Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II

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Deletion of calponin 2 in mouse fibroblasts increases myosin II-dependent cell traction force M. Moazzem Hossain, Guangyi Zhao, Moon-Sook Woo, James H-C Wang, and Jian-Ping Jin Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00856 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 15, 2016

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Deletion of calponin 2 in mouse fibroblasts increases myosin II-dependent cell traction force M Moazzem Hossain†, Guangyi Zhao‡, Moon-Sook Woo†, James H-C. Wang‡, Jian-Ping Jin*,† †

‡

Department of Physiology, Wayne State University School of Medicine, Detroit, MI 48201

Departments of Orthopedic Surgery and Bioengineering, University of Pittsburg, Pittsburg, PA 15213

Running title: Calponin 2 regulates cell traction force Funding sources: This work was supported in part by National Institutes of Health grants HL086720 and AR048816 to JPJ, and AR061395 and AR065949 to JHW. *Correspondence should be addressed to: Dr. J.-P. Jin, Department of Physiology, Wayne State University School of Medicine, 5374 Scott Hall, 540 E. Canfield, Detroit, MI 48201. Tel: 313-577-1520 Fax: 313-577-5494, Email: [email protected] Keywords: Calponin 2, cell traction force, actin cytoskeleton, adhesion, motility

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Abbreviations used: CTF - cell traction force FBS - fetal bovine serum KO - knockout mAb - monoclonal antibodies MEF - mouse embryonic fibroblasts NCM -net contractile moment PBS - phosphate buffered saline RMST - root mean square traction SDS-PAGE - SDS-polyacrylamide gel electrophoresis TBS - Tris buffered saline TSE - total strain energy WT - wild-type


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Abstract Cell traction force (CTF) plays a critical role in controlling cell shape, enabling cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three calponin isoforms. Among them, calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts and fibroblasts, and plays a role in regulating cytoskeleton activities such as cell adhesion, migration and cytokinesis. Knockout (KO) of the gene encoding calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of calponin 2 in modulating CTF. In the present study, we examined fibroblasts isolated from Cnn2 KO and wild type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured on polyacrylamide gel substrates embedded with fluorescent beads to measure root-mean square traction, total strain energy, and net contractile movement. The results showed that calponin 2-null fibroblasts exhibit greater traction force than that of WT cells. Adherent calponin 2-null fibroblasts de-adhered faster than WT control during mild trypsin treatment, consistent with increased CTF. Blebbistatin, an inhibitor of myosin II ATPase, is more effective on altering cell morphology when calponin 2 is present in WT fibroblasts than that on Cnn2 KO cells, indicating their additive effects on inhibiting myosin motor activity. The novel finding that calponin 2 regulates myosin-dependent CTF in non-muscle cells demonstrates a mechanism to control cell motility-based functions.


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Introduction Cell motility is essential in life activities. During movement, cells generate force in the cytoskeleton through interactions with the extracellular matrix. Measurement of these forces enables better understanding of the cellular mechanisms that regulate cell motility. The actin cytoskeleton plays a central role in myosin ATPase-generated cellular mechanical force and motility-based functions, including cell division,1 migration2 and contraction.3 During adhesion and migration, cells exert a cell traction force (CTF) that is essential for them to move and also plays critical roles in controlling cell morphology4 and maintaining cellular homeostasis in various physiological and pathological processes, such as angiogenesis, development, wound healing, and cancer metastasis.5-9 Cell traction force microscopy (CTFM) is a technique that quantitatively determines the traction force generated by individual cells or a group of cells10. An advantage of CTFM is to directly measure the traction force of cells to indicate the “cause” (the traction force) of cell movement, unlike other techniques that measure the “effect” (cell movement itself) of cell movement11. A commonly employed approach for CTFM is to use a polyacrylamide gel substrate embedded with fluorescent beads, which has an elasticity to allow measurement of a wide range of CTF in a linear and elastic manner12. The following variables are measured: the root mean square traction (RMST) calculated as the mean traction exerted by whole cells13;; the net contractile moment (NCM) that is a coordinate invariant scalar measure of the cell’s contractile “strength”;; and the total strain energy (TSE) transferred from the cell to the substrate through elastic distortion, which is another measure of contractile strength14. Calponin is an abundant actin filament-associated protein first found in smooth muscle.15 In vertebrates, calponin is present in three isoforms, calponin 1, 2 and 3 (previously named h1, h2 and h3 (acidic) calponins, which are encoded by three homologous genes.16-18 Tissue distribution of the three calponin isoforms is different. Calponin 1 encoded by Cnn1 gene is specifically expressed in differentiated smooth muscle cells with a function in modulating


