Microcystin-LR and bisindolylamaleimide IV were purchased from EMD Biosciences (La Jolla, CA). Myristoylated-PKC ζ peptide antagonist (N
-Myristoyl-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu), R18 14-3-3 peptide antagonist, and glutathione S
-transferase (GST)-14-3-3ζ recombinant protein were purchased from BIOMOL (Plymouth Meeting, PA). GSH Sepharose beads were from Amersham Biosciences (GE HealthCare Bio-Sciences, Piscataway, NJ). K18 mAb (Ks 18.04) for Western blotting was purchased from Research Diagnostics (Flanders, NJ). K18pSer33 antibody was kindly provided by Dr. Bishr Omary (Stanford University, CA). K8/18 polyclonal antibody for immunoprecipitation was produced in rabbits using keratins from a rat liver preparation as antigen (Toivola et al., 1997
). PKC ζ mAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies for immunofluorescence, goat anti-mouse and goat anti-rabbit fluorescein, and rhodamine-tagged immunoglobulins were obtained from Molecular Probes (Eugene, OR). Secondary antibodies for immunoblotting were peroxidase-labeled goat anti-mouse or goat anti-rabbit immunoglobulins (Bio-Rad Laboratories, Hercules, CA). Protein A/G agarose beads were purchased from Santa Cruz Biotechnology.
Cells derived from a human lung adenocarcinoma (A549) were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM supplemented with 10% FBS, 2 mM l
-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated in a humidified atmosphere of 5% CO2
/95% air at 37°C. A549 cells expressing a dominant negative PKC ζ (Dada et al., 2003
) were propagated in complete DMEM supplemented with G418.
A549 cells were cultured on 25 × 75 × 1-mm glass slides and subjected to continuous laminar flow to generate shear stress (30 dyn/cm2) using the FlexCell Streamer Device (FlexCell Streamer Device; FlexCell International, Hillsborough, NC).
A549 cells grown on glass slides were rinsed three times in PBS and fixed in either methanol (−20°C) for 5 min or 3.7% formaldehyde at room temperature (RT) for 9 min. After formaldehyde fixation, cells were permeabilized with 1% Triton X-100 in PBS for 30 min. Cells were then washed three times with PBS and processed for indirect immunofluorescence as previously described (Ridge et al., 2005
). After staining, the glass slides were washed in PBS and mounted in gelvatol containing 100 mg/ml Dabco (1,4-diazabicyclo [2.2.2] octane; Sigma-Aldrich, St. Louis, MO; Prahlad et al., 1998
). Images of fixed, stained preparations were taken with a Zeiss LSM 510 microscope (Carl Zeiss, Thornwood, NY; Prahlad et al., 1998
). Intensity modulated display (IMD) images were processed using the Ratio Imaging tool in MetaMorph Software (Universal Imaging, Downingtown, PA). Changes in KIF structure were evaluated blind by two different individuals, who compared immunofluorescence images of static control and shear-stressed cells.
Transmission Electron Microscopy
Ultrastructural observations of cytoskeletal preparations were performed as described previously (Sivaramakrishnan et al., 2008
). For preservation of IFs, cells on coverslips were extracted with PEM buffer (100 mM Pipes, pH 6.9, 1 mM MgCl2
, 1 mM EGTA) containing 1% Triton X-100, 0.4 M NaCl, 4% polyethylene glycol, and 1 mg/ml DNase 1 (Sigma-Aldrich, St. Louis, MO) for 10 min at RT. To remove any residual actin, all preparations were treated with recombinant gelsolin NH2
-terminal domain. Cells were then fixed with 2% glutaraldehyde, stained with 0.2% uranyl acetate, and processed by critical point drying followed by rotary shadowing with platinum and carbon (Sivaramakrishnan et al., 2008
). Replicas were removed from the coverslips and transferred to copper grids as described (Sivaramakrishnan et al., 2008
) for observation by transmission electron microscopy.
Cell Viability Assay
AEC apoptosis was assessed by annexin V staining (Roche Diagnostics, Alameda, CA) and DNA nucleosomal fragmentation ELISA, as previously described. Briefly, A549 cells were exposed to shear stress, and then the cells in the supernatant and attached to the dish were collected for determination of apoptosis. Annexin V–stained cells were assessed under a fluorescence microscope (Eclipse TE200; Nikon, Melville, NY) by two investigators who were blinded to the experimental protocol. Lactate dehydrogenase released from control and shear-stressed cells was measured using a commercially available assay (Cytotoxicity Detection Kit, Roche Pharmaceuticals, Indianapolis, IN).
