Mitochondrial fission and bioenergetics mediate human lung fibroblast durotaxis

Pulmonary fibrosis is characterized by stiffening of the extracellular matrix. Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we found primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness–regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a dynamin-related protein 1/mitochondrial fission factor–dependent (DRP1/MFF-dependent) pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. These findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a potentially new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of human idiopathic pulmonary fibrosis.

For cell migration assay on stiffness-gradient gels, lung fibroblasts were serum-starved for 24 hours and then were seeded on stiffness-gradients gels in the presence or absence of 2 µM p259 or TAT-carrier control peptides, 1 mM 2-DG, and 5 µM Oligomycin A. To reduce fibroblast proliferation and aid cell migration, 0.5% FBS were added to lung fibroblasts cultured on stiffness-gradient gels. Plasmids or siRNAs were transfected into 1 x 10 6 lung fibroblasts by a Nucleofector 2b device (Lonza, Basel, Switzerland) according to the manufacturer's protocol.
Briefly, cell pellets were resuspended in 100 µl room-temperature Nucleofector Solution R and 2 µg DNA or 100 nM siRNA. Cell suspension was transferred into a cuvette, and electroporation was carried out using Nucleofector Program V-001. 500 µl of the pre-equilibrated culture medium was immediately added to the cuvette. Transfected cells were gently transferred to the prepared hydrogels and cultured until harvesting.

Quantitative analysis of mitochondria
Quantifications with the Mitochondrial Analyzer were performed based on standard confocal fluorescence microscopy and the open-source image analysis platform ImageJ/Fiji as described previously (2). Briefly, confocal micrographs were processed and thresholded, and the resulting binary images were used as inputs for measuring the mitochondrial area. For network connectivity analysis, the binarized mitochondria were converted into topological skeletons. The skeleton map was used to calculate the number of branches, branch lengths, and branch junctions in the skeletonized network.

Quantitative real-time PCR
Total RNA was extracted with the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). 1 μg total RNA was reversely transcribed into cDNA with a cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA). Quantitative PCR reactions were carried out in a Bio-Rad iCycler (Bio-Rad Laboratories, Hercules, CA). Each sample was run in triplicate. Relative quantification was calculated using the comparative CT method. Delta CT values of target gene were normalized to GAPDH and subjected to statistical analysis. The ratio of distal transcripts/common transcripts was calculated as ΔCT = (CTdistal -CTGAPDH) -(CTcommon -CTGAPDH) = CTdistal -CTcommon.

Immunoblot and densitometry analysis
Cell lysates containing 10 -20 µg total proteins were loaded onto multiple SDSpolyacrylamide gels under reducing conditions. Each gel loaded with the same samples with the same amount of cell lysates was used for detection of a specific target protein. After electrophoresis, proteins were electrophoretically transferred from the gels to nitrocellulose at 100 V for 1.5 hr at 4°C. Membranes were blocked in casein solution (1% casein, 25 mM Na2HPO4, pH 7.1) for 1 hr at room temperature. Primary antibodies were diluted in TBS-T and casein solution (1:1) at a working concentration recommended by manufactures. Membranes were incubated with primary antibodies at room temperature for 1 hr. After extensive washing, membranes were incubated with peroxidase-conjugated secondary antibodies (0.1 µg/ml) diluted in TBS-T for 1 hr at room temperature.
Immunodetection was carried out by chemiluminescence. Blot images were scanned. Bands were quantified by ImageJ (NIH, Bethesda).
An equal number of living cells (1 x 10 5 cells per chamber) were suspended in 500 µl of serumfree medium and then plated in the upper chamber of transwell inserts that feature a tissue culture-treated polyester (PET) membrane for optimal cell attachment (VWR, Radnor, PA, USA). Culture medium with 10% FBS used as chemoattractant was added to the lower chambers. Cells were cultured in a humidified incubator at 37°C with 5% CO2. Cell migration was measured at 7 hours to minimize the potential effect of differential matrix stiffness on fibroblasts proliferation. Transwell inserts were washed twice with phosphate-buffered saline.
Cells on the inside of the transwell inserts were gently removed using moistened cotton swabs, and cells on the lower surface of the membrane were then stained with 0.5% crystal violet for 30 min. The transwell inserts were washed twice with PBS to remove unbound crystal violet and then air-dried. The migrated cells were observed and imaged under a Nikon Eclipse TS2 microscope equipped with DS-Fi3 camera. Inserts were eluted in 400 µl 33% acetic acid for 10 min. The eluents were transferred to a 96-well microplate and absorbance was measured at 590 nm using a plate reader. Cell number was calculated based on a standard curve plotted from a series of numbers of human lung fibroblasts that migrated on transwells.

Measurements of ATP levels
ATP levels were measured using a luminescent ATP detection assay kit according to the manufacturer's protocol (Cayman, Ann Arbor, MI, USA). Luminescence was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The ATP concentration was determined by interpolation within the ATP standard reference. The relative ATP levels were corrected by total DNA.

Mechanical testing by AFM microindentation
The

Flow cytometry
Single cell suspensions were pelleted and washed with PBS three times. Flow cytometry was performed on a LSRII Flow Cytometer (BD Biosciences, San Jose, CA). Data were processed using FACSDiva software (BD Biosciences).

