Proc. Natl. Acad. Sci. USAVol. 93, pp. 9182-9187, August 1996Medical Sciences

The differential adhesion forces of anterior cruciate and medialcollateral ligament fibroblasts: Effects of tropomodulin, talin,vinculin, and a-actininK.-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 ofCalifornia 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-modulin (Tmod), talin, vinculin, and a-actinin on ligamentfibroblast adhesion. The anterior cruciate ligament (ACL),which lacks a functional healing response, and the medialcollateral ligament (MCL), a functionally healing ligament,were selected for this study. The micropipette aspirationtechnique was used to determine the forces needed to separateACL and MCL cells from a fibronectin-coated surface. De-livery of exogenous tropomodulin, an actin-filament cappingprotein, into MCL fibroblasts significantly increased adhe-sion, whereas its monoclonal antibody (mAb) significantlydecreased cell adhesiveness. However, for ACL fibroblasts,Tmod significantly reduced adhesion, whereas its mAb had noeffect. mAbs to talin, vinculin, and a-actinin significantlydecreased the adhesion of both ACL and MCL cells withincreasing concentrations of antibody, and also reduced stressfiber formation and cell spreading rate as revealed by immu-nofluorescence microscopy. Disruption of actin filament andmicrotubule assembly with cytochalasin D and colchicine,respectively, also significantly reduced adhesion in ACL andMCL cells. In conclusion, both ACL and MCL fibroblastadhesion depends on cytoskeletal assembly; however, thisdependence differs between ACL and MCL fibroblasts inmany ways, especially in the role of Tmod. These results addyet another possible factor in explaining the clinical differ-ences in healing between the ACL and the MCL.

Cell-cell and cell-extracellular matrix adhesion play fundamen-tal roles in physiological processes such as wound healing,immune surveillance, thrombosis, and hemostasis. In thewounded ligament, fibroblasts embedded in the amorphoushealing tissue matrix of ligaments have been found to migrateinto damaged sites. This migration, which regulates woundclosure during the healing process, involves adhesion eventsand the remodeling of the internal cytoskeleton through thereorganization of actin filaments and other cytoskeletal pro-teins (1, 2). Studies have shown the importance of cytoskeletalproteins in the adhesion and motility of different cell types(1-7). Recent evidence indicates that the capping proteingelsolin, at the barbed ends of actin filaments, has strikingeffects on cell motility by influencing actin-filament assembly(8, 9).Tropomodulin (Tmod) is a capping protein found at the free

(pointed) ends of actin filaments, and its cDNA sequence hasbeen determined (10, 11). It is known to be involved inactin-filament assemblage, and it has been shown that Tmodinteracts with the N terminus of tropomyosin (TM) andinhibits TM binding to actin by blocking the association ofTMalong actin filaments (12, 13). TM polymerizes in a head-to-tail

fashion along the grooves of the actin double helix, stiffeningthe filament and regulating the interaction between actin andother actin-binding proteins (12). Tmod may, therefore, reg-ulate the length and/or organization of actin filaments bydifferential binding to TM. In fibroblasts, hTM5 (one of theTM isoforms) is present not only in the more stable structuresof stress fibers, but also in the ruffled regions where the F-actinstructures are rapidly changing (14). We found previously in amonocytic cell line that Tmod increases cell adhesion strengthto fibronectin (FN), whereas the antibody against Tmoddecreases adhesion strength (15).

In this study we investigate the differences between anonhealing ligament, the adult anterior cruciate ligament(ACL), and a functionally healing ligament, the medial col-lateral ligament (MCL) (16, 17) by characterizing the roles ofseveral cytoskeletal proteins in ligament fibroblast adhesion.The purpose of the study is to determine the effects of a newactin-filament capping protein, Tmod, and three integrin/actin-filament bridging proteins (talin, vinculin, and at-actinin)on ACL and MCL fibroblast adhesion behavior (Fig. 1).Experiments were performed using the micropipette single-cell aspiration technique to compare the adhesion forces ofnormal ACL and MCL cells with those ofACL and MCL cellselectroporated with monoclonal antibodies (mAbs) againsttropomodulin, talin, vinculin, and a-actinin. In addition, weexamined the effects of agents that disrupt actin filament andmicrotubule assemblage [cytochalasin D (C.D.) and colchicine(C.C.), respectively] on adhesion behavior as well as thedifferences in the organization of stress fibers between ACLand MCL fibroblasts by immunofluorescence microscopy. Todetermine the effects of cytoskeletal assembly on cell adhe-sion, experiments were performed using a FN base. FN waschosen because of its significance in ligament healing, not onlyby providing a provisional submatrix for fibroblast migrationand ingrowth but also by acting as a linkage for woundcontraction in the healing ligament (6).

