Rhotekin protein, human

Plasmids and generation of stable transfectants. Wi-26 cells (SV40-transformed human lung fibroblasts) transfected with the cDNA coding for CA forms of RhoA (RhoA-Q63L), Rac1 (Rac1-Q61L) and Cdc42 (Cdc42-Q61L) in pIRESpuro vector (Clontech, Palo Alto, Calif.) were subcloned by limited dilution and amplified as reported elsewhere [16 ]. The cloned cells (hereafter referred to as RhoA-QL, Rac1-QL and Cdc42-QL) and the parental line transfected with the empty vector (hereafter referred to as control) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 200 mM glutamine and a mixture of antibiotics (streptomycin, penicillin, puromycin) at 37°C under 5% CO₂ except where otherwise stated. All products were from Seromed/Biochrom (Berlin, Germany).
GTPase pull-down assays. Confluent cells were serumstarved for 24 h and lysed as previously described [16 ]. Active GTPases were pulled-down from the lysates (500 µl) using either the GST-PBD fusion protein with the Cdc42- and Rac1-binding region of PAK-1B, or the GST-RBD fusion protein with the RhoA-binding region of rhotekin [17 , 18 (link)]. The total lysate (40 µl) and pull-down fractions were separated by SDS-PAGE on 15% acrylamide gels under reducing conditions. Proteins were transferred to nitrocellulose membranes and immunodetected with mouse monoclonal primary antibodies against either RhoA (Sc-418; Santa Cruz, Calif.), Rac1 (clone 05-389; Upstate Biotechnology, Lake Placid, N. Y.) or Cdc42 (clone 610928; Transduction Laboratories, San Diego, Calif.), followed by secondary horseradish peroxidase-conjugated antibodies (DAKO, Glostrup, Denmark). The signals were visualized using ECL (Amersham Biosciences Europe, Freiburg, Germany). Band intensities were quantified with NIH Scion image software and the relative amount of active, GTP-bound GTPase was normalized to the total content of GTPase in the lysate.
Immunofluorescence staining. Cells grown on glass coverslips for 24 h in complete medium were fixed with freshly prepared 2% paraformaldehyde in phosphate-buffered saline, pH 7.4 (PBS) for 15 min, permeabilized with ice-cold 0.2% Triton X-100 in PBS for 1 min and incubated with 3% bovine serum albumin (BSA, fraction V; Serva, Heidelberg, Germany) for 1 h. The cells were labeled with either affinity-purified rabbit antiserum against FHL2 [19 (link)] or mouse monoclonal antibody F-VII against vinculin (a gift from Dr. M. Glukhova, Institut Curie, Paris, France), followed by Cy3-conjugated secondary antibodies against rabbit or mouse immunoglobulins (Jackson, distributed through Dianova). Fibrillar actin was visualized with FITC-conjugated phalloidin (Sigma-Aldrich, Deisenhofen, Germany). The coverslips were mounted on histoslides and the stainings were observed by laser scanning confocal microscopy (Leica, Heidelberg, Germany) with single-channel excitation. Confocal images were acquired and stored using the Leica confocal software and mounted using Adobe Photoshop.
Cell adhesion and spreading assays. Multiwell tissue culture plates (96 wells; Costar, Bodenheim, Germany) were coated with collagen I (20 µg/ml; Seromed-Biochrom), laminin 1 (20 µg/ml; kindly provided by Dr. R. Timpl, Max-Planck Institut für Biochemie, Martinsried, Germany), fibronectin (40 µg/ml; Chemicon) or laminin 5 (5 µg/ml [20 (link)]). After saturation of the wells with 1% BSA, equal numbers of control, Cdc42-QL, Rac1-QL and RhoA-QL cells were seeded in triplicate for 15, 30 and 60 min in FCS-free DMEM. At the end of the experiments, non-adherent cells were removed by washing with PBS and adherent cells were fixed, stained with crystal violet and the extent of adhesion was quantified by colorimetry as previously reported [21 (link)]. To monitor cell spreading, photographs of adherent cells were taken from triplicate wells with a phase contrast microscope (Axiovert S100TV; Zeiss) equipped with a monochromatic digital camera (PowerShot G5; Canon, Tokyo, Japan). Spreading was quantified by visual counting of round (no visible cytoplasm) and spread (visible cytoplasm) cells in a population of at least 100 adherent cells on each photograph. Data were expressed as average of triplicate measurements ± SD.
Cell migration assays. Confluent cells were suspended by 0.05% trypsin and 0.02% EDTA in PBS, centrifuged and resuspended in FCS-containing DMEM at high cell density (10⁶ cells/ml). Aliquots of the cell suspension (10 µl) were deposited as colonies in the center of duplicate wells (one colony/well, 24-well tissue culture plates; Costar) and the cells were allowed to attach at 37 °C in a humidified incubator. After 1 h, the colonies were washed with PBS and the wells were filled with 400 µl of FCS-free medium. At this time point (T0), a first photograph of each colony was taken with an inverted phase contrast microscope (Zeiss Axiovert S100TV, Leipzig, Germany) equipped with a CCD camera (Xillix MicroImager, Richmond, Canada) and further photographs of the colonies were captured automatically every 10 min for the next 800 min. Images were stored and processed using Openlab software (Improvision, Heidelberg, Germany). The sequences of images were converted to Quick Time movies to analyze cell migration tracks using Dynamic Image Analysis System software (Solltech, Oakdale, Iowa). Twenty tracks were analyzed under each condition and extracted migration parameters included cell velocity (speed in µm/min) and directed migration index defined by the ratio between the linear and the absolute distances covered by a cell during the time of recording. An index of 1 indicates that a cell moves following a straight path while a value of 0 indicates random movement. Differences between clones were analyzed by Student’s t test and were considered to be significant at p < 0.05.
Collagen gel contraction. Cells were seeded in triplicate at a density of 1.5×10⁵ cells/ml into 32-mm bacteriological dishes (2 ml/dish; Renner, Dannstatt, Germany) in DMEM supplemented with 10% FCS, Na-ascorbate (50 µg/ml), antibiotics and 0.3 mg/ml of newborn calf skin, acid-extracted collagen I (IBFB-Pharma, Leipzig, Germany) as previously described [22 (link)]. The cultures were placed at 37°C to allow collagen polymerization (an intrinsic and specific property of collagen I) and gradual lattice contraction was monitored by measuring the gel diameter of triplicate set ups at successive time points up to 48 h. Data were expressed as average of triplicate measurements ± SD. Phase contrast photographs of gelembedded fibroblasts were taken using an Olympus IX-81 microscope equipped with a monochromatic digital camera.