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smooth muscle contractility.19 Calponin 2 encoded by Cnn2 gene is expressed in both smooth muscle20 and many non-muscle cell types including epidermal keratinocytes,21,22 lung alveolar cells,23 endothelial cells,24,25 fibroblasts,26,27 myeloid blood cells27, platelets,28 osteoclasts,29 myoblasts30 and prostate cancer cells.31 Calponin 3 encoded by Cnn3 gene is found in smooth muscle32 and brain with a connection to neuronal regeneration and growth.33 It also expresses in lymphocytes,34 trophoblasts35 and myoblasts36 with a potential role in cell fusion. Based on studies of calponin 1, an established biochemical function of calponin is the inhibition of myosin ATPase in smooth muscle cells.19 Amino acid sequences of the calponin isoforms are largely conserved, indicating their conserved functions. Previous studies observed that calponin 2 stabilizes actin stress fibers,22 inhibits cytokinesis,37 and slows down cell migration.27 Calponin binds multiple proteins, including F-actin and tropomyosin10 that are key components of the cytoskeleton. The actin filaments play central functions in cell motility and contractility, whereas tropomyosin stabilizes the actin filaments in non-muscle cells.38 Therefore, calponin may participate in regulating cell adhesion and motility by inhibiting myosin motor- based cytoskeleton activities, analogous to that of troponin in striated muscles.16,19 The function of calponin in regulating cell motility is a new area of research. Cell structure and motility are affected by mechanical forces exerted from the surrounding environment including stiffness of the substrate on which the cell adheres.39-43 The gene expression and protein turnover of calponin 2 are both regulated by mechanical tension in the cytoskeleton.22,23,30 The effects of calponin on cell adhesion and migration involve mechanical interactions with the surrounding cells and tissue matrix. We previously demonstrated that knockout (KO) of Cnn2 gene in mice increased the motility of macrophages27 and fibroblasts.31 Decreased calponin 2 was associated with weakened substrate adhesion of prostate cancer cells31 and platelets.28 These findings suggest a role of calponin 2 in regulating CTF. In the present study, we investigated this hypothesis by using primary fibroblasts isolated from Cnn2 KO and wild type (WT) mice. CTFM studies showed that deletion of calponin 2 in


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Cnn2 KO cells significantly increased CTF. Calponin 2 and blebbistatin, an inhibitor of myosin II ATPase, exhibit additive effects. The results demonstrate that calponin 2 is an inhibitor of myosin II motor-generated CTF, indicating a novel mechanism to regulate cell adhesion and motility-based functions.