MATERIALS AND METHODSCell Cultures. Human ligament fibroblasts were obtained

from ACL and MCL explants from five subjects (one femaleand four male adults, 30-52 years old). Fibroblasts wereharvested at autopsy within 6-24 hr after death and isolatedaccording to a prescribed protocol (18) and showed no dif-ferences in viability and adhesion behavior among subjects (19,20). The cells were grown in Dulbecco's modified Eagle'smedium (DMEM)/10% fetal calf serum, supplemented with

Abbreviations: Tmod, tropomodulin; FN, fibronectin; ACL, anteriorcruciate ligament; MCL, medial collateral ligament; TM, tropomyosin;C.D., cytochalasin D; C.C., colchicine.§To whom reprint requests should be addressed at: Departments of

I Orthopaedics and Bioengineering, University of California at SanDiego, La Jolla, CA 92093-0412.

9182

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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matrix

FIG. 1. Schematic drawing of cytoskeleton-integrin-extracellularmatrix complex. Tmod, located at the pointed end of actin filaments,and the bridging proteins (talin, vinculin, and a-actinin), locatedbetween actin filaments and integrins, are shown.

nonessential amino acids (0.10 mM), L-glutamine (4 mM),penicillin (100 units/ml), streptomycin (100 ,ug/ml), and fun-gizone (0.25 ,ug/ml) (BioWhittaker). Cultures were main-tained at 37°C and 5% C02/95% air. At confluency, fibro-blasts were trypsinized, washed, and resuspended for experi-ments. Passages 2-6 of fibroblast cultures were used.

Electroporation of Fibroblasts. Electroporation (21) wasused to deliver Tmod (25 and 50 ,tg/ml), mAbs against Tmod(mAb Tmod-204, 25 and 50 ,tg/ml), talin (200 and 385 ,ug/ml),vinculin (30 and 100 ,g/ml), and a-actinin (50 and 100 ,tg/ml)into fibroblasts. mAbs against Tmod, talin (Sigma), vinculin(Sigma), and a-actinin (Sigma) are anti-human antibodiesfrom mice and albumin antibody (control) is an anti-humanantibody developed in rabbits. The selection of antibodyconcentrations was based on cell concentrations, stoichiome-try, and spatial distributions of the involved molecules avail-able in literature: (i) talin was found to aggregate withf31-integrin in a 1:1 ratio at focal contact sites during the veryearly stages of fibroblast adhesion, whereas vinculin (0.5:1ratio with f31-integrin) and a-actinin (0.2:1 ratio with 13i-

integrin) were less abundant at focal contacts (2, 22, 23); (ii)vinculin, which has a total endogenous concentration of 100,ug/ml (24), binds to talin in a 1:3 molar ratio (25); and (iii)a-actinin, which has a total endogenous concentration of 1mg/ml (24), is found to be more abundant in stress fibers as an

actin-filament crosslinking protein than at focal contacts (26).Electroporation was performed using a T820 electroporationsystem (BTX, San Diego) at a setting (1 pulse, 99 ,usec pulselength, 300 volts) that yields 90% cell viability (trypan blueexclusion). A negative control in the absence of antibody orprotein was used to monitor the nonspecific effects of thetreatment. Cells electroporated with anti-albumin antibody(150 ,tg/ml, control for mAbs) or albumin (50 ,tg/ml, controlfor Tmod) were used to normalize values in subsequentadhesion studies. Cells were used 2-3 hr after electroporation,allowing time for the cells to recover and for the exogenousproteins or mAbs to bind.Western Blot Analysis. Western blot analyses were per-

formed on fibroblasts before and after electroporation with 25and 50 ,ug/ml of Tmod into the cells to verify the successfuldelivery of protein. After electroporation, 106 cells weredissolved in an SDS/solubilization solution (1.4 M 2-mercap-toethanol/4% SDS/50 mM Tris, pH 6.8), and the total pro-teins were separated by SDS/10% PAGE (27). Protein profileswere transblotted onto nitrocellulose paper (28) and probedwith an mAb against Tmod (mAb Tmod-204, unpublishedwork), followed by a goat anti-mouse IgG secondary antibodyconjugated with horseradish peroxidase (1:3000) (Bio-Rad).

