Chemicals and antibodies
DME medium was obtained from Nissui (Tokyo, Japan) or Sigma (St. Louis, MO). FCS was purchased from GIBCO BRL Life Technologies (Paisley, Scotland) or Sigma. Other tissue culture reagents were purchased from GIBCO. All lipids were from Sigma. Glutaraldehyde (70% stock in water, EM grade), paraformaldehyde (PFA), and organic solvents were purchased from Nacalai Tesque (Kyoto, Japan). Anti-β-actin mouse mAb (A5441), anti-β-tubulin mouse mAb (T4026), anti-vinculin mouse mAb (V9131), anti-mouse IgG FITC conjugate (F2012), anti-rabbit IgG FITC conjugate (F1262), anti-rabbit IgG TRITC conjugate (T5268), and TRITC labeled phalloidin were purchased from Sigma. Anti-mouse IgG rhodamine conjugate (AP124R) was obtained from Chemicon International (Temecula, CA). Affinity-purified polyclonal antibodies (95/1) were used for the identification of moesin by immunoblotting and for immunofluorescence. The affinity-purified polyclonal antibodies against threonine558-phospho-moesin were prepared as described previously . Actin was prepared as described previously . Tubulin was purchased from Cytoskeleton (Denver, CO).
NIH 3T3 fibroblasts were grown in DME medium containing 1000 mg/liter glucose, supplemented with 10% FCS, 4 mM L-glutamine, penicillin (100 IU/ml), and streptomycin (100 μg/ml), at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were washed with PBS containing 1 mM CaCl2 and 1 mM MgCl2 (PBS(+)), detached by treating with 0.05% trypsin in PBS or 0.02% EDTA in PBS. The suspended cells were washed with PBS, resuspended in growth medium, and plated on FCS-coated glass coverslips for microscopy, or onto 35-mm or 100-mm Falcon plastic culture dishes (Beckton Dickinson, Lincoln, NJ) or FCS-coated glass culture dishes for biochemical experiments. In some cases, detached cells were resuspended in growth medium without washing.
Scanning electron microscopy
For the observation of cellular surface, cells were rinsed with PBS(+) and fixed with PBS(+) containing 2% glutaraldehyde (dilute 70% glutaraldehyde just before use) at 4°C for 1 hour. In most references, cells were rinsed with PBS (although PBS, in some references, means PBS that contains CaCl2 and MgCl2), but we preferred PBS(+) because retraction of cells were observed in a few minutes when the cells were exposed to PBS even briefly. Samples were then washed with distilled water, dehydrated in a graded series of ethanol, transferred into t-butylalchohol, and dried in Freeze Dryer ES-2030 (Hitachi, Japan). Dried samples were coated with Pt-Pd in Ion Sputter E-1030 (Hitachi, Japan) for 120 seconds, and were examined under an S-4200 scanning electron microscope (Hitachi, Japan) at 15 kV. Micrographs were taken at 0° or 60° tilt.
Fixation and extraction for microscopy
Cells on glass coverslips were rinsed with PBS(+), fixed and/or extracted by one of the procedures listed in Table 1. Methanol-free formaldehyde solution can be obtained from commercial sources, but it has to be used within a week and is more expensive than PFA powder . Although formaldehyde and PFA were used in papers as listed in Table 1 and formaldehyde solutions may have been obtained from commercial sources, for our study a formaldehyde aqueous stock solution was prepared by dissolving 20 g of PFA powder in 90 ml of double distilled water. The mixture was heated to 60-65°C and the pH was adjusted to 7.3 with NaOH. The solution was diluted with water to 100 ml (final concentration 20%) and stored at 4°C for not more than one week. In earlier work, formaldehyde solutions may have been prepared from formalin that contains 37% formaldehyde and 8∼ 15% methanol. Unless noted as formalin, formaldehyde solutions were solely prepared here as described above. Therefore, formaldehyde and PFA are essentially indistinguishable. Buffers were prepared as 2× or 5× stock solutions. Magnesium-and calcium-free PBS was used unless noted otherwise in the references. When detailed descriptions were lacking, specimens were treated as listed in Table 1.
Confocal laser scanning microscopy
Blocking of nonspecific binding sites and incubation with antibodies basically followed procedures detailed in references, but cells were stained as follows when descriptions were lacking. After the cells were soaked in 1% BSA/PBS for 30 min, they were treated with the primary antibodies in 1% BSA/PBS for 1 h. The cells were then washed with PBS three times, followed by incubation with secondary antibodies in 1% BSA/PBS for 1 h. For the detection of actin filaments, 100 ng/ml TRITC-phalloidin was mixed with the second antibody solution. For double staining, secondary antibodies that did not cross-react with each other were chosen. Cells were washed three times in PBS for 5 min and rinsed with water at room temperature, mounted in PermaFluor (Shandon, Pittsburgh, PA) and observed under a laser scanning confocal microscope (MRC-1024, Bio-Rad Laboratories, Richmond, CA). Confocal sections were taken with an iris aperture of 2.0. Images were processed using Adobe Photoshop software.
