Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction (original) (raw)

Cells. Lmna+/+ and Lmna–/– mouse embryo fibroblasts were maintained in DMEM (Invitrogen Corp., Carlsbad, California, USA) containing 10% FCS (HyClone Laboratories, Logan, Utah, USA) and penicillin/streptomycin (Invitrogen Corp.).

Nuclear strain experiments. Cells were plated at 900 cells/cm2 on fibronectin-coated silicone membranes in DMEM supplemented with 10% FCS followed by serum starvation for 48 hours in DMEM containing insulin, transferrin, and selenium (ITS) supplement (Sigma-Aldrich, St. Louis, Missouri, USA). Preceding the strain experiments, cells were incubated with Hoechst 33342 nuclear stain (1 μg/ml; Molecular Probes Inc., Eugene, Oregon, USA) in DMEM plus ITS for 20 minutes. Membranes were placed on a custom-made strain device mounted on an Olympus IX-70 microscope (Olympus America Inc., Melville, New York, USA), and biaxial strain was applied in a stepwise fashion. Membrane and nuclear strain was computed on bright field and fluorescence images using a custom-written image-analysis algorithm. Normalized nuclear strain was defined as the ratio of nuclear strain to membrane strain to compensate for small variations in applied membrane strain (range 17.4–19.8%).

Magnetic bead microrheology. Cells were plated on 35-mm polystyrene dishes (Corning-Costar Corp., Corning, New York, USA). The following day, cells were incubated with fibronectin-coated paramagnetic beads (Dynal Biotech Inc., Lake Success, New York, USA) for 30 minutes. To minimize nuclear effects, only beads attached more than 5 μm from the nucleus were selected for analysis. A sinusoidal force (amplitude 0.6 nanonewtons [nN], frequency 1 Hz, offset 0.6 nN) was applied through a magnetic trap, and bead displacement was monitored using a digital camera (Roper Scientific Inc., San Diego, California, USA). Displacement amplitudes were computed using custom-written MATLAB (The MathWorks Inc., Natick, Massachusetts, USA) algorithms. In separate experiments, smaller (2 μm) fibronectin-coated polystyrene beads (Bangs Laboratories Inc., Fishers, Indiana, USA) were incubated together with the magnetic beads for 1 hour to adhere to the cell surface. Cells containing single magnetic beads and several polystyrene beads were subjected to a brief force pulse (2.5 nN for 3 seconds). Using custom-written MATLAB algorithms, maximal induced magnetic and polystyrene bead displacements were computed and expressed in cylindrical coordinates (r, θ) with the magnetic bead at the origin and θ = 0 for the force direction. The induced strain field can be described by an analytical cell mechanics model proposed by Bausch et al. (20) expressing the radial component _u_r of the induced bead displacement as a function of the applied force F, cell stiffness μ*, the characteristic cut of radius κ−1, the distance from the magnetic bead center r, and the polar angle θ:

Equation1

where _Κ_0 and _Κ_1 are modified Bessel functions of the second kind (order 0 and 1, respectively) and using κ1 = [(1 – ς)/2]1/2κ. The parameters μ* and κ were obtained by fitting equation 1 to the bead displacement data using the GraphPad Prism 4.0 robust curve fit function (GraphPad Software, San Diego, California, USA) and assuming ς = 0.5 for incompressible media and a magnetic bead contact radius of 2 μm.

The magnetic trap calibration was performed as described previously (21). In brief, magnetic beads suspended in viscous solution were monitored while being attracted to the magnetic trap operated at various currents. The applied force as a function of current and distance from the magnetic trap was then computed based on Stokes’ law.

Microinjection. Cells were plated on fibronectin-coated glass dishes (WillCo Wells BV, Amsterdam, The Netherlands) or silicone dishes and incubated overnight. Microinjections were performed using an Eppendorf microinjector with Eppendorf Femtotips (Eppendorf AG, Hamburg, Germany). In each dish, 20–50 cells were injected with Texas Red–labeled 70-kDa dextran (Molecular Probes Inc.) dissolved at 10 mg/ml in PBS (Invitrogen Corp.), either into the cytoplasm (injection pressure 500 hectopascals [hPa], injection time 0.6 seconds) or into the nucleus (injection pressure 10, 100, 500, and 1,500 hPa, injection time 0.6 seconds). Following microinjection, cells were washed in HBSS (Invitrogen Corp.), and intracellular localization of dextran–Texas Red was recorded under a fluorescent microscope. Selected silicone membranes were subjected to constant biaxial strain (about 32% for 30 minutes) or cyclic biaxial strain (10% at 1 Hz for 24 hours), and localization of Texas Red–labeled dextran in strained and control cells was analyzed on a fluorescence microscope (Olympus America Inc.).

Strain experiments. Strain stimulation was carried out as previously described (22). In brief, cells were plated on fibronectin-coated silicone membranes (2,500–3,300 cells/cm2). After 72 hours of serum starvation, cells were subjected to biaxial cyclic strain (4% or 10% at 1 Hz). For chemical stimulation, cells were incubated with IL-1β (25 ng/ml; R&D Systems Inc., Minneapolis, Minnesota, USA) or PMA (200 ng/ml, Sigma-Aldrich) in DMEM plus ITS.