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Materials and Methods The development of Cnn2 KO mouse line and PCR genotyping were described previously.27 All animal protocols used in our study were approved by the Institutional Animal Care and Use Committee of Wayne State University. Isolation and culture of adult mouse primary fibroblasts Fibroblasts were isolated as described previously44 from leg muscles of adult WT and Cnn2 KO mice.27 Briefly, WT and Cnn2 KO mice were euthanized and muscle tissues from both hind legs were isolated under sterile conditions. Using a pair of sharp scissors, the tissues were minced in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5% pancreatin and 0.125% trypsin, triturated using a 10 mL pipette, and incubated at 37°C for 6 min. The trituration and incubation steps were repeated once and the crude tissue digest was filtered by passing through a 100 µm mesh. The filtrate containing isolated cells were collected by centrifugation at 200 x g for 5 min, suspended in DMEM containing 20% fetal bovine serum (FBS), 2 mM L- glutamine, 100 U/mL penicillin and 50 U/mL streptomycin, and incubated in tissue culture dishes at 37°C in 5% CO2. After 1 hr, floating cells not attached to the dish were removed to selectively culture the adherent cells. The cells were passed when reaching 80-90% confluence. The second and third passages of cells were used for experiments. Isolation and culture of mouse embryonic fibroblasts From Cnn2+/- heterozygote parents, 13.5-day embryos were collected to isolate primary fibroblasts from individual embryos as described.45 After removing the head and innards, the embryo was washed with sterile phosphate buffered saline (PBS) and minced with a pair of sharp scissors in 1 mL 0.25% trypsin, 1 mM EDTA. After incubation at 37°C for 30 min, the digestion was stopped by adding 4 mL of culture media (DMEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 50 U/mL streptomycin). Cells were dissociated from the embryo tissue


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by pipetting up and down for 20 times. Additional 6 mL culture media was added to the cells suspension for plating on a 100 mm tissue culture dish. Genotype of each embryo was determined using PCR on genomic DNA isolated from the head tissue. Cells from Cnn2-/- and WT embryos were passed after 3 days of culture at 80-90% confluence. Second passages of the mouse embryonic fibroblasts (MEF) were used for experiments after Western blot verification of the Cnn2 KO and WT genotypes. Isolation and culture of neonatal mouse skin fibroblasts Adapted from a standard method (Current Protocols in Molecular Biology, 2001), primary skin fibroblasts were isolated from 4 days old WT and Cnn2 KO mice. From animals euthanized after PCR genotyping, back skin was dissected under sterile conditions and incubated with 0.25% trypsin at 37°C for 1h. The epidermal layer was removed mechanically with forceps and the dermis was washed with DMEM, cut into small pieces and incubated with 2 mL of 700 U/mL collagenase I at 37°C for 2h with agitation every 20 min. 2 mL of ice-cold culture media (DMEM, 20% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 50 U/mL streptomycin) was then added before a vigorous vortex. The cell suspension was passed through a 100 µm mesh and the cells were spun down at 150x g for 10 min. The cell pellet was re-suspended in the culture media and seeded in a 100-mm tissue culture dish for incubation at 37°C in a CO2 incubator. The media was changed after 24h and changed again 48 hr later. Five days after the original plating, the cells (P0) at 80-90% confluence were treated with 0.5% dispase in DMEM at 37°C for 30 min to remove keratinocyte contamination before passing to three 100-mm culture dish. At near confluence, the cells were made into frozen stocks (P1). Cells recovered from the frozen stock were cultured to near confluence and passed once (P2) for use in experiments. Anti-calponin antibodies


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9 A mouse monoclonal antibody (mAb) CP22 and a rabbit polyclonal antiserum RAH2

generated by immunization with purified mouse calponin 2 were used in the present study. Details for the antibody preparation and characterization have been described previously.22,46 SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting Total protein from primary cultures of mouse fibroblasts was extracted by lysis of monolayer cells in SDS-PAGE sample buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.1% bromophenol blue, pH 8.8) after washing with PBS. The 2% SDS in the buffer aims for a rapid inactivation of endogenous proteases. The protein extracts were heated at 80°C for 5 min to solubilize proteins and clarified by centrifugation in a microcentrifuge at the maximum speed for 5 min. The supernatant was loaded on 12% gel in Laemmli buffer and electrophoresed at a constant current of 25 mA per Bio-Rad mini-gel.22 The protein bands resolved were visualized by staining with Coomassie Blue R250. Duplicate SDS-gels were electrically blotted to nitrocellulose membranes using a semi- dry transfer apparatus (Bio-Rad, Hercules, CA) at 5 mA/cm2 for 15 min. After blocking with 1% bovine serum albumin (BSA) and 0.05% Tween-20 in Tris buffered saline (TBS), the membrane was incubated with anti-calponin antibody RAH2 in TBS containing 0.1% BSA and 0.05% Tween-20 under gentle rocking at 4°C overnight. The membrane was then washed with TBS containing 0.05% Tween-20, and further incubated with alkaline phosphatase-labeled anti-rabbit IgG (Sigma) secondary antibody at room temperature on a rocker for 1 hr. The membrane was washed again as above, followed by development in 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chromogenic substrate solution to visualize the calponin bands.22 Immunofluorescence microscopy


