Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function blocks adhesion of endothelial cells to fibronectin. A and B, uninfected (control), syntaxin 6-cyto-, syntaxin 16-cyto-, siSTX6- or siSTX16-expressing HUVECs were cultured on fibronectin-coated surfaces in complete medium. Western blotting was performed to assess relative levels of endogenous syntaxin 6, syntaxin 16, syntaxin 6-cyto, syntaxin 16-cyto, or tubulin in cell lysates. A representative blot is shown (n = 5 independent experiments). C, HUVECs were transfected with siRNAs against STX6 or STX16. After 72 h of transfection, cells were fixed and co-immunostained using anti-syntaxin 6 and -syntaxin 16 Abs. Fluorescence images of representative cells from randomly chosen fields are shown, and fluorescence (excluding nuclei) was quantified and used to calculate the percentage of cells showing ≥90% reduction in syntaxin 6 or syntaxin 16 relative to untreated (control) cells. D and E, uninfected HUVECs (Control), and HUVECs stably expressing syntaxin 6-cyto, syntaxin 16-cyto, siSTX6, or siSTX16 were trypsinized and seeded in adhesion buffer onto plates coated with extracellular matrix proteins. After the cells were allowed to adhere for 20, 40, and 60 min, nonadherent cells were removed and cells were subjected to fixation and staining with crystal violet. Dye was extracted and absorbance was assessed as an indicator of the number of bound cells. The number of cells bound in the controls was set as basal adhesion (100%). E, control and syntaxin 6-cyto-expressing HUVECs were either left untreated (no Ab) or were pretreated with the indicated integrin blocking Abs for 10 min prior to seeding onto fibronectin-coated plates. Values represent relative change in adhesion normalized to an arbitrary value of 100% for controls. Values in D and E represent mean ± S.D. from four independent experiments; p ≤ 0.003 in D, p ≤ 0.005 in E.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Expressing, Cell Culture, Western Blot, Transfection, Fluorescence, Stable Transfection, Staining, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Syntaxin 6, α5 integrin, and β1 integrin co-localize at EEA1-positive early endosomes in endothelial cells. HUVECs cultured in complete medium on fibronectin-coated surfaces were fixed, permeabilized, incubated with the indicated primary Abs, incubated with appropriate secondary Abs, and imaged by confocal fluorescence microscopy. A-C, representative confocal z-stacks, with enlarged insets highlighting colocalization between syntaxin 6 and EEA1, integrin α5, and integrin β1. In each case, arrowheads indicate colocalization between endosome-associated syntaxin 6 and each protein; arrows indicate lack of colocalization between Golgi-localized (perinuclear) syntaxin 6 and each protein. Confocal z-planes corresponding to the PM (dorsal- and ventral-most) are not included in B and C to avoid interference of signal from cell surface with signal from intracellular structures. D, signal overlap in images such as those were used to quantitate the extent to which syntaxin 6 was colocalized with each protein within EEs. Values represent mean ± S.D. (n = 50 cells for each condition, from 5 separate experiments; p ≤ 0.05). Scale bar represents 5 μm.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Cell Culture, Incubation, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function decreases α5β1 integrin levels, both cell surface-localized and total in endothelial cells. Uninfected (Control), syntaxin 6-cyto-, syntaxin 16-cyto-, siSTX6-, or siSTX16-expressing HUVECs were cultured on fibronectin-coated surfaces in complete medium. A, cells were pre-treated with Abs against α5 and β1 integrin at 4 °C prior to fixation and labeling with Alexa 488-labeled secondary Ab. Samples were then imaged by epifluorescence microscopy; representative images show cell surface levels of α5 and β1 integrins. B and C, cells were fixed and permeabilized, labeled with Abs against α5 or β1 integrin, and then labeled with the appropriate fluorescently tagged secondary Ab. Representative images obtained by epifluorescence microscopy show localization of total cell-associated α5 and β1 integrins. D, quantification of total cellular α5 and β1 integrin in syntaxin 6-cyto-, syntaxin 16-cyto-, siSTX6-, or siSTX16-expressing cells. Epifluorescence images were acquired and total cell-associated fluorescence was quantified by image analysis. Values represent relative change in the levels of α5 or β1 integrin normalized to an arbitrary value of 100% for untreated controls. Results are expressed as mean ± S.D. (n = 70 cells for each condition, from 3 separate experiments; p ≤ 0.001). E and F, Western blotting to assess α5 and β1 integrin levels in cell lysates. A representative blot is shown. G, α5 and β1 integrin band densities from E and F were quantified; values represent relative levels of α5 and β1 after normalization to the arbitrary value of 100 for uninfected cells (Control). (mean ± S.D.; n = 3; p ≤ 0.005). Scale bars represent 5 μm.