Confocal fluorescence microscopy and time-lapse microscopy
Cells cultured on PA gels were loaded with 50nM MitoTracker Red CMXRos (Invitrogen, Eugene, OR, USA) for 45 min to stain mitochondria. Then, cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, and incubated with phalloidin CF488A (Biotium, Fremont, CA, USA) to stain actin filaments. Nuclei were stained with DAPI (Thermo Fisher Scientific, Waltham, MA). Fluorescent signals were detected using a confocal laserscanning microscope Zeiss LSM510 confocal microscope (Oberkochen, Germany) equipped with a digital color camera (Oberkochen, Germany). All fluorescent images were generated using sequential laser scanning with only the corresponding single wavelength laser line, activated using acousto-optical tunable filters to avoid cross-detection of either one of the fluorescence channels. The area of mitochondria was measured using ImageJ (NIH, Bethesda).
Time-lapse imaging of single lung fibroblast migration on stiffness-gradient gels was conducted using a Lionheart FX automated imaging system from BioTek (Winooski, VT), with images collected at 30-min intervals for 24 hours at 37°C and 5% CO2. Images were acquired using the Gen5 software, and the resulting pictures were further handled with ImageJ.

Lung histology and immunofluorescent staining
Masson trichrome staining was performed to evaluate collagen deposition in mouse lung Supplemental Figure 6: Tracking single human lung fibroblast migration on stiffnessgradient hydrogels by time-lapse microscopy. A, Primary human lung fibroblasts were cultured on stiffness-gradient PA gels. Single cell movement was recorded for 24 hours by a Lionheart FX automated microscope. Scale bar = 1000 µm; B, Cell tracking and trajectory analysis was performed using the MTrackJ plugin in ImageJ. The distance of durotactic migration (231 µm) was determined as described previously.
Supplemental Figure 7: Effects of p259 peptides on ATP production. Primary human lung fibroblasts were cultured on soft and stiff PA gels in the presence of p259 peptides or the control TAT peptides. The levels of ATP produced by cells were determined by luminescent ATP detection assays. Results were corrected by total DNA. Bar graphs represent mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA.
Supplemental Figure 8: Effects of p259 peptides, oligomycin, and 2DG on single human lung fibroblast durotaxis. Primary human lung fibroblasts were cultured on stiffness-gradient PA gels in the presence of p259 peptides or the control TAT peptides (A), Oligomycin or DMSO (vehicle for Oligomycin ) (B), and 2DG or PBS (vehicle for 2DG) (C). Single cell movement was recorded for 24 hours by a Lionheart FX automated microscope. Cell tracking and trajectory analyses were performed using the MTrackJ plugin in ImageJ. The distance of durotactic cell migration under each condition was determined as described previously. Bar graphs represent mean ± SD per cell from 30 individual cells isolated from 3 independent human subjects (n = 10 cells per subject) under each condition. Scale bar = 1000 µm. A two-tailed Student's t test was used for comparison between groups.
Supplemental Figure 9: Evaluation of DRP1 and MFF expression in (myo)fibroblasts in normal and fibrotic lungs of human and mice. Frozen lung tissue sections from normal and fibrotic human and mouse lungs were subjected to co-immunofluorescence staining of DRP1/Drp1, MFF/Mff, and normal fibroblast marker (NPNT/Npnt) and myofibroblast marker (αSMA/αSma). Co-localization of DRP1/MFF expression with (myo)fibroblasts was evaluated by Pearson's correlation coefficient analyses. Scale bar = 20 µm.
Supplemental Figure 1: Evaluation of mitochondrial mass by flow cytometry analysis. Human lung fibroblasts cultured on soft and stiff PA gels were stained by MitroTracker Green. The intensity of green fluorescence, a measure of mitochondrial mass (3,4), was determined by flow cytometry analysis. Results are mean ± SD of three independent experiments. A two-tailed Student's t test was used for comparison between groups. Supplemental Figure 4 Supplemental Figure 4: Evaluation of cell attachment and viability of pre-conditioned human lung fibroblasts in transwells. Human lung fibroblasts were cultured on soft and stiff PA gels for 72 hours. Pre-conditioned cells were detached from PA gels by trypsin. An equal number of pre-conditioned living lung cells were seeded in transwells and incubated for 2 hours. Cell attachment and viability were evaluated by crystal violet staining followed by colorimetric assays. Results are mean ± SD of three independent experiments. A two-tailed Student's t test was used for comparison between groups. Supplemental Figure 6: Tracking single human lung fibroblast migration on stiffnessgradient hydrogels by time-lapse microscopy. A, Primary human lung fibroblasts were cultured on stiffness-gradient PA gels. Single cell movement was recorded for 24 hours by a Lionheart FX automated microscope. Scale bar = 1000 µm; B, Cell tracking and trajectory analysis was performed using the MTrackJ plugin in ImageJ. The distance of durotactic migration (231 µm) was determined as described previously.

Supplemental Figure 7
Supplemental Figure 7: Effects of p259 peptides on ATP production. Primary human lung fibroblasts were cultured on soft and stiff PA gels in the presence of p259 peptides or the control TAT peptides. The levels of ATP produced by cells were determined by luminescent ATP detection assays. Results were corrected by total DNA. Bar graphs represent mean ± SD of three independent experiments. Statistical analysis was performed by one-way ANOVA.
Supplemental Figure 8: Effects of p259 peptides, oligomycin, and 2DG on single human lung fibroblast durotaxis. Primary human lung fibroblasts were cultured on stiffness-gradient PA gels in the presence of p259 peptides or the control TAT peptides (A), Oligomycin or DMSO (vehicle for Oligomycin ) (B), and 2DG or PBS (vehicle for 2DG) (C). Single cell movement was recorded for 24 hours by a Lionheart FX automated microscope. Cell tracking and trajectory analyses were performed using the MTrackJ plugin in ImageJ. The distance of durotactic cell migration under each condition was determined as described previously. Bar graphs represent mean ± SD per cell from 30 individual cells isolated from 3 independent human subjects (n = 10 cells per subject) under each condition. Scale bar = 1000 µm; A two-tailed Student's t test was used for comparison between groups.