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Vinculin, and A-Actinin K.-L Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9182-9187, August 1996 Medical Sciences The differential adhesion forces of anterior cruciate and medial collateral ligament fibroblasts: Effects of tropomodulin, talin, vinculin, and a-actinin K.-L. PAUL SUNG*tt§, LI YANG*t, DARREN E. WHITTEMOREt, YAN SHI*, GANG JIN t, ADAM H. HSIEH*t, WAYNE H. AKESON*, AND L. AMY SUNGt¶ *Departments of Orthopaedics and tBioengineering, tCancer Center, and TCenter for Molecular Genetics, Institute for Biomedical Engineering, University of California at San Diego, La Jolla, CA 92093-0412 Communicated by Y C. Fung, University of California at San Diego, La Jolla, CA, May 28, 1996 (received for review April 8, 1996) ABSTRACT We have determined the effects of tropo- fashion along the grooves of the actin double helix, stiffening modulin (Tmod), talin, vinculin, and a-actinin on ligament the filament and regulating the interaction between actin and fibroblast adhesion. The anterior cruciate ligament (ACL), other actin-binding proteins (12). Tmod may, therefore, reg- which lacks a functional healing response, and the medial ulate the length and/or organization of actin filaments by collateral ligament (MCL), a functionally healing ligament, differential binding to TM. In fibroblasts, hTM5 (one of the were selected for this study. The micropipette aspiration TM isoforms) is present not only in the more stable structures technique was used to determine the forces needed to separate of stress fibers, but also in the ruffled regions where the F-actin ACL and MCL cells from a fibronectin-coated surface. De- structures are rapidly changing (14). We found previously in a livery of exogenous tropomodulin, an actin-filament capping monocytic cell line that Tmod increases cell adhesion strength protein, into MCL fibroblasts significantly increased adhe- to fibronectin (FN), whereas the antibody against Tmod sion, whereas its monoclonal antibody (mAb) significantly decreases adhesion strength (15). decreased cell adhesiveness. However, for ACL fibroblasts, In this study we investigate the differences between a Tmod significantly reduced adhesion, whereas its mAb had no nonhealing ligament, the adult anterior cruciate ligament effect. mAbs to talin, vinculin, and a-actinin significantly (ACL), and a functionally healing ligament, the medial col- decreased the adhesion of both ACL and MCL cells with lateral ligament (MCL) (16, 17) by characterizing the roles of increasing concentrations ofantibody, and also reduced stress several cytoskeletal proteins in ligament fibroblast adhesion. fiber formation and cell spreading rate as revealed by immu- The purpose of the study is to determine the effects of a new nofluorescence microscopy. Disruption of actin filament and actin-filament capping protein, Tmod, and three integrin/ microtubule assembly with cytochalasin D and colchicine, actin-filament bridging proteins (talin, vinculin, and at-actinin) respectively, also significantly reduced adhesion in ACL and on ACL and MCL fibroblast adhesion behavior (Fig. 1). MCL cells. In conclusion, both ACL and MCL fibroblast Experiments were performed using the micropipette single- adhesion depends on cytoskeletal assembly; however, this cell aspiration technique to compare the adhesion forces of dependence differs between ACL and MCL fibroblasts in normal ACL and MCL cells with those of ACL and MCL cells many ways, especially in the role of Tmod. These results add electroporated with monoclonal antibodies (mAbs) against yet another possible factor in explaining the clinical differ- tropomodulin, talin, vinculin, and a-actinin. In addition, we ences in healing between the ACL and the MCL. examined the effects of agents that disrupt actin filament and microtubule assemblage [cytochalasin D (C.D.) and colchicine Cell-cell and cell-extracellular matrix adhesion play fundamen- (C.C.), respectively] on adhesion behavior as well as the tal roles in physiological processes such as wound healing, differences in the organization of stress fibers between ACL immune surveillance, thrombosis, and hemostasis. In the and MCL fibroblasts by immunofluorescence microscopy. To wounded ligament, fibroblasts embedded in the amorphous determine the effects of cytoskeletal assembly on cell adhe- healing tissue matrix of ligaments have been found to migrate sion, experiments were performed using a FN base. FN was into damaged sites. This migration, which regulates wound chosen because of its significance in ligament healing, not only closure during the healing process, involves adhesion events by providing a provisional submatrix for fibroblast migration and the remodeling of the internal cytoskeleton through the and ingrowth but also by acting