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Expressing, Cell Culture, Labeling, Epifluorescence Microscopy, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function reduces endocytic recycling of α5 integrin and increases its ubiquitination and degradation in lysosomes. A, uninfected (control) and syntaxin 6-cyto- or syntaxin 16-cyto-infected HUVECs were surface biotinylated. Samples were then incubated at 16 °C for 30 min, to allow biotinylated α5β1 integrin to accumulate in EEs. Biotin remaining at the surface was removed by treatment with MesNa and quenching of MesNa with iodoacetamide (0-min time point); samples were further incubated at 37 °C for the indicated periods and, at each time point shown, subjected to a second MesNa treatment and then assessed for recycling of internalized integrin. After each time point, the cells were lysed and the amount of biotinylated integrin was determined by capture ELISA, using an Ab against α5 integrin. The fraction of internalized integrin recycled back to the PM is expressed as a percentage of surface-labeled protein originally internalized (from the 0-min chase time point). B, uninfected and syntaxin 6-cyto-infected cells were surface biotinylated and assessed for α5 integrin expression as in A, but were not subjected to a second treatment with MesNa. The fraction of internalized integrin remaining (i.e. PM-associated due to recycling + intracellular) after various chase periods is expressed as the percentage of surface-labeled internalized (0-min chase time point). Values in A and B are mean ± S.D. from four independent experiments; p ≤ 0.005 in A, p ≤ 0.05 in B. C and D, uninfected (Control) and syntaxin 6-cyto- or syntaxin 16-cyto-infected HUVECs after 12 h of infection were electroporated with (i) α5-integrin-GFP plasmid (in C) or (ii) a mixture of α5-GFP-integrin and HA-ubiquitin plasmids. After 12 h, the cells were cultured in the presence of 300 μm leupeptin for 12 h (in D). After 24 h of electroporation, cell lysates were prepared and incubated with anti-GFP Ab coupled to beads. Relative levels of α5-GFP-integrin in the cell lysates were determined by immunoprecipitating (IP) with anti-GFP Ab, and immunoblotting (WB) with anti-α5 integrin Ab. Ubiquitination was detected by immunoblotting against anti-HA Ab. Relative enrichment of β1 integrin and tubulin in cell lysates (10% input) was detected using Abs against β1 integrin and tubulin.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Infection, Incubation, Enzyme-linked Immunosorbent Assay, Labeling, Expressing, Plasmid Preparation, Cell Culture, Electroporation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function leads to change in trafficking of α5β1 integrins from the early endosomes toward the degradation versus recycling pathway. A and B, uninfected (control), syntaxin 6-cyto or syntaxin 16-cyto-infected HUVECs were labeled with anti-β1 integrin Ab at 10 °C, followed by incubation at 16 °C for 30 min to allow internalized Ab-β1 integrin complexes to accumulate in EEs. The remaining surface-bound Ab was removed by washing with low pH buffer. These samples were then subjected to chase at 37 °C for the indicated time periods before being fixed and permeabilized. Intracellular Ab-β1 integrin complexes and EEA1 were detected using the Alexa 488- and Alexa 594-labeled secondary Abs, respectively. Samples were then imaged by confocal fluorescence microscopy, for intracellular localization of the Ab-β1 integrin complexes, EEA1 (early endosomes), and GFP-lgp120 (lysosomes). C, colocalization of Ab-β1 integrin complexes with EEA1 and GFP-lgp120 was quantitated from images such as those shown in A and B. Values represent mean ± S.D. (n = 50 cells for each condition from 5 separate experiments; p ≤ 0.05). Scale bar represents 5 μm.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Infection, Labeling, Incubation, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function in endothelial cells blocks migration and recruitment of active FAK to focal adhesions. Uninfected (control), syntaxin 6-cyto-, syntaxin 16-cyto-, siSTX6-, or siSTX16-expressing HUVECs were grown on fibronectin-coated surfaces were serum starved for 2–3 h before being used for experiments. A, directional migration of HUVECs toward fibronectin in a Boyden chamber assay, with the medium in the lower well lacking serum. The number of migrating cells was normalized to that in uninfected controls. B, cells were stimulated with serum-containing medium for 10 min and then co-stained with Abs against phospho-Tyr397-FAK and vinculin to visualize focal adhesion sites. C, cells treated as in B were used to prepare lysates. Proteins were subjected to SDS-PAGE and immunoblotting with anti-FAK and anti-phospho-Tyr397-FAK Abs. Relative levels of tubulin in cell lysate confirms that protein loading in each well was equal. D, quantification of FAK and pFAK band densities; values represent ratios of pFAK to total FAK after normalization to the arbitrary value of 100 for uninfected HUVECs (control). Values represent mean ± S.D. (n = 3; p ≤ 0.005). Scale bars represent 5 μm.