Microfilament and Microtubule-Disrupting Agents andTreatment. The actin-filament disrupting agent, C.D. (29) andthe microtubule-disrupting agent, C.C. (30), both obtainedfrom Sigma, were used at final concentrations of 0.5 and 0.1,uM, respectively, sufficient to inhibit cytoskeletal assemblywithin the time course of our experiments. Fibroblasts weretreated 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 roomtemperature for 15 min followed by addition of C.D. or C.C.to the medium and the measurement of adhesion strengths for45 min thereafter. These two treatments are intended toelucidate the effects of actin-filament- and microtubule-disrupting agents before and after the formation of stress fibersand adhesion plaques during cell attachment and spreading.Control experiments were conducted without any agent.

Micropipette Chamber/Adhesion Force Measurements.The detailed coating procedure of 5 ,ug/ml FN on cover glasseswithin micropipette chambers and the micropipette-micromanipulation system used for the measurement of ad-hesion forces has been described (19, 31, 32). Glass micropi-pettes (prepared using a Flaming Brown Model P-87 puller,Sutter Instruments, Novato, CA), with an internal tip radius of1.5-3.0 ,um, were used and the adhesion characteristics weremeasured under direct microscopic observation in conjunctionwith a video recording system. The force (product of aspirationpressure and the cross-sectional area of the micropipette tip)required to separate the cell from the FN coat was measuredby stepwise increases in aspiration pressure followed by re-traction of the pipette. Cells were chosen at random andadhesion data were collected for 45 min following an initial15-min seeding time at room temperature.

Stress Fiber Visualization. Immunofluorescence stainingand epifluorescence (Nikon Diaphot-TMD-EF) microscopywere used to characterize stress fiber formation during adhe-sion and spreading of control cells and cells introduced withmAbs against talin (385 ,ug/ml), vinculin (100 gg/ml), anda-actinin (100 ,ug/ml) on FN-coated glass. Cells were fixedwith 3.7% (vol/vol) formalin for 20 min, permeabilized with0.5% Triton X-100 in PBS for 30 min, and incubated for 20 minat room temperature with 0.17 ,AM rhodamine phalloidin (Mo-lecular Probes), which binds to F-actin in the microfilaments.

Statistics. Comparisons between controls and treated groupswere performed using the unpaired Student's t test (mean) todetermine if any significant differences (P < 0.05) exist.

RESULTSAdhesion Experiments. The effects of mAbs against four

important actin-associated proteins, Tmod (25 and 50 Ag/ml),talin (200 and 385 ,ug/ml), vinculin (30 and 100 ,ug/ml), anda-actinin (50 and 100 jig/ml), on the adhesion of ACL andMCL fibroblasts to FN (5 ,ug/ml)-coated surfaces were inves-tigated. The normalized forces required to detach the cellsfrom the chemically well-defined surfaces are shown in Fig. 2.Anti-Tmod (50 ,ug/ml) significantly reduced MCL cell adhe-sion by 18% (P < 0.002), but had no effect on ACL celladhesion. A lower concentration of anti-Tmod (25 p,g/ml) wasunable to affect the adhesion of either ACL or MCL cells.Adhesion of MCL cells was significantly reduced by anti-talinat both concentrations of 200 Ag/ml (31% reduction, P < 0.01)and 385 jig/ml (20% reduction, P < 0.05), whereas adhesionof ACL cells was significantly reduced only by 385 ,ug/ml ofanti-talin (29% reduction, P < 0.015) and not 200 ,ug/ml (10%reduction, P > 0.2), suggesting that a talin threshold levelexists. There was no significant difference in adhesion forcebetween MCL cells introduced with 200 and 385 ,ug/ml ofanti-talin. ACL cell adhesion was significantly reduced byanti-vinculin and anti-a-actinin at both concentrations (43%reduction by 30 and 100 ,ug/ml anti-vinculin, P < 0.0001; 32%

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p0.48

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0.4

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0-Ant-ibody Tmod talin vinculin oz-actinin(tgIml) 250 50 200 385 30 100 50 100

FIG. 2. Effect of mAbs against Tmod, talin, vinculi'n, and a-actininon the adhesion of ACL (Upper) and MCL (Lower) fibroblasts to anFN-coated (5 tkg/ml) surface. Two concentrations of mAbs used wereindicated beneath each mAb. Adhesion forces were normalized tocontrol (anti-albumin antibody at 150 4g/ml). Error bars are SEM.Number of cells measured: control ACL (n = 48) and MCL (n =87),anti-Tmod ACL (n = 79) and MCL (n = 97), anti-talin ACL (n =49)and MCL (n = 93), anti-vinculin ACL (n = 69) and MCL (n =96),and anti-a-actinin ACL (n = 82) and MCL (n = 85). *, denotessignificant difference with respect to the control.

