Myosin II antibodies inhibit the resorption activity of isolated rat osteoclasts

Cell Motility and the Cytoskeleton 17:25&263 (1990)

Myosin II Antibodies Inhibit the Resorption Activity of Isolated Rat Osteoclasts

Masahiko Sat0 and William Grasser

Department of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania

We present microinjection data in support of an indirect approach by which cytoplasmic protein interactions important in the processes of bone resorption can be elucidated. Three polyclonal antibodies (M1 , M3, M5) raised against myosin I1 from perfused rat liver differently affected the actin-activated Mg ATPase of myosin 11. These antibodies microinjected into isolated rat osteoclasts affected osteoclast morphology and activity in bone resorption. M1, which completely inhibited myosin ATPase activity at a antibody:myosin ratio of 10: 1, initially promoted the extensioniretraction motility of lamellipodia but eventually reduced the spread area of osteoclasts along the substrate after 20 hr. M3, which inhibited ATPase activity by 70% , had similar effects; however, M5, which weakly inhibited ATPase activity, neither promoted extensioniretraction nor reduced spread area of osteoclasts. Immunofluorescence showed that these antibodies removed myosin I1 from the majority of actin filaments in injected osteoclasts. Because antibodies that did not bind to a myosin I1 column had little effect on the extensioniretraction of lamellipodia or the osteoclast spread area, these data suggest that myosin I1 participates in the stabilization of osteoclast lamellipodia along the substrate. M 1 injection strongly inhibited injected osteoclasts from excavating resorption lacunae in bone slices, compared to control antibody. M3 and M5 were less effective but also inhibited bone resorption. These data show that myosin I1 is functionally important in bone resorption and that the osteoclast-differentiated activity of bone resorption is a more sensitive assay for myosin activity than lamellipodia motility or cell morphology.

Key words: myosin, microinjection

INTRODUCTION Baron et al., 1985, 1989; Blair et al., 1986, 19891. Os-

Osteoclasts are large, multinucleate cells responsi- ble for excavating bone in vertebrates [Vaes, 1988; Sat0 and Rodan, 1989; Chamber, 19891. Bone resorption pro- ceeds by the intricate coordination of attachment to bone, polarization of the cell in response to attachment, secretion of lysosomal enzymes, and active lamellipodia motility. Resorbing osteoclasts firmly adhere to bone by forming an organelle-free “clear zone” rich in actin filaments that may be linked to vitronectin-like receptor- containing podosomes at the osteoclast/substrate inter- face [King and Holtrop, 1975; Marchisio et al., 1984; Zambonin-Zallone et al., 1989; Davies et al., 1989; Sato et al., in press]. The clear zone surrounds a highly in- vaginated membrane region, a “ruffled border,” where secretion of acid and proteases occurs [Vaes, 1968;

0 1990 Wiley-Liss, Inc.

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teoclasts active in resorption are highly dynamic with ruffling, extension/retraction of lamellipodia and active translocation over the bone surface [Hancox and Boo- throyd, 1961; Kanehisa et al., 19881.

The motility data and the intricate actin cytoskeleton [Zambonin-Zallone et al., 19831 suggested possible roles for actin-binding proteins in the several processes coordinated to achieve bone resorption. This hypothesis

Received May 4, 1990; accepted August 3, 1990

Address reprint requests to Masahiko Sato, Dept. of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Laboratories, West Point. PA 19486.

Myosin Antibodies Inhibit Resorption 251

MATERIALS AND METHODS Osteoclast Isolation

Osteoclasts were isolated from the long bones of 1-3 day old rats (Sprague-Dawley) by methods described by Chambers and Magnus [ 19821 and Arnett and Demp- ster [1987]. Rat osteoclasts were used because in our hands yields varied from 300-1,400/neonatal rat compared to 20-300/neonatal or weanling mouse or rabbit. Dead osteoclasts were easily distinguished by the absence of intact organelles (nuclei, nucleoli, vacuoles, plasma membrane), by the lack of motility, or by their disappearance with time. Some osteoclasts were stained for TRAP and acid hematoxylin (Sigma, St. Louis, MO). All chemicals were from Sigma unless otherwise stated. All experiments were conducted within 24 hr of cell isolation.