Cell Cycle Analysis
Cell cycle analysis was determined in A549 cells that were suspended in 100 μl PBS and fixed by adding 900 μl ice-cold 70% ethanol (overnight, 4°C). Cells were centrifuged (200 × g, 5 min) and subsequently resuspended in PBS containing 0.1% Triton X-100 (Sigma Chemicals), 0.2 mg/ml RNase (Qiagen, Chatsworth, CA), and 50 μg/ml propidium iodide (Molecular Probes) and incubated at 37°C for 30 min. Fluorescence-activated cell sorting (FACS) was performed using the MoFlo High-Performance Cell Sorter (Dako North America, Carpinteria, CA) to determine the proportion of cells in G0, G2/M, and S phases.
Cells were lysed in buffer containing 0.2% Triton X-100 in PBS solution with protease inhibitors (phenyl methyl sulfonate, 100 μg/ml; leupeptin, 2 μg/ml; and N-tosyl-l-phenylal-anine chloromethyl ketone [TPCK], 100 μg/ml) and phosphatase inhibitors (sodium orthovanadate [Na3VO4], 1 mg/ml and 0.126 μg/μl of DNASEI; Sigma Chemicals). Cells were homogenized using a 26-gauge syringe needle (Becton Dickinson, Franklin Lakes, NJ) and were incubated on ice for 45 min to obtain total cell lysates. Protein concentrations were determined using the Standard Bradford Assay (Bio-Rad Laboratories). Samples containing equal amounts of protein were then centrifuged at 10,000 × g for 5 min at 4°C. The pellet constitutes the filamentous keratin fraction, whereas the supernatant is the soluble keratin fraction. All samples were solubilized in boiling Laemmli buffer, loaded on 10% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with primary antibodies as follows: 1) K18pSer33 mAb (1:100 in TBS; gift from Dr. Bishr Omary at Stanford University, CA); 2) K18 mAb (1:100 in TBS), clone Ks 18.04, Research Diagnostics; 3) PKC ζ mAb (1:250 in TBS) from Santa Cruz Biotechnology. Membranes were washed three times with TBS containing 0.1% Tween for 30 min, then incubated with secondary antibodies coupled to horseradish peroxidase (in dilutions recommended by supplier), and visualized using enhanced chemiluminescence (Amersham Biosciences).
Cells were lysed in immunoprecipitation buffer (IP buffer) containing 0.2% Triton X-100 in PBS with protease and phosphatase inhibitors (see above). Cells were homogenized using a 26-gauge needle and incubated on ice for 45 min to obtain total cell lysates. Samples containing equal amounts of protein were sonicated on ice and mixed with IP buffer. Samples were centrifuged at 10,000 × g for 15 min to pellet insoluble K8/18. The supernatant was then mixed with antibodies to K8/18 and incubated on a rotator overnight at 4°C. Samples were again centrifuged at 10,000 × g for 15 min to remove any filamentous K8/18, mixed with protein A/G agarose beads (Santa Cruz Biotechnology) and incubated for 90 min at 4°C on a rotator. Protein A/G beads were spun down and washed three times with IP buffer followed by three washes with cold PBS containing the phosphatase inhibitor. Bead suspensions were then incubated for 120 min in 1× Laemmli buffer at 37°C to release K8/K18 from beads.
PKC Translocation Assay
After the exposure of A549 cells to shear stress, cells were scraped into a lysis buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 5 μg/ml trypsin inhibitor, and 20 μM leupeptin and homogenized for 2 min. Lysates were then centrifuged at 1000 × g
for 10 min to obtain nuclear and supernatant fractions (P1). The supernatant fraction was further centrifuged at 100,000 × g
for 60 min to obtain the membrane fraction (P2) and cytosol fraction (S). The P2 fraction was suspended in lysis buffer containing 0.1% Triton X-100 for 20 min and centrifuged (16,000 × g
, 20 min, 4°C) to separate the detergent-insoluble and -soluble material. Twenty to 50 μg of cytosolic and membrane fractions were then subjected to immunoblotting using isozyme-specific anti-PKC antibodies. Specificity of membrane fractionation was determined by histone H1, a nuclear protein that mainly localizes in the P1 nuclear fraction, and MEK-1, a marker of cytosol protein, present in the S fraction but not in the P1 or P2 fractions (Correa-Meyer et al., 2002
; Ridge et al., 2002
In Vitro Kinase Assay
Cells were lysed and homogenized as described earlier and incubated on ice for 45 min to obtain total cell lysates. Protein concentrations were determined using the Standard Bradford Assay (Bio-Rad Laboratories). Equal protein amounts were then centrifuged at 10,000 × g for 5 min at 4°C. The pellet was solubilized by sonication in lysis buffer (defined earlier) to obtain purified KIF. Either purified KIF or recombinant keratin 18 was added along with recombinant PKC ζ (EMD Biosciences) to reaction buffer containing 10 mM MgCl2, 250 μM EGTA, 400 μM CaCl2, diacylgylcerol 30 μg/μl (Sigma Chemicals), phosphatidylserine 25 μg/μl (Sigma Chemicals), BSA 0.1 μg/μl (Sigma Chemicals), and 100 μM ATP (containing trace 32γ-ATP) in 20 mM Tris HCl, pH 7.4. The reaction mixture was incubated for 30 min at 30°C followed by quenching with 1× Laemmli buffer. Samples were then loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes. 32P activity associated with the keratin 18 band was measured using a Phosphor Imager (Cyclone, PerkinElmer, Norwalk, CT). Protein loading and phosphorylation was checked with antibodies to K18 and K18pSer33.
