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Role of the ß1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia

¹ Department of Microbiology, Umeå University, 901 87 Umeå, Sweden
² Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, 751 23 Uppsala, Sweden
³ Max Planck Institute for Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18A, 82152 Martinsried, Germany

View larger version (25K): [in a new window]   Fig. 1. ß1-integrin-mediated internalization of Yersinia, and integrin attachment to invasin. Cells were infected with Yersinia at a calculated bacteria:cell ratio of 150:1 for 30 minutes at 37°C, and thereafter fixed and stained for extracellular and total cell-associated bacteria, respectively. (A) The percentage of cell-associated bacteria located intracellularly is shown. (B) The total number of bacteria associated to one cell is shown. The values represent the means±s.e.m. of at least five separate experiments. (C) The indicated cells were allowed to bind to microtiter plates coated with GST-invasin (solid line), GST alone (dotted line) or vitronectin for 1 hour. The number of cells adhering to vitronectin (coated at 10 µg/ml) represents 100% of attached cells.

View larger version (57K): [in a new window]   Fig. 2. Tyrosine phosphorylation of focal contact proteins. The GD25ß1A and GD25ß1B cell lines were allowed to attach to invasin for 60 minutes and then lysed in RIPA buffer. The lysates were immunoprecipitated for FAK, p130Cas or paxillin, and the precipitated proteins were analyzed by western blotting using antibodies specific for phosphotyrosine. As a loading control, the membranes were stripped and incubated with protein-specific antibodies.

View larger version (32K): [in a new window]   Fig. 3. ß1 constructs transfected into GD25 cells. The conserved NPXY motifs and the threonine-rich region are indicated.

View larger version (29K): [in a new window]   Fig. 4. Internalization of Yersinia and invasin-attachment by GD25 cells expressing different ß1A-integrin mutants. Cells were infected with Yersinia at a calculated bacteria:cell ratio of 150:1 for 30 minutes at 37°C, and thereafter fixed and stained for extracellular and total cell-associated bacteria respectively. (A) The percentage of cell-associated bacteria located intracellularly is shown. (B) The total number of bacteria associated to one cell. The values represent the means±s.e.m. of at least five separate experiments. (C) The indicated cells were allowed to bind to microtiter plates coated with GST-invasin (solid line), GST alone (dotted line) or vitronectin for 1 hour. The number of cells adhering to vitronectin (coated at 10 µg/ml) represents 100% of attached cells.

View larger version (45K): [in a new window]   Fig. 5. Distribution of the different ß1-integrin variants in cells grown in culture. The indicated cells were seeded on glass cover slips and incubated overnight in culture medium after which they were fixed and stained by anti-ß1 -integrin antibodies followed by fluorescein-conjugated secondary antibodies. The specimens were analyzed using a fluorescence microscope, and images were taken using a CCD camera. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 6. Distribution of the different ß1-integrin variants in cells adhering to invasin. GD25 cells expressing different variants of the ß1-integrin were allowed to spread on GST-invasin-coated glass cover slips for 3 hours. The cells suspended in serum-free DMEM were pretreated with cycloheximide (25 µg/ml) and GRGDS (0.1 mg/ml). After fixation, the cells were double-stained with anti-ß1-integrin (Aa-Ka) and anti-vinculin (Ab-Kb) antibodies or with anti-ß1-integrin (Ac-Kc) and anti-phosphotyrosine (Ad-Kd) antibodies. The specimens were analyzed using a fluorescence microscope and images were taken using a CCD camera. Bar, 10 µm.