is based on data from other systems in which actin- binding proteins were shown to participate in adhesion complexes [Burridge et al., 19871, secretion [Creutz et al., 19881, and various types of cell motility [Warrick and Spudich, 1987; Stossel, 19881. Amongst the actin binding proteins, myosin is perhaps the best characterized and is the only member of this class of proteins capable of generating force at the expense of ATP, as in cell shape change, translocation of cells, movement of organelles, and anchoring of cells to substrate [Isenberg et al., 1976; Kreis and Birchmeir, 1980; Warrick and Spudich, 1987; Korn and Hammer, 1988; Stossel, 1988; Adams and Poilard, 19891.

There are 2 major classes of myosins which include the conventional 2-headed myosin I1 found in muscle and nonmuscle cells and the single-headed myosin I origi- nally identified in Acanthamoeba [Pollard and Korn, 19731. Both can perform work (mechanochemical transduction), crosslink and superprecipitate actin filaments, and are characterized by actin-activated ATPase activities [Warrick and Spudich, 1987; Conzelman and Mooseker, 1987; Korn and Hammer, 19881. Perhaps the best demonstration of the involvement of myosin in cell shape change was the inhibition of cytokinesis with myosin polyclonal antibodies [Mabuchi and Okuno 1977; Kiehart et al., 19821 and by the use of antisense RNA [Knecht and Loomis, 19871 or by recombination-mediated gene disruption [DeLozanne and Spudich, 19871.

An obstacle to the biochemical investigation of mammalian osteoclastic activity is the inability to purify sufficient quantities of cells for most types of experimentation. At least two approaches have emerged to over- come this problem. The first is to purify or differentiate precursors into multinucleate giant cells which stain for tartrate-resistant acid phosphatase (TRAP), respond to calcitonin, and resorb bone [Takahashi et al., 19881. There are some questions about this approach [Hattersley and Chambers, 1989; Chambers, 19891, but the chicken system appears to be promising in yielding precursors that can be induced in culture to resorb bone [Alvarez et al., 1989; Prallet et al., 19891. Unfortunately, a debate also exists as to possible physiological differences between avian and mammalian systems because the former may be unresponsive to calcitonin [Nicholson et al., 1986, 1987; Arnett and Dempster, 1987; DeVernejoul et al., 1988; Hunter et al., 19891. An alternative approach that we advocate here is the biochemical characterization of antigens from cells which may share the protein of interest, followed by microinjection of functional antibodies into osteoclasts to probe the participation of the antigen in osteoclastic activity. We present data in support of this indirect approach and show that antibody- induced depletion of cytoplasmic myosin I1 from osteoclast actin filaments inhibits bone resorption.

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Myosin Purification and Antibody Production Myosin I1 was purified [Pollard, 1982b3 from per-

fused (0.9% NaCl, 4°C) rat liver which was ground with a Potter Elvenheim homogenizer (10 X ) into Buffer A (0.34 M sucrose, 2 mM EGTA, ImM DTT, 1mM ATP, 0.1 mM benzamidine, 1 mM PMSF, 0.5 kg/ml leupeptin, 1 .O pg/ml pepstatin A, 10 mM imidazole, pH 7.0). This suspension was centrifuged at 150,000 g, 70 min, 2"C, and myosin was purified from the supernatant by chromatography on a A-800 column (Amicon, Dan- vers, MA), ammonium sulfate precipitation, gel filtration on sephacryl S-300 (Pharmacia, Piscataway, NJ), actin pelleting with rabbit skeletal actin (0.2 mg/ml), gel filtration on a 0.6 M KI discontinuous sephacryl S-300 column otherwise equilibrated in KC1 buffer (0.6 M KC1, 1 mM DTT, 0.5 mM ATP, 10 mM imidazole, pH 7.0) and hydroxyapatite column (Biorad, New York). Fractions were assayed by conductivity, A295, 7.5% Laemmli [1970] gels, and Mg ATPase activity.

