes enriched for gp91 phox ; red arrowheads, phagosomes negative for gp91 phox . BF, brightfield. Scale bars: 10 µm. (C) Quantification of the phagosomal enrichment of gp91 phox from panel B (individual donors shown; one-way ANOVA with Dunnett's test; representative confocal images of gp91 phox KD are shown in panel F). ns, not significant. (D) Quantification of the phagosomal enrichment of EEA1 and LAMP1 (individual donors shown; representative confocal images are shown in Fig. S4B ). (E) Quantification of the enrichment of gp91 phox and p67 phox to zymosan-containing phagosomes in dendritic cells with knockdown of gp91 phox or stx7 ( n =3 donors; one-way ANOVA with Tukey's test). (F) Representative confocal images for data presented in E. Yellow arrowheads, phagosomes enriched for gp91 phox (green) and p67 phox (magenta); red arrowheads, phagosomes negative for gp91 phox and p67 phox . BF, brightfield. Scale bar: 10 µm. (G) Confocal images of zymosan-pulsed dendritic cells with VAMP8, stx7, stx12 or SNAP23 knockdown. Zymosan particles were labeled with ROS-sensitive OxyBURST that becomes fluorescent upon oxidation (shown in rainbow lookup table). Scale bar: 10 µm. (H) Intraphagosomal ROS production calculated from the fluorescence intensities shown in panel G (normalized to those with non-targeting siRNA; one-way ANOVA with Dunnett's test; individual donors shown). Results show mean±s.e.m.

Article Snippet: Primary antibodies used were: mouse-IgG1 anti-gp91 phox (D162-3, MBL), rabbit serum anti-SNAP23 (111202, Synaptic Systems), rabbit serum anti-stx8 (110083, Synaptic Systems), rabbit serum anti-stx7 (110072, Synaptic Systems), mouse-IgG1 anti-stx7 (sc-514157, Santa Cruz), rabbit serum anti-stx12 (299022, Synaptic Systems), rabbit serum anti-VAMP8 (104302, Synaptic Systems), rabbit serum anti-Vti1b (164002, Synaptic Systems), rabbit serum anti-stx5 (110053, Synaptic Systems), rabbit serum anti-LAMP1 (L1418, Sigma), rabbit serum anti-TGN38 (sc-27680, Santa Cruz), mouse-IgG1 anti-EEA1 (610456, BD Biosciences), rabbit serum anti-p67phox (07-002, Merck Millipore), rabbit monoclonal-IgG anti-GAPDH (2118, Cell Signaling Technology), mouse-IgG1 anti-FITC (200-602-037, Jackson ImmunoResearch Laboratories) and mouse-IgG1 isotype control (400102, Biolegend) antibodies.

Techniques: Western Blot, Immunolabeling, Labeling, Fluorescence