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#1 glass cover slips in 24-well culture plates and cultured at 37°C in 5% CO2 for 18 hrs. Adherent cells on coverslips were rinsed in PBS, fixed in cold acetone for 30 min and blocked with 1% BSA in PBS in a humidified box at room temperature for 30 min. The cover slips were incubated with anti-calponin 2 mAb CP22 at 4°C overnight. After washing in PBS containing 0.05% Tween-20, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (Sigma) and tetramethylrhodamine isothiocyanate (TRITC)- conjugated phalloidin (Sigma) (for F-actin) at room temperature for 1 hr. After final washes with PBS containing 0.05% Tween-20, the coverslips were mounted on glass slides and viewed using a confocal fluorescence microscope to examine the cellular localization of calponin 2 and its relationship to the actin cytoskeleton. Polyacrylamide gel culture substrate for CTFM Two-dimensional gel substrate was prepared as described previously11,47 by making thin layers of polyacrylamide gel on the glass bottom of a MatTek culture dish (Ashland, MA). Prior to casting the gel, the dishes were pre-treated with 0.1 N NaOH and air-dried, treated with 3- aminopropyltrimethoxysilane for 5 min, rinsed three times with double distilled water, incubated with 0.5% glutaraldehyde for 30 min, washed three times again with double distilled water, and dried at 60°C. The treated dishes were stored at room temperature and used within one week. Two layers of polyacrylamide gel were casted on the treated glass bottom (14 mm diameter) of the culture dishes. The lower layer was ~120 μm thick, and the upper layer was ~60 μm thick and contained 0.2 μm yellow-green fluorescent micro-beads (Ex/Em = 505/515, Molecular Probes, Eugene, OR) at 1/200 dilution. 0.5 µL TEMED and 5 µL of 10% ammonium persulfate were added to 1 mL gel stock solution containing 5% acrylamide and 0.225% bis- acrylamide immediately before use. The lower layer was fabricated by adding 11 μL of the acrylamide mix to the center of the glass bottom for polymerizing at room temperature for 2 hrs.


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The upper layer was then prepared by adding 5 μL of the gel stock containing fluorescent beads on top of the lower layer and covering with a circular cover slip (12 mm diameter). The dishes were then placed upside down at 4°C for 2 hrs to allow the fluorescence beads to settle on the cover slip (thus at the surface of the gel substrate). After polymerization at room temperature for 30 min, the cover slip was carefully removed and the polyacrylamide gel surface was washed with double distilled water. The gel substrate was activated by treating with 100 μL of 1 mM sulfo-SANPAH (Pierce, Rockford, IL) in 50 mM HEPES buffer under UV light for 9 min followed by three washes with double distilled water. The polyacrylamide gel substrates were then coated with 180 μL of 100 μg/mL type I collagen at 4°C overnight and thoroughly washed three times with PBS prior to seeding cells. The Young’s modulus of the polyacrylamide gel substrate was 8-kPa, and the Poisson’s ratio was 0.48,48 which responds to a wide range of forces in a linear and elastic manner.12 Acquiring CTFM images The culture dishes holding polyacrylamide gel substrates were sterilized with UV irradiation for 30 min. Cnn2 KO and WT adult mouse leg muscle fibroblasts and MEF were seeded at 5 x 103 cells/dish, allowed to attach and spread for 24 hrs in DMEM containing 20% FBS. An area on the polyacrylamide gel substrate with an adequate density of adherent cells was selected for CTFM analysis. Two sets of microscopic images were obtained for CTF analysis. Images of individual cells with the fluorescent beads embedded in the underneath gel substrate were first taken using a fluorescence microscope equipped with a CCD camera. The cells were then removed by adding 100 μL of 1 N NaOH to the well that contained 2 mL culture media and fluorescence images of the same area were recorded again. The following parameters were then computed by comparing the positions of the fluorescence beads before and after removing the cells: a)