Antibodies (MI, M3, M5) to myosin were raised by injecting weanling New Zealand rabbits with cytoplasmic myosin I1 at 20 pg/ml (M3, M5) or injecting the heavy chain (50 pg) from 7.5% Laemmli gels (Ml) with Freund's adjuvant to generate antibodies. The IgG fractions were purified by protein A affinity chromatography from antisera (MI, M3, M5) and nonspecific rabbit serum (Beckman, Fullerton, CA). Myosin-specific IgGs were eluted with 3 M K-thiocyanate, Na phosphate buffer pH 7.0 from a column in which liver myosin I1 was linked to cyanogen bromide-activated Sepharose 4B (Pharmacia).

Blots and ATPases Proteins in sucrose extracts of primary cultures

from rat long bones were examined for reactivity to MI , M3, and M5 by immunoblotting [Kiehart et al., 1984;

252 Sat0 and Grasser

Towbin et al., 19791. The effect of antibodies on the Mg and actin-activated ATPases of cytoplasmic myosin was examined by the method of Pollard [1982a]. Briefly 50 pg/0.5 ml (0.2 pM) myosin was incubated with about 2 pM of rabbit IgG, M I , or M3 in 15 mM imidazole, pH 7.0, 60 mM KCI, 2 mM ATP, and 2 mM MgSO, in the presence or absence of 1 mg/0.5 ml actin filaments. The actin was purified by gel filtration from rabbit acetone powder [MacLean-Fletcher and Pollard, 19801.

Microinjection arid Scrap Loading Microinjection was performed by the method of

Kiehart [1982]. Briefly, micro-needles with 1 p,m tips were pulled from Omega dot capillaries (0.6 mm outer diameter with internal glass fiber (Glass Co. of America, Bargaintown, NJ) using a vertical pipette puller (model 730, David Kopf Inst., Tujunga, CA). Antibodies were concentrated to 5 mg/ml with a RCF-Confilt (Bio-Mo- lecular Dynamics, Beaverton, OR), and needles were backloaded with 1-3 p1 of a 5 mg/ml antibody solution in microinjection buffer (10 mM imidazole, pH 6.8, 60 mM potassium acetate). In some cases, a fluorescent tracer, tetramethyl rhodamine dextran (10,000 MW, Mo- lecular Probes, Eugene, OR) at 2 mg/ml was included to help identify injected cells. Loaded needles were mounted on a Narishige micromanipulator (MO- 102, Optical Apparatus Co. Inc., Ardmore, PA) which was mounted on a Nikon Diaphot with 2 0 x ELWD phase contrast optics. Osteoclasts were injected at an angle of about 10" while maintaining a constant, low flow rate out of the tip. Needles were kept in osteoclasts until about 10% of the cell was injected as estimated from the re- sulting cell swelling. If osteoclasts are similar to macrophages in which 0.7-1.5% of the protein is myosin (about 1 pM [Hartwig and Stossel, 1975]), then the molar ratio of antibody to myosin would be about 3:l in osteoclasts [see Honer et al., 1988; Sinard and Pollard, 19891.

Buffer, myosin antibodies, or rhodamine-conjugated goat anti-mouse IgG antibodies (RGAM, Cappel, Malvern, PA) were scrape loaded into osteoclasts by a modification of the method of McNeil et al. [1984; Mc- Neil and Warder, 19871. Osteoclasts (300-1,000/well) were aliquoted into 6-well plates (Costar, Cambridge, MA) for 30-40 rnin at 37°C. Attached cells were washed 2 X with 4°C Ca-Mg-free PBS (CMF-PBS) and then incubated in CMF-PBS with 45% D,O (Aldrich, Milwau- kee, WI) for 6-10 rnin on ice until osteoclasts retracted lamellipodia. This solution was gently replaced with 50 pl of desired injection buffer and cells were scraped off with 2-4 strokes of a rubber policeman. Cells were im- mediately resuspended in 0.5 ml of complete 199 media (Gibco, New York) and aliquoted (0.4 ml) onto a 6 X 6

X 0.1 mm longitudinal slice of bovine femur cut from the diaphysis and (0.1 ml) onto a No. 1 coverslip in 2 ml of complete 199 media. Bone slices with osteoclasts were supplemented with 1 ml of media 199 and incubated at 37"C, C0,-air for 24 hours while osteoclasts on coverslips were counted and examined for fluorescence with a Nikon Microphot after a 4-6 hour incubation.