Live Cell Imaging
Green fluorescent protein (GFP)-K18 stably expressing A549 cells were cultured to ~30% confluence on Bioptechs coverslips (Butler, PA) for 48 h before the experiments. Coverslips were placed into cell-imaging medium (DMEM without phenol red, Hanks' F12 medium, and 0.5 M Tris, pH 7.5 combined 6:3:1) for 60 min at 37°C (Note: this medium is used throughout imaging experiment). PKC ζ antagonist (1 μM final concentration, BIOMOL) was then added to the media for 30 min at 37°C. Coverslips were then sealed into a Bioptechs FCS2 chamber with serum-free media containing appropriate PKC ζ inhibitor (Note: PKC ζ inhibitors remain in the media throughout the experiment for both unsheared and shear stress conditions). The Bioptechs FCS2 chamber maintains the cells and media at 37°C. Unsheared (CT) cells were incubated for 120 min at 37°C. Shear stress (SS) cells were subject to flow through the chamber to yield an average laminar shear stress of 15 dyn/cm2 for 120 min at 37°C. Finally, the Bioptechs chamber was mounted onto the stage of an LSM 510Meta confocal microscope (Carl Zeiss) whose stage and objective preequilibrated at 37°C by an air stream stage incubator (model ASI 400; Nevtek, Burnsville, VA). For SS cells flow was continued through the chamber for the entire duration of the experiment. No flow was passed through the chamber for the CT cells. To ensure the health of the cells for the duration of the experiment, samples of media were taken from the Bioptechs chamber at the start and end of the experiment to measure pH, pCO2 and pO2 using Novamed Blood Gas Analyzer. No significant changes in pH, pCO2 or pO2 were observed in the cell imaging medium from the start to the end of experiment.
Fluorescence Recovery After Photobleaching
Fluorescence recovery after photobleaching (FRAP) was carried out with the LSM 510Meta confocal microscope (Carl Zeiss). Phase-contrast images of cells were taken before and immediately after FRAP to ensure that there were no significant changes in cell shape or position. Bar-shaped regions were bleached using the area-scan function at 488 nm (50% power, 1% attenuation), and recovery of fluorescence was monitored (7% power, 10% attenuation) using the time-series function at 30-min intervals for up to 4 h.
Position, length, and intensity measurements were made on digitized confocal images using Metamorph image analysis software (Universal Imaging). Pixel values were converted to distance using confocal calibration bars. Analyses of the dynamic properties of GFP-K18 fibrils were restricted to cells that showed no obvious shape changes for the duration of the FRAP experiment (3–4 h). FRAP half-times (t1/2) were calculated by monitoring the gray-scale pixel values (intensity measurements) across the bleached GFP-K18 fibrils.
GST-14-3-3 ζ Binding Assay
A549 cells cultured on 25 × 75 × 1-mm glass slides for 3 d were subjected to continuous laminar flow to generate shear stress (30 dyn/cm2) for 60 min using the FlexCell Streamer Device. Unstressed (CT) and SS cells were lysed in 1% NP-40 buffer with protease inhibitors (phenylmethyl sulfonate, 100 μg/ml; leupeptin, 2 μg/ml; and TPCK, 100 μg/ml), phosphatase inhibitors (Na3VO4, 1 mg/ml) and 0.126 μg/μl of DNASEI (Sigma Chemicals). Cells were homogenized using a 26-gauge syringe needle (Becton Dickinson) and were incubated on ice for 45 min to obtain total cell lysates. Protein concentrations were determined using the Standard Bradford Assay (Bio-Rad Laboratories). One hundred micrograms of protein from CT and SS conditions was sonicated (Branson Sonifier, Danbury, CT) to solubilize KIF followed by centrifugation to obtain supernatant fraction (10,000 × g for 5 min). Alternatively recombinant K18 (rK18) was phosphorylated in vitro by recombinant PKC ζ (see below). Supernatant fractions from CT, SS, and rK8 were mixed with 2 μg of recombinant GST-14-3-3ζ and incubated overnight on rotary shaker (4°C). GST-14-3-3ζ was recovered with GSH-Sepharose beads, and washed, and proteins bound to GST-14-3-3ζ were separated on 10% SDS-PAGE, transferred, and immunoblotted with anti-K8/K18 and anti-14-3-3ζ antibodies.
Comparisons were performed using the unpaired Student's t test. One-way ANOVA with Tukey's test was used to analyze the data. p < 0.05 values were considered significant.