as a linkage for wound reorganization of actin filaments and other cytoskeletal pro- contraction in the healing ligament (6). teins (1, 2). Studies have shown the importance of cytoskeletal proteins in the adhesion and motility of different cell types MATERIALS AND METHODS (1-7). Recent evidence indicates that the capping protein gelsolin, at the barbed ends of actin filaments, has striking Cell Cultures. Human ligament fibroblasts were obtained effects on cell motility by influencing actin-filament assembly from ACL and MCL explants from five subjects (one female (8, 9). and four male adults, 30-52 years old). Fibroblasts were Tropomodulin (Tmod) is a capping protein found at the free harvested at autopsy within 6-24 hr after death and isolated (pointed) ends of actin filaments, and its cDNA sequence has according to a prescribed protocol (18) and showed no dif- been determined (10, 11). It is known to be involved in ferences in viability and adhesion behavior among subjects (19, actin-filament assemblage, and it has been shown that Tmod 20). The cells were grown in Dulbecco's modified Eagle's interacts with the N terminus of tropomyosin (TM) and medium (DMEM)/10% fetal calf serum, supplemented with inhibits TM binding to actin by blocking the association of TM along actin filaments (12, 13). TM polymerizes in a head-to-tail Abbreviations: Tmod, tropomodulin; FN, fibronectin; ACL, anterior cruciate ligament; MCL, medial collateral ligament; TM, tropomyosin; C.D., cytochalasin D; C.C., colchicine. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Departments of payment. This article must therefore be hereby marked "advertisement" inI Orthopaedics and Bioengineering, University of California at San accordance with 18 U.S.C. §1734 solely to indicate this fact. Diego, La Jolla, CA 92093-0412. 9182 Downloaded by guest on September 29, 2021 Medical Sciences: Sung et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9183 Microfilament and Microtubule-Disrupting Agents and Treatment. The actin-filament disrupting agent, C.D. (29) and the microtubule-disrupting agent, C.C. (30), both obtained from Sigma, were used at final concentrations of 0.5 and 0.1 ,uM, respectively, sufficient to inhibit cytoskeletal assembly within the time course of our experiments. Fibroblasts were treated with C.D. and C.C. by either (i) pre-incubation-30- min incubation at 37°C prior to cell seeding or (ii) post- incubation-cell seeding in the micropipette chamber at room temperature for 15 min followed by addition of C.D. or C.C. to the medium and the measurement of adhesion strengths for 45 min thereafter. These two treatments are intended to elucidate the effects of actin-filament- and microtubule- disrupting agents before and after the formation of stress fibers and adhesion plaques during cell attachment and spreading. matrix Control experiments were conducted without any agent. FIG. 1. Schematic drawing of cytoskeleton-integrin-extracellular Micropipette Chamber/Adhesion Force Measurements. matrix complex. Tmod, located at the pointed end of actin filaments, The detailed coating procedure of 5 ,ug/ml FN on cover glasses and the bridging proteins (talin, vinculin, and a-actinin), located within micropipette chambers and the micropipette- between actin filaments and integrins, are shown. micromanipulation system used for the measurement of ad- hesion forces has been described (19, 31, 32). Glass micropi- nonessential amino acids (0.10 mM), L-glutamine (4 mM), pettes (prepared using a Flaming Brown Model P-87 puller, penicillin (100 units/ml), streptomycin (100 ,ug/ml), and fun- Sutter Instruments, Novato, CA), with an internal tip radius of gizone (0.25 ,ug/ml) (BioWhittaker). Cultures were main- 1.5-3.0 ,um, were used and the adhesion characteristics were tained at 37°C and 5% C02/95% air. At confluency, fibro- measured under direct microscopic observation in conjunction blasts were trypsinized, washed, and resuspended for experi- with a video recording system. The force (product of aspiration ments. Passages 2-6 of fibroblast cultures were used. pressure and the cross-sectional area of the micropipette tip) Electroporation of Fibroblasts. Electroporation (21) was required to separate the cell from the FN coat was measured used to deliver Tmod (25 and 50 ,tg/ml), mAbs against Tmod by stepwise increases in aspiration pressure followed by re- (mAb Tmod-204, 25 and 50 ,tg/ml), talin (200 and 385 ,ug/ml), traction of the pipette. Cells were chosen at random and vinculin (30 and 100 ,g/ml), and a-actinin (50 and 100 ,tg/ml) adhesion data were collected for 45 min following an initial into fibroblasts. mAbs against Tmod, talin (Sigma), vinculin 15-min seeding time at room temperature. (Sigma), and a-actinin (Sigma) are anti-human
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