Bone Slice Resorption Assay Prior to incubation with osteoclasts, bone slices (6

X 6 X 0.1 mm) [see Arnett and Dempster, 1987; Sat0 and Grasser, 19901 were hydrated in complete 199 media in a 48 well plate (Costar) overnight. After 24 hours with scrape-loaded osteoclasts , slices were devitalized by ul- tra-sonication in filtered 5% NaOCl, (J.T. Baker, VWR Scientific) for 1-5 minutes. Slices were fixed in 2.5% glutaraldehyde, 0.1 M cacodylate pH 7.4 (EM supplies, Fort Washington, PA), dehydrated through ethanol (40, 70, 100%) and then infiltrated for 40 rnin with 1:l eth- ano1:Peldri I1 (Ted Pella, Inc., Redding, CA). After incubation in 100% Peldri 11, solidified samples were evac- uated overnight to sublimate off the Peldri 11. Slices were rotary shadowed with gold (DV-502A Denton Vacuum, Cherry Hill, NJ) and then examined on a JEOL JSM 840 at 3 KV accelerating voltage.

Immunofluorescence, Image Processing and Statistics

Osteoclasts were processed for immunofluorescence by modification of previous methods [Osborn and Weber, 1982; Schliwa and Van Blerkom, 19811. Cover- slips with osteoclasts were briefly rinsed in buffer S (60 mM pipes pH 6.9, 25 mM hepes, 10 mM EGTA, 2 mM MgSO,) at 37"C, then fixed for 2 rnin in buffer S + 10% paraformaldehyde, pH 7.0. Cells were permeabilized in buffers S + 0.5% Triton X-100 and then rinsed. Spec- imens were incubated for 30 rnin with the appropriate antibody or rhodamine-phalloidin (Molecular Probes) and followed by 30 min in fluorescein-conjugated goat anti-rabbit antibody (Cappel).

Quantitation of resorbed areas (from SEM negatives/Polaroid 55 film) and of osteoclasts (35 mm negatives) was achieved through digital image processing (Magiscan 2A, Joyce Loebl, New York) of video images (Newvicon tube, Image Technology Corp., New York) fed through a NTSC/PAL digital standards converter (CEL P156, James Grunder and Assoc., Inc. Mission, KA). Images of osteoclasts in culture were recorded in real time onto a Sony VO-5800H video cassette recorder. Statistical significance was assessed by unpaired t test analysis with RS/1 (BBN Research Systems, Cambridge, MA).


TABLE I. Myosin ATPase Activity 2 Antibodies*

Mg + F Actin

Myosin 32.3 ? 3.2 61.4 & 3.5 Myosin + IgG 36.7 k 5.3 52.4 * 1.9

15.8 ? 0.7 Myosin + M3 IgG Myosin t M5 IgG 31.7 f 4.1 48.9 k 2.0

Myosin + M1 IgG 4.3 ? 0.9 0 13.0 & 2.2

Fig. 1. Electron microscopy of the myosin antigen. The cytoplasmic myosin purified from rat liver appeared as two-headed structures with a long tail on replicas of specimens rotary shadowed with platinum- carbon. These structures are reminiscent of the two-headed dimers observed previously for myosin I1 and skeletal muscle myosin [Korn and Hammer, 19881 but with smaller dimensions of 14.8 ? 5.4 nm (heads), 141 * 16 nm (tails). This preparation was used to generate antibodies M1, M3, and M5. Scale: 150 nm.