reduction by 50 ~tg/ml anti-a-actinin, P < 0.005; 36% reduc-tion by 100 jig/ml anti-a-actinin, P < 0.002), whereas MCL celladhesion demonstrated a concentration dependence for anti-vinculin and anti-a-actinin, showing reduced adhesion only atthe higher concentration of 100 gg/ml (11% reduction by 30~tg/ml anti-vinculin, P > 0.4; 20% reduction by 100 txg/mlanti-vinculin, P < 0.05; 4% reduction by 50 jitg/ml anti-a-actinin, P> 0.5; 36% reduction by 100 ttg/ml anti-a-actinin,P < 0.005). There was no significant difference in adhesionforce between ACL cells electroporated with 30 and 100 jtg/mlof anti-vinculin, and also between ACL cells introduced with50 and 100 iLg/ml of anti-a-actinin. Adhesion forces betweenthe negative control cells that received no protein or antibodyduring electroporation and the control cells that received acontrol protein, albumin or its antibody, were not significantlydifferent, indicating that neither albumin nor its antibody haveany effect on ACL and MCL fibroblast adhesion to FN and thatthey may serve as a negative control for the adhesion studies.

In addition to the mAb to Tmod, a recombinant humanTmod was also used in evaluating the effects of Tmod on 'theadhesion of the ligament fibroblasts. Western blot analysis ofACL and MCL cells before and after electroporation withTmod (25 pLg/ml and 50 .tkg/ml) demonstrated that electro-poration was successful in delivering the exogenous Tmod intoboth types of cells. Human Tmod is a 40.6-kDa protein (11)with a Mr of 43,000 (33). Compared with control, densitometermeasurements of the treated groups showed that Tmod con-tent was 21% (ACL cells) and 64% (MCL cells) higher in cellselectroporated with 25 .tLg/ml of Tmod and 55% (ACL cells)and 101% (MCL cells) higher in cells electroporated with 50jitg/ml of Tmod. ACL and MCL cell adhesion was measuredand found to be affected differently after delivering exogenous

Table 1. Effect of Tmod on ACL and MCL fibroblast adhesion toan FN-coated (5 j,g/ml) surface

ACL normalized MCL normalizedadhesion, force adhesion, force

Treatment (± SEM) (± SEM)

Tmod (25 t,g/ml) 0.69 + 0.05* (n = 57) 1.14 + 0.09 (n = 87)Tmod (50 ,ug/ml) 0.70 ± 0.06* (n = 38) 1.79 + 0.12* (n = 71)

n, Number of cells.*P < 0.05.

Tmod into cells (Table 1). ACL cells electroporated with 25 or50 ,ug/ml of Tmod showed a decrease in adhesion by '30%with respect to control. In contrast, MCL cells showed a 79%increase in adhesion relative to control at 50 ,tg/ml of Tmod,but no significant change with respect to the control at 25,ug/ml. The differential effects of electroporation of Tmod onthe adhesion between these two types of cells strongly suggestdistinct molecular mechanisms acting within the ACL andMCL cells.The effects of disrupting actin-filament assembly with 0.5

,uM C.D. and microtubule assembly with 0.1 ,uM C.C. on theadhesion of ACL and MCL fibroblasts to FN is shown in Fig.3. Pre-incubation treatment with C.D. significantly reducedACL and MCL cell adhesion force by 91% and 84% (P <0.0001), respectively. Post-incubation treatment with C.D. wasless effective, but still significantly reduced the adhesion forcefor ACL and MCL cells by 27% and 34% (P < 0.01),respectively. Pre-incubation treatment with C.C. significantlyreduced the adhesion force of ACL and MCL cells by 20%

1.5 -

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0

0u:

c/i

0"0N-

zA

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a. Cytochalasin D

ACL* MCL

* *

*W

0

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FIG. 3. Effect of C.D. (0.5 ,uM, a) and C.C. (0.1 ,tM, b) on theadhesion of the two types of ligament fibroblasts to an FN-coated (5,ug/ml) surface. Adhesion forces were normalized to a control withoutreagent treatment. Error bars are SEM. Number of cells measured:C.D.-control ACL (n = 67) and MCL (n = 57), pre-incubation; ACL(n = 37) and MCL (n = 40), post-incubation; ACL (n = 78) and MCL(n = 25). C.C.-control ACL (n = 103) and MCL (n = 71),pre-incubation; ACL (n = 99) and MCL (n = 111), post-incubation;ACL (n = 57) and MCL (n = 31). *, Significant difference with respectto the control.