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root mean square traction (RMST), the mean of traction through the whole cell;; b) net contractile moment (NCM), a coordinate invariant scalar measure of the cell’s contractile strength;; and c) total strain energy (TSE), the energy transferred from the cell to the elastic distortion of the substrate and another measure of contractile strength. Calculation of these parameters was done using the method by Butler et al.14 Fibroblast de-adhesion assay To investigate the effect of calponin 2 deletion on the dynamics of actin cytoskeleton reorganization, Cnn2 KO and WT mouse fibroblasts was examined for de-adhesion during mild trypsin digestion. The cells were seeded in 6-well plates at 1 x 105 cells per well and cultured for 24 hrs. The monolayer of adherent cells was washed with PBS and treated with a low concentration of trypsin-EDTA solution (0.025% trypsin, 0.1 mM EDTA) for 10 min. The digestion was stopped by adding FBS to a final concentration of 10%. Phase-contrast microscopic images were taken in multiple randomly selected areas to determine the percentage of round-up cells and evaluate the de-adhesion velocity.49 Effect of blebbistatin treatment on CTF and sensitivity test To investigate the biochemical mechanism by which calponin 2 regulates cell traction force, CTFM was repeated with treatment of blebbistatin, an inhibitor of myosin II ATPase.50 10 μM blebbistatin treatment for 30 min was determined as an optimal condition that applies an inhibition of myosin II ATPase with a minimally change in cell morphology. Cnn2 KO and WT mouse adult fibroblasts and MEF treated under this condition were analyzed with CTFM as described above. To test the sensitivity of Cnn2 KO and WT mouse neonatal skin fibroblasts and MEF to blebbistatin, cells were seeded in 6-well culture plate a 2.5 x 104 cells/well for growing on plastic and on 8-kPa gel substrate (the same stiffness as that used in CTFM studies), cultured for 24


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hr, and treated with 1, 3 and 10 μM blebbistatin. Phase-contrast images of the cells were taken at 15, 30, 45, 60, 90 and 120 min of the treatment to quantify the effect on the spreading area of cells. The spreading area of cells at different concentration of blebbistatin was plotted against the time of treatment to assess the effect of calponin 2 deletion on the sensitivity of cells to blebbistatin. Data analysis All experiments were repeated three times or more and the data are presented as mean ± SE. Statistical analysis was performed using unpaired two-tail Student’s t-test. Differences between two samples were considered significant when P was less than 0.05.