*ATPase activities (nmol/min mg) were determined by using 50 Fgi 0.5 ml of purified liver myosin in a buffer containing 15 mM imidazole, pH 7.0, 60 mM KC1, 2 mM ATP, 2 mM MgSO, plus or minus 1 mg/0.5 ml actin from rabbit skeletal muscle at 37°C. Where speci- fied, rabbit antibodies or nonspecific IgG from MI preimmune serum (Ml, M3, or M5) were included at a 10-fold molar excess ( 2 pM) to myosin (0.2 FM).

A B C D E F G H I J K Transmission Electron Microscopy

Liver myosin at 1 mg/ml was diluted to 20-50 pg/ml in 0.6 M ammonium acetate buffer, pH 7.0, and 33% glycerol. Samples were sprayed onto mica with an atomizer (Effa spray mounter, Fullam, Schenectady , NY), dried in 1.3 X ton vacuum, and rotary shadowed at 6" with platinum-carbon (Bench top turbo, Den- ton Vacuum, C h e w Hill, NJ), as described by Fowler and Aebi [ 19831. Electron micrographs of replicas were taken on a Philips CM12 at 80 kV.

200.

116. 95

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RESULTS 55-

The antigen from perfused rat liver used to generate functional antibodies to probe myosin activity in osteoclasts was identified as a myosin I1 by gels, electron microscopy, and ATPase activity. Rotary shadowing of the antigen revealed two headed monomers characteristic of myosin I1 with heads of 14.8 ? 5.4 nm and tails of 141 t 16 nm (Fig. 1). M g t t - and actin-activated ATPase activities which are properties of myosin I1 [Korn and Hammer, 19881 were measured as shown in Table I. Polyclonal antibodies were raised to this antigen and the IgG fraction for three antibodies (MI IgG, M3 IgG, and M5 IgG) were observed to have different effects on the Mg- and actin-activated ATPase activities of liver myosin 11. At a 1O:l molar ratio, M1 IgG inhibited both ATPase activities by >88%, while M3 IgG inhibited them by 66% and 75%, respectively. M5 IgG had little effect on either ATPase activity as did nonspecific rabbit IgG (Table I).

The three myosin antibodies were compared for their ability to recognize myosin on blots of liver preparations and extracts of long bone cell cultures (Fig. 2A-K). Affinity-purified M 1 IgG (Affi M 1 ) recognized the 200 K heavy chain of myosin on blots of liver extract and a similar band on blots of long bone cultures. M 1,

Fig. 2. Cross-reactivity of myosin antibodies. Antibodies to liver myosin reacted to the 200K heavy chain of rat liver preparations and a similar band in extracts of osteoclast cultures which were isolated from the long bones of neonatal rats. Lanes A and F correspond to Coomassie-stained standards. Lanes E D correspond to liver samples incubated in flow through IgG, affinity-purified M1 (Affi Ml), and nonspecific rabbit IgG, respectively, stained with horseradish perox- idase reaction product. Lane E corresponds to silver-stained extract from ostcoclast cultures. Lanes G-K correspond to osteoclast extract incubated with Affi M1, M1 IgG, M3 IgG, M5 IgG, and nonspecific rabbit IgC, respectively, followed by incubation with '251-protein A and autoradiography. In primary cultures, osteoclasts typically com- prised 1% or less of the adherent cell number, but because osteoclasts are significantly larger than mononuclear cells (see Fig. 3), their protein contribution per cell is probably greater.

M3, and M5 IgGs (not affinity purified) recognized a similar band but also additional bands (about 70, 65K) were observed. Flow through IgGs and nonspecific IgGs from rabbits did not recognize bands above background on blots of either liver or long bone cultures.

Living osteoclasts were microinjected with these antibodies and changes in morphology were examined as a function of time. Osteoclasts injected with Affi M1 or


0 hr. 6 hr. 20 hr.