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Ii111 III lil iIIIIII Ib

FIG. 4. Epifluorescence micrographs of ACL (I) and MCL (II) fibroblasts seeded to an FN-coated (5 j,g/ml) coverglass. Actin filaments werestained with rhodamine phalloidin to visualize the spatial organization and formation of actin filaments into bundles (stress fibers). Seeding times:(a) 1 hr, (b) 4 hr, (c) 24 hr, and (d) 1 hr with mAb against a-actinin (I-d) and talin (II-d).

(P < 0.01) and 29% (P < 0.0005), respectively. Post-incubationtreatment with C.C. significantly reduced MCL cell adhesionforce by 23% (P < 0.05), but had no significant effect on ACLcell adhesion.

Visualization of Actin Filaments. Fig. 4 shows epifluores-cence micrographs of actin-filament bundles (stress fibers) inACL (Fig. 4I) and MCL fibroblasts (Fig. 411) seeded to FN (5,g/ml) for different time periods (1, 4, and 24 hr, labeled as

a, b, and c, respectively). At the beginning of cell seeding to FN,the stress fibers at the edge of both ACL and MCL cellsemanated radially, whereas the inner stress fibers were ran-

domly organized. After 1 hr, the inner stress fibers appearedcircumferential and in parallel rings (Fig. 4 I-a and II-a). Dueto cell elongation, the fibers then became elliptical, while

remaining parallel after 4 hr (Fig. 4 I-b and II-b). Stress fibersorganized into nearly parallel lines after 24 hr (Fig. 4 I-c andII-c). The changes in stress fiber orientation in ACL cellsoccurred more quickly than in MCL cells, but stress fibers inMCL cells appeared thicker than in ACL cells. The spacesbetween stress fibers in MCL cells were also wider than in ACLcells. The effect of mAbs against a-actinin in ACL cells (Fig.4I-d) and against talin in MCL cells (Fig. 4II-d) are also shown.Stress fiber formation and cell attachment area were reducedin ACL and MCL cells introduced with mAbs against talin,vinculin, and a-actinin compared with control cells.

DISCUSSIONThis study investigated the roles of various cytoskeletal pro-teins-Tmod, talin, vinculin, a-actinin, actin, and tubulin-in

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ACL and MCL fibroblast adhesion to an FN-coated surface.The major findings of this study are as follows: (i) Adhesion ofACL and MCL fibroblasts is influenced by the actin-filamentassembly and cytoskeletal integrity, and requires that cellspossess intact stress fibers for their normal attachment prop-erties; (ii) the actin-filament pointed-end capping protein,Tmod, influences cell adhesion behavior differently betweenACL and MCL fibroblasts; (iii) adhesion force and stress fiberformation are functions of the intact linkage between actinfilaments and integrin receptors by the bridging proteins talin,vinculin and a-actinin; and (iv) ACL and MCL fibroblastsdiffer in their sensitivity to mAbs against the same cytoskeletalproteins.We believe that this study is the first to characterize,

quantitatively, adhesion forces of human ligament fibroblastsinfluenced by the assembly of cytoskeletal proteins. By exam-ining intrinsic differences in adhesion between ACL and MCLfibroblasts in response to the disruption of cytoskeletal pro-teins, we seek to provide important insights to the ligamenthealing process. Previous studies in several laboratories haveshown differences in cell morphology (34), procollagen pro-duction rate (35), and adhesion characteristics between ACLand MCL cells in culture (18-20, 36-39). Our study shows thatthe cytoskeletal proteins have striking effects on ACL andMCL cell adhesion and on the formation of stress fibers duringcell spreading. A disruption in the cytoskeleton-integrin bridg-ing complex results in significantly reduced adhesion. Thisreduction in adhesion is most likely a result of decreasedformation of stress fibers and cell attachment area seen infibroblasts introduced with mAbs against talin, vinculin, anda-actinin. Our results indicate that the cytoskeletal proteinstalin, vinculin, and ac-actinin all influence the adhesion ofACLand MCL fibroblasts to FN; however, their roles appear to becell-type specific. By performing experiments using differentconcentrations of mAbs against these three proteins, wedetermined that ACL fibroblasts are more dependent onvinculin and a-actinin for adhesion to FN than MCL fibro-blasts. ACL fibroblasts require lower concentrations of anti-vinculin and anti-a-actinin than MCL fibroblasts to reduceadhesion force significantly. On the other hand, talin plays amore prominent role in MCL fibroblast adhesion to FN thanin ACL fibroblasts.By disrupting the assembly of actin filaments with C.D.,