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Results Deletion of calponin 2 in mouse fibroblasts increases cell traction force Western blot analysis of total protein extracts showed significant levels of calponin 2 in fibroblasts isolated from the adult leg muscles as well as day 13.5. embryos of WT mice while it is absent in cells of Cnn2 KO mice (Fig. 1A). Confocal microscopic images of adult mouse primary fibroblasts showed that calponin 2 was co-localized with F-actin stress fibers (Fig. 1B), consistent with its role in regulating actin filament-mediated cellular functions.22,23,25,27-29,31 Analysis of phase-contrast and fluorescent microscopic images of fibroblasts from adult WT and Cnn2 KO mice cultured on type I collagen-coated thin layer of polyacrylamide gel with embedded fluorescent beads revealed higher CTF in Cnn2 KO fibroblasts than that in WT cells (Fig. 2). The constrained traction ﬁeld computed from the beads movement, and the direct computation of tractions from the Fourier decomposition of the displacements are shown in Fig. 2B and 2D. Quantification of CTF parameters determined that Cnn2 KO fibroblasts exerted significantly higher RMST, NCM and TSE (Fig. 3A, 3B and 3C, respectively) than that in the WT fibroblasts. The shape index (SI) of Cnn2 KO and WT cells was not significantly different (Fig. 3D). Studies of Cnn2 KO and WT MEF produced similar results (Fig. 4). Cnn2 KO mouse fibroblasts exhibit faster de-adhesion than that of WT cells A previous study observed that cells with faster round-up velocity during trypsin treatment had higher CTF.49 To further demonstrate the role of calponin 2 in the regulation of CTF in mouse fibroblasts, the percentage of adherent cells rounded-up by 10 min of mild trypsin treatment was examined to show that Cnn2 KO mouse fibroblasts de-adhered significantly faster than that of WT cells (Fig. 5). It was reported that the de-adhesion time during trypsin-treatment of adherent cells inversely correlated with cell contractility. Inhibition of myosin motor with blebbistatin treatment


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delayed the trypsin treatment-produced de-adhesion and reduced the stiffness of cellular structure.49 Therefore, although the trypsin de-adhesion test is not specific to a particular cell signaling pathway, it is informative to compare cytoskeleton reorganization dynamics and intrinsic contractile force in the presence or absence of calponin 2’s inhibitory effect on myosin motor activity. The result indicates that deletion of calponin 2 in mouse fibroblasts increases cell traction force and the dynamics of cytoskeleton reorganization. Effect of calponin 2 deletion on increasing CTF is myosin II motor-dependent Treatment of mouse primary fibroblasts with high concentrations of blebbistatin, an inhibitor of myosin II ATPase,50 resulted in cell deformation shown with a slacked morphology (data not shown). Under a mild blebbistatin treatment condition which does not cause drastic change in morphology (10 µM 30m min), CTF was significantly decreased in both Cnn2 KO and WT cells (Fig. 6) as compared with that in the absence of blebbistatin (Fig. 3). This result was consistent with a previous report that treatment of NIH-3T3 cells with 10 μM blebbistatin reduced CTF by 13 fold.51 The quantification of CTF parameters showed diminished RMST, NCM and TSE in Cnn2 KO and WT cells while no significant change in cell shape (SI) and spreading area for both adult mouse fibroblasts (Fig. 7) and MEF (Fig. 8). The effect of blebbistatin on diminishing the difference in CTF between Cnn2 KO and WT fibroblasts demonstrate that calponin 2 regulates CTF via inhibiting myosin II ATPase and motor activity. Quantitative analysis of the dose-dependence and time course of blebbistatin’s effect on mouse fibroblasts in adherent culture showed that Cnn2 KO fibroblasts were significantly less sensitivity to blebbistatin than that of WT cells (Fig. 9). The results demonstrate that whereas the spreading area of WT MEF was as expected larger than that of Cnn2 KO cells on 8-kPa gel substrate prior to blebbistatin treatment, it began to decrease earlier (at 45 min) and to a large degree with a trend of responding to lower concentrations of blebbistatin than that of Cnn2 KO cells (Fig. 9A). At 120 min, WT and Cnn2 KO MEF showed similar spreading areas and


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sensitivity to blebbistatin concentrations. The same pattern was found for MEF on plastic substrate although the WT and Cnn2 KO cells both showed earlier responses (at 30 min) as compared with the time course on gel substrate (Fig. 9B). Very similar results were obtained for WT and Cnn2 KO neonatal skin fibroblasts (Fig. 9C and D). The lower sensitivity of Cnn2 KO fibroblasts to blebbistatin indicates a less endogenous inhibition of myosin II ATPase when calponin 2 is absent, supporting that calponin 2 regulates CTF via inhibiting myosin II motor activity.