DsRed/peroxiredoxin-2 localization. Cells were plated on fibronectin-coated glass dishes or fibronectin-coated silicone membranes. Following overnight incubation, cells were transfected with a CMV promoter–driven DsRed/peroxiredoxin-2 fusion construct (CLONTECH Inc., Palo Alto, California, USA) using FuGENE 6 (F. Hoffman–La Roche Ltd., Basel, Switzerland) and incubated for 24 hours. Selected silicone membranes were subjected to constant (about 19% for 60 minutes) or cyclic biaxial strain (10% at 1 Hz for 3 hours), and localization of DsRed-labeled peroxiredoxin-2 in strained and control cells was analyzed on a fluorescence microscope (Olympus America Inc.).

Flow cytometry. For cell viability assays, propidium iodide (2 μg/ml, Sigma-Aldrich) was added to the dishes after 24 hours of strain application. Cells were collected and analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter Inc., Fullerton, California, USA), counting 10,000–30,000 events in each group. Thresholds for propidium iodide incorporation were determined based on negative (no propidium iodide staining) and positive (cells permeabilized by 50% ethanol) controls. Apoptotic and necrotic cell fractions were measured in similar experiments using the Vybrant Apoptosis Assay Kit no. 3 (Molecular Probes Inc.).

Northern and Western analyses. Expression of iex-1 and egr-1 mRNA was assessed by Northern analysis as described previously (23). Protein expression was analyzed by Western analysis of nuclear and cytoplasmic cell fractions using antibodies against NF-κB p65 (antibody does not recognize p50 or p105), IκBα (both from Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), and actin (Sigma-Aldrich). Additional immunoblotting was performed on whole cell lysates using specific antibodies against total ERK1/2 (Santa Cruz Biotechnology Inc.) and phospho-p44/p42 MAP kinase (Cell Signaling Technology, Beverly, Massachusetts, USA). After incubation with HRP-conjugated secondary antibody (Bio-Rad Laboratories Inc., Hercules, California, USA), specific bands were visualized by enhanced chemiluminescence (PerkinElmer Inc., Boston, Massachusetts, USA).

Luciferase experiments. Cells were transfected with plasmids for NF-κB–controlled luciferase expression and SV40-regulated β-gal (Promega Corp., Madison, Wisconsin, USA) using FuGENE 6 (F. Hoffman–La Roche Ltd.). Following transfection, cells were serum starved in DMEM plus ITS medium for 48 hours, followed by overnight stimulation with PMA (200 ng/ml) or IL-1β (25 ng/ml). Luciferase assays were quantified in a Victor2 Multilabel Counter (Perkin Elmer Inc.). Results were normalized for β-gal activity and expressed as percent WT control.

Immunohistochemistry. Cells were plated on untreated or fibronectin-coated chamber slides and serum-starved for 24–72 hours followed by stimulation with IL-1β. Cells were fixed in 4% paraformaldehyde or methanol, washed in PBS, and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated overnight with primary rabbit antibody anti–NF-κB (p65; Santa Cruz Biotechnology Inc.) at 4°C or Alexa Fluor 568 phalloidin (A-12380; Molecular Probes Inc.) for 1 hour at 25°C, followed by 1 hour of incubation with secondary FITC- or TRITC-conjugated antibodies.

Cellular protein fractions and electrophoretic mobility shift assay. Nuclear extracts were prepared as described previously (24) with the following modifications. Cells were washed with ice-cold PBS and lysed in buffer A, which consisted of 0.1% Triton X-100, 10 mM EDTA, 10 mM EGTA, 10 mM KCl, 10 mM HEPES, 1 mM DTT, 0.5 mM PMSF, and protease inhibitor cocktail (P-8340; Sigma-Aldrich). After centrifugation at 1,200 g for 10 minutes, the supernatant was stored as the cytoplasmic cell fraction, while the nuclear pellet was washed once in PBS, resuspended in buffer C (1 mM EDTA, 1 mM EGTA, 0.4 M NaCl, 20 mM HEPES, 5 mM MgCl2, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, and protease inhibitor cocktail), and incubated at 4°C for 10 minutes. The nuclear extract was centrifuged for 10 minutes at 4°C at 12,000 g, and the supernatant was used for Western analysis and electrophoretic mobility shift assay. NF-κB–specific oligonucleotides (Promega Corp.) were end-labeled using T4 polynucleotide kinase and [γ-32P]ATP (DuPont NEN Research Products, Boston, Massachusetts, USA). Nuclear extracts were preincubated for 10 minutes in binding buffer followed by 20 minutes of incubation at room temperature with labeled oligonucleotide. Samples were separated on a 4% native polyacrylamide gel. For competition studies, an excess (50 times) of unlabeled oligonucleotide was used, and for the supershift assay, the nuclear extracts were incubated with 2 μg of anti-p50 or anti-p65 antibody (Santa Cruz Biotechnology Inc.).

Statistical analysis. All experiments were performed at least three independent times. Data are expressed as mean ± SEM. Statistical analysis was performed using PRISM 4.0 and INSTAT software (GraphPad Software Inc., San Diego, California, USA). The data were analyzed by unpaired t test (allowing different SDs),one-way ANOVA, or the Mann-Whitney test in case of nonparametric distribution. A two-tailed P value below 0.05 was considered significant.