Evaluation of crosslinking by polypeptide analysis after fixation and extraction
Subconfluent cultures of NIH3T3 cells on 3.5 cm plastic culture dishes, were rinsed with PBS(+), fixed with 4% paraformaldehyde in PBS(+) at room temperature or 37°C, or fixed and/or extracted by one of the procedures listed in Table 1. For some experiments, cells after fixation were treated twice with 0.5 mg/ml NaBH4 in PBS for 10 min at room temperature. After rinsing with PBS, the any material remaining on the dish was extracted with 200 μl of 1× SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 10% 2-mercaptoethanol, pH 6.8) at room temperature for 30 min in preparation for SDS-PAGE. The dissolved material was recovered and heated at 95°C for 10 min and resolved by SDS-PAGE on a 9% polyacrylamide gel. Polypeptides were stained with Coomassie Brilliant blue or immunologically stained with antibodies after transfer to nitrocellulose or polyvinylidenedifluoride membranes.
Extraction for western blotting
NIH 3T3 cells were detached from the dishes in the presence of 1 μM calyculin A, but not staurosporine and pervanadate. In order to exclude the effect of attachment on the extractability of proteins, suspended cells were used as a control sample. The cells were cultured on 10 cm plastic culture dishes as described above. The subconfluent cells were washed three times with PBS(+), once with PBS, detached by treating with 0.02% EDTA in PBS. The suspended cells were collected, centrifuged at 1,000 × g for 5 min, and resuspended with PBS. The suspensions were divided and incubated with or without 1 μM calyculin A, 1 μM staurosporine, or 100 μM pervanadate in 200 μl of PBS(+) for 10 min at 37°C. The cells were extracted for 5 min on ice with 200 μl of 2× detergent extraction buffer (2% of Triton X-100 or DOTMAC, 50 mM Tris-HCl, 10 mM EGTA, 200 mM NaCl, 2 mM MgCl2, protease inhibitor cocktail [20 μg/ml aprotinin, 20 μM E-64, 200 μM leupeptin, 200 μM p-amidinophenylmethanesulfonyl fluoride], 2 μM calyculin A, 10 mM sodium pyrophosphate, 2 μM staurosporine, 20 μM phalloidin, and 20 μg/ml taxol). The lysates were centrifuged at 15,800 × g for 5 min at 4°C. The resulting pellets were solubilized in 150 μl of 1× SDS sample buffer. Total lysate was prepared by adding 150 μl of 1× SDS sample buffer to the pelleted cells after drug treatment described above. All samples were heated at 95°C, sonicated and resolved by SDS-PAGE on a 9% polyacrylamide gel electrophoresed under reducing conditions as described . β-Actin, moesin, and phosphorylated moesin were detected by immunoblotting and the enhanced chemiluminescence detection system.
Lipid analysis of detergent-resistant components
When NIH 3T3 fibroblasts were treated with Triton X-100 lysis buffer, most of the cells detached from the culture dish. This made it impossible to quantitatively analyze lipids in the detergent-insoluble fraction of attached growing cells. Therefore, the cells were suspended using EDTA before lipid analysis and extracted with detergent extraction buffer as described above. The lysates were centrifuged at 15,800 × g for 5 min at 4°C. The supernatant was discarded and the pellet was washed with PBS. The lipids were extracted with 0.5 ml of chloroform/methanol (2:1, v/v) in an ultrasonic bath for 30 min at 37°C from the pellet. After centrifugation at 15,800 × g for 5 min, the supernatant was concentrated under nitrogen and spotted on a 10 × 10 cm Silica gel thin layer glass plate (LHP-K, Whatman). Phospholipids were separated by chromatography in 100:75:7:4 of chloroform:methanol:acetic acid:water. Plates were stained with 40% sulfuric acid in water. L-α-phosphatidylethanolamine, L-α-phosphatidylcholine, L-α-phosphatidyl-L-serine, L-α-phosphatidylinositol, L-α-phosphatidylinositol 4,5-biphosphate, sphingomyelin were identified by using a standard solution.
When the cells were treated with DOTMAC lysis buffer, they did not detach and, therefore, lipids could be extracted directly from the cells on glass culture dishes with 1 ml of chloroform/methanol (2:1, v/v) after treatment with the detergent extraction buffer. The solvents and residues were collected in a tube and sonicated in an ultrasonic bath as described above. After centrifugation at 15,800 × g for 5 min, lipids were analysed from the supernatant as described above.
Transmission electron microscopy
The phalloidin-stabilized α-actin filaments (1 μM) were incubated in buffer F (5 mM Tris-HCl, pH 7.5, 0.5 mM Na2ATP, 2 mM MgCl2, 140 mM NaCl, 0.2 mM DTT, 0.2 mM CaCl2, 0.005% sodium azide) with or without 0.1 % DOTMAC for 10 min at 25°C. The taxol-stabilized β-tubulin (1 μM) was also incubated in PEM buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH6.8) containing 1 mM GTP with or without 0.1 % DOTMAC for 10 min at 25°C. 10 μl of samples were adsorbed for 60 s to carbon-coated 400 mesh/inch copper grids that were rendered hydrophilic by glow discharge for 15 s in air at low pressure. The grids were washed with 100 mM KCl and stained for 60 s on six drops of 1% uranyl acetate. Microscopy was performed on a HITACHI H-8100 electron microscope at an accelerating voltage of 100 kV.