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Discussion Calponin is an abundant protein in smooth muscle cells and in multiple types of non- muscle cells. The biological functions of calponin are not fully understood.17,18 Extensive studies have been carried out to decipher the biochemical function of calponin in smooth muscle contraction.18,19 While calponin 1 is specifically expressed in smooth muscle cells, calponin 2 is found in both smooth muscle37 and many non-muscle cell types, including fibroblasts.26,27 Our previous studies have demonstrated the inhibitory effects of calponin 2 on cell migration, proliferation, phagocytosis and platelet adhesion.27,28,31,37 In the present study, we characterized the function of calponin 2 in regulating cell traction force using primary Cnn2 KO mouse fibroblasts27 as a physiologically informative experimental system. Traction force exerted by cells on their extracellular environment mediates many cellular functions such as cell adhesion,52-54 differentiation,55-57 embryogenesis,58,59 cancer metastasis9,60,61 and wound healing.62,63 In cancer cells, increased CTF increases metastatic potential.9 Our previous studies showed that the gene expression and protein level of calponin 2 in adherent cells both positively correlate to substrate stiffness, reflecting mechanical tension regulations.22,23,30 In the present study, we found that primary fibroblasts from Cnn2 KO mice exhibit higher traction force than that of WT fibroblasts (Figs. 3 and 4), indicating an inhibition of CTF by calponin 2. This finding provides a novel insight into the molecular mechanism by which calponin 2 regulates cell motility.27,31 Cultured cells adherent to a substrate represent many cell types with biological and medical importance. For example, macrophages, epithelial cells, and fibroblasts are all adherent cells in vivo and generate strong traction force.64 In these cells, myosin II is the primary motor protein that generates CTF.51,65 Calponin 1, the smooth muscle specific isoform of calponin, has been extensively characterized in in vitro biochemical and biophysical studies as an inhibitor of myosin ATPase, regulating the function of actin filaments to modify smooth muscle contractility.18,19 Our present study, on the other hand, is the first report for the role of calponin 2


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in regulating non-muscle myosin II motor activity in live cells. The finding that calponin 2 plays a role in regulating myosin II motor-generated CTF is analogous to the function of calponin 1 in regulating smooth muscle contractility. Blebbistatin is a potent drug that inhibits myosin II50 and acts by binding to the large cleft of the motor domain of myosin and interfering with ADP dissociation and Pi release.50,66,67 Using blebbistatin as a tool to identify cytoskeleton motor activities, we found that blebbistatin significantly decreased CTF and diminished the difference between Cnn2 KO and WT mouse fibroblasts (Fig. 9). This observation indicates that the effect of calponin2 is myosin motor dependent. The deletion of calponin 2 decreased the sensitivity of cells to blebbistatin treatment. This finding demonstrates that calponin 2 as an endogenous inhibitor of myosin II ATPase regulates CTF on the same target, i.e., myosin II motor activity. The effects of calponin 2 and blebbistatin are additive, indicating parallel mechanisms where calponin inhibits the activation of myosin by actin and blebbistatin directly inhibits myosin ATPase. Therefore, they are non-competitive inhibitors. The deletion of calponin 2 removes an endogenous inhibition to permit a higher level of myosin activation, which results in a higher resistance to blebbistatin due to the need of an increased blebbistatin inhibition for reaching the level of effect produced by blebbistatin-calponin double inhibition (Fig. 9). It is worth noting that calponin binds F-actin with high affinity and inhibits the actin- activated myosin ATPase68-71 and motor activity,72-74 whereas blebbistatin directly binds and inhibits myosin motor domain.15,66 Therefore, their additive inhibitory effects on CTF have two different components: The effect of calponin 2 is an endogenous regulatory inhibition to lower the degree of myosin II motor activation, whereas blebbistatin is a toxin that kills myosin ATPase and motor activity. This difference is critical for calponin 2 to function as a physiological modulator of cell traction force and motility in contrast to the effect of blebbistatin on paralyzing all myosin motor-based functions.