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Migration, Expressing, Boyden Chamber Assay, Staining, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Inhibition of syntaxin 6 function blocks integrin activation, alters Rac1 distribution, reduces active Rac1, and slows spreading of endothelial cells on fibronectin. Uninfected HUVECs (control), syntaxin 6-cyto- or syntaxin 16-cyto-infected, or siSTX6- or siSTX16-expressing HUVECs were cultured on fibronectin-coated surfaces in complete medium. HUVECs were grown on fibronectin-coated surfaces. A, samples were then labeled with the HUTS21 Ab, which recognizes β1 integrin in its active conformation. B, Rac1 localization was assessed by staining fixed, permeabilized cells with anti-Rac1 Ab. Images were acquired by epifluorescence microscopy, and line scans were generated using Metamorph image analysis software. For clarity of presentation, representative syntaxin 6-cyto-infected cell edge was marked by a white dashed line. C, lysates were prepared and subjected to a GST-PBD pulldown assay, followed by blotting with Rac1 Ab to detect active Rac1. The relative levels of total Rac1 and tubulin in cell lysates (10% input) were detected by immunoblotting with Abs. D, the intensity of the band of active Rac1 was assessed after a GST-PBD pulldown assay and quantitated and normalized to that of the control. Values represent mean ± S.D. (n = 3; p ≤ 0.003). E, cells were trypsinized and then allowed to spread on a fibronectin-coated glass surface at 37 °C for the indicated times. Following fixation, permeabilization, and staining with Alexa 488-labeled phalloidin, the cells were imaged by epifluorescence microscopy. Representative images are shown, representative images of siSTX6- and siSTX16-treated samples are included under supplemental Fig. 4. F, quantitation of the cell surface areas of individual cells, based on epifluorescence images of cells described in E. Values represent mean ± S.D. (cells = 300, from 3 separate experiments; p ≤ 0.003). Scale bars represent 5 μm.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Inhibition, Activation Assay, Infection, Expressing, Cell Culture, Labeling, Staining, Epifluorescence Microscopy, Generated, Software, Western Blot, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Migration on Fibronectin Is Regulated by Syntaxin 6-mediated ?5?1 Integrin Recycling *

doi: 10.1074/jbc.M111.260828

Figure Lengend Snippet: Proposed itineraries of α5β1 integrin in endothelial cells with and without syntaxin 6 function. 1, α5 and β1 integrin complexes are co-internalized from the cell surface. 2, the complexes are transported to EEA1-positive EEs. 3, α5β1 complexes are transported through the perinuclear recycling compartment (PNRC) (recycling pathway) for delivery to the PM. Loss of syntaxin 6 function blocks the transport of α5β1 integrin through the recycling pathway. In the absence of syntaxin 6 function: 4, the pool of ubiquitinated α5 integrin within the EE increases, and α5β1 complexes are transported to LEs/lysosomes for degradation; and 5, levels of α5β1 integrin at the cell surface are reduced, leading to impaired cell adhesion, migration, and spreading on fibronectin.

Article Snippet: The antibodies (Ab) used in this study were obtained from the following companies and institutions: the anti-β1 integrin mouse monoclonal antibody (mAb; MAB1981) and anti-α5 rabbit polyclonal Ab (AB1928) used in immunofluorescence assays, Millipore-Chemicon; the anti-syntaxin 6 (610636) and anti-β1 integrin (for immunoblotting) mAbs, BD Transduction Laboratories (NJ); the P5D2 mAb against human β1-integrin developed by Dr. Elizabeth A. Wayner (Fred Hutchinson Cancer Research Center), the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa; Alexa Fluor-conjugated secondary antibodies, Invitrogen and Molecular Probes (Eugene, OR); FITC-conjugated secondary antibodies, Jackson ImmunoResearch Laboratories, Inc.; anti-integrin β1 (clone P4C10) and anti-integrin β3 (clone B3A) mAbs for blocking, Chemicon (Millipore, MA); heterodimeric antibodies against αvβ3 (clone LM609) and α5β1 (clone JBS5), Chemicon International (Temecula, CA); and mouse anti-human CD29 mAb (clone HUTS-21) and anti-focal adhesion kinase (FAK) mouse mAb, BD Biosciences (San Diego, CA).

Techniques: Migration