adhesion force can be drastically decreased by 80-90% whenACL and MCL cells are pre-incubated with the agent beforecell attachment. A previous study strongly suggested theinvolvement of the cytoskeleton in the regulation of integrin-mediated cell adhesion after it was found that C.D. abolishedchimeric receptor adhesion to immobilized fibrinogen (40).Therefore, the reduction in adhesion force observed in ourexperiments may be caused by a loss of adhesion function ofthe 131-integrin receptor for FN. It is possible that this loss infunction could stem from a conformational change in theintegrin receptor brought on by the disruption in the associ-ation between the actin filaments of the cytoskeleton and theintegrin receptor. Another possibility is that actin filaments areanchored to the integrin receptor by bridging proteins. Theassembly of many actin filaments into stress fibers requires thatthe barbed ends of the actin filaments be anchored to theintegrin receptors. This condensed area of integrin receptorsand stress fibers is built up to form a functional adhesionplaque. Any disruption in stress fiber assembly will disruptadhesion plaque formation. Microtubule assembly also ap-pears to be an important factor in fibroblast adhesion to the FNmolecule, but not as essential as actin-filament assembly. Thismechanism, however, is not yet completely understood. Onereport showed a close functional correlation between micro-tubules and microfilaments with respect to cytoskeletal activityin leukocytes (41).

The role of Tmod in ACL and MCL fibroblast adhesion isless clear. For the two concentrations of Tmod used, MCL celladhesion depended on concentration (being affected only atthe higher concentration of 50 ,tg/ml Tmod), whereas ACLcell adhesion was affected similarly at both concentrations.This seems to indicate a threshold difference in Tmod con-centration between ACL and MCL cells. Fibroblasts from theMCL had increased adhesion with Tmod and decreased ad-hesion with anti-Tmod, similar to the findings from otheradhesion studies performed on white blood cells in our labo-ratory (15). It is assumed that the capping of actin filaments atthe pointed end (nonpreferred end of polymerization) withTmod serves in accelerating the polymerization of actin fila-ments at the barbed end near the focal contacts. This allows forgreater bundling of actin filaments into stress fibers which, inturn, results in greater adhesion. Fibroblasts from the ACL, onthe other hand, showed a much different effect of Tmod onadhesion behavior compared with MCL fibroblasts. This find-ing is consistent with past findings on the contrasting intrinsicproperties between ACL and MCL fibroblasts regarding theiradhesion and migration behavior (18-20) and also consistentwith new findings on intracellular differences. During the earlyperiod of adhesion, ACL fibroblasts lack the elevation inintracellular calcium observed in MCL fibroblasts (19), andthis may account for the differences seen in stress fiberformation rate and size between ACL and MCL fibroblasts(Fig. 4). Overall, we found ACL fibroblast adhesion to be lesssensitive than MCL fibroblast adhesion to calcium fluctuations(unpublished data). Signal pathway experiments we conductedalso reveal differences in the roles of A-kinase, G-kinase, andthe Ca2+/phospholipid pathway in cell adhesion between ACLand MCL fibroblasts (unpublished data). This may also havean impact on actin polymerization, cytoskeletal protein phos-phorylation, calcium- regulated stress fiber assembly, andpossibly the actin-filament capping function of Tmod duringadhesion.

This investigation concentrates only on cellular adhesionforces and the topographic distribution of actin filaments(stress fibers), and, therefore, primarily reflects cell adhesionbehavior. It would be of interest to examine the cell signaltransmission through the integrin receptor during cell adhe-sion to the extracellular matrix, the process of phosphorylationof cytoskeletal proteins, and the assembly of stress fibers.Further study on the signal pathways of ACL and MCLfibroblasts would help establish the molecular basis of thebiophysical behavior of cell adhesion, migration, and prolif-eration.

We thank Dr. Shu Chien for his constant support in our scientificresearch. This research was supported by National Institutes of HealthGrants AR34264 and HL43026 and American Cancer Society GrantIM-648.

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