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19 The fact that deletion of calponin 2 increases the velocity of cell de-adhesion (Fig. 5)

also reflects a higher myosin II motor activity and increased dynamics of cytoskeleton reorganization. This observation is consistent with the role of calponin 2 in stabilizing the actin cytoskeleton.22 Based on the experimental data, our current hypothesis is that increased myosin motor activity and contractility due to the deletion of calponin 2 will increase the dynamics of actin cytoskeleton to slow down cell adhesion and to promote migration. On the other hand, the building-up of cytoskeleton tension by myosin motor depends on adhesion. While the size and stability of focal adhesions is enhanced by myosin-based contraction forces where blebbistatin often had a negative effect by inactivating myosin motor, calponin 2 imposes a modulatory and restrictive inhibition of myosin motor to slow down the kinetics without reducing the maximum capacity of cell adhesion.31 In calponin 2-null cells, higher myosin activity and contractile force slows down the formation of stable adhesion29,31 and the higher cytoskeleton dynamics may produce smaller spreading area in adherent cultures. The reduced cell dimension will then produce less total tension in the cytoskeleton to reach an equilibrium. This hypothesis is supported by our recent observation that calponin 2 is not concentrated at focal adhesions but co-localized with myosin in stress fibers70 and worth further investigating. Fig. 10 illustrates a model derived from our knowledge of calponin regulation and function in cell traction force and motility. The inhibition of myosin ATPase and motor activity by calponin 2 decreases CTF and cell motility to facilitate cell adhesion, increase cell dimensions in monolayer cultures and produce higher tension in the cytoskeleton22,23,27,29,31,80. The increased cytoskeleton tension further increases calponin 2 gene expression as a positive feedback. In the meantime, the inhibition of myosin motor activity decreases CTF to reduce the tension in the cytoskeleton as a negative feedback thus to maintain an equilibrium. Cnn2 KO or down- regulation will function in opposite directions.


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20 Living cells dynamically change structure and function through gene regulation and

posttranslational protein modification in response to mechanical signals.75-79 Calponin is a cytoskeleton regulatory protein responsive to changes in the mechanical environment.22,23,30 The substrate stiffness and other extracellular mechanical force signals will influence the set point of this equilibrium depending on the cell type and their native tissue environments, an interaction between cells and the mechanical environment that merits further investigations. Acknowledgements We thank Hui Wang for mouse genotyping and technical assistance and Ahila Manivannan for preparing frozen stocks of neonatal mouse skin fibroblasts.


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21

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(Figure Legends) Figure 1. Calponin 2 is abundant in adult and embryonic mouse fibroblasts. (A) Total protein extracts of adult muscle fibroblasts and MEF isolated from WT and Cnn2 KO mice were examined with SDS-PAGE and Western blotting using anti-calponin 2 antibody RAH2. Significant amounts of calponin 2 were detected in WT mouse fibroblasts but not in Cnn2 KO cells. (B) Adherent cultures of WT mouse adult fibroblasts were stained with anti-calponin 2 mAb CP22 together with phalloidin staining of F-actin. The result showed that calponin 2 co- localizes with the actin filaments. Figure 2. Cell traction force microscopy measurement of adult wild type and Cnn2 KO mouse fibroblasts. The phase contrast and CTF microscopic images showed examples of WT (A, B) and Cnn2 KO (C, D) mouse fibroblasts. Figure 3. Deletion of calponin 2 increases CTF of adult mouse fibroblasts. CTFM data showed that the stress data (Pa and pJ) normalized to individual cell areas (µm2), Cnn2 KO cells had significantly higher root mean square traction (RMST) (A), net contractile moment (NCM) (B), and total strain energy (TSE) (C) than that of WT cells (*P

Deletion of Calponin 2 in Mouse Fibroblasts Increases Myosin II

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