Fig. 3. Osteoclast morphology after MI microinjection. Living osteoclasts injected with Affi MI (row A) or MI IgG (rows B, C) were examined by phase contrast and fluorescence microscopy within 5 minutes of the time of injection (0 hours), 6 hours later, and 20 hours later. Tetramethylrhodamine dextran (10,000 MW) at 2 mg/ml was

included as fluorescent tracer. Extensionhetraction of lamellipodia was observed but osteoclasts did not appear to translocate significantly with respect to the mottled pattern of the petri dish as observed for 20 hours. Scale: 100 pm.

M1 IgG at a concentration of 5 mg/ml showed dynamic extension and retraction of the lamellipodia (see Fig. 3A-C). However, these cells translocated less than 1 cell diameter/20 hours, as seen by examining the osteoclasts in relation to the mottled pattern of the petri dish in Figure 3. In addition, a slow decrease in the cell area associated with the substrate became apparent at 6 and 20 hours after injection. Osteoclasts injected with more M 1 as judged by fluorescent intensity (Fig. 3B) retracted to more compact cells in less time than osteoclasts injected with less antibody (Fig. 3C).

Osteoclasts injected with buffer alone, control antibodies, or M5 IgG were much less active in extension/ retraction of lamellipodia, as seen by comparing the se-

of flow through IgGs (Fig. 4A), buffer alone (Fig. 4B), M5 IgG (Fig. 4C), rhodamine goat anti-mouse antibodies (RGAM), and nonspecific rabbit IgGs did not induce

- ries in Figure 4 with the M1 series in Figure 3. Injection

substantial changes in cell morphology over the same time period (see also Fig. 5) . Control-injected osteoclasts also were not observed to translocate very far compared to neighboring mononuclear cells (see osteoclast with respect to scratch in series of Fig. 4B). These data suggest an inverse relationship between myosin I1 ATPase activity and the extension/retraction activity of osteoclast lamellipodia [see Honer et al., 19881.

Digital image processing of the spread area of injected osteoclasts confirmed that M 1 induced significant morphological changes as compared to other antibodies and controls. Figure 5A compares the time-dependent changes in spread area of 3 osteoclasts of about lo4 pm2 which were injected with affinity-purified M1, flow through IgG or M5 IgG. Additional microinjection data (Fig. 5B) normalized with respect to the osteoclast spread area at the time of injection (0 hour) show that only myosin antibodies from M1 (or M3, data not

0 hr.


6 hr. 20 hr.

X

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Fig. 4. Osteoclast morphology after microinjection of control soh- tions. Living osteoclasts injected with flow through IgG (Flow thru, row A), buffer plus rhodamine dextran (Dex, row B), or M5 IgG (M5, row C) were examined within 5 min of injection (0 hours), 6

hours later, and 20 hours later. Limited extensioniretraction motility of larnellipodia were observed and all of these osteoclasts retained extensive lamellipodia at the substrate after 20 hours. Scale: 100 pm.

shown) induced a 50% reduction in cell area as followed for 1 day. Osteoclasts that reduced spread area by more than 50% tended to detach and were lost. Flow through IgG and nonspecific rabbit IgG had little effect on osteoclast area. M5 IgG induced a 20% reduction in area, but so did buffer ? fluorescent tracer, tetramethylrhodamine dextran (l0,OOO MW), and RGAM. These data suggest a relationship between myosin I1 ATPase activity and the spread area of osteoclasts along the substrate.

Injected osteoclasts could also be followed with some difficulty without tracer molecules, because no more than five osteoclasts were present per grid square in culture. The presence or absence of tracer did not significantly influence the effect of M1 on osteoclast area (Fig. 5B). Osteoclasts smaller than 5 x lo3 pm2 were not analyzed because the halo of phase contrast images [Francon, 1961; Inoue, 19861 and interference from the

grid pattern of culture plates made quantitation of smaller osteoclasts with 20 X objectives imprecise.

Fluorescence microscopy of injected osteoclasts showed that cell shape imprecisely documents the effect of antibodies on osteoclasts because M1 Ab altered both myosin distribution and cytoplasmic organization before reducing the osteoclast spread area. As seen by comparing Figure 6A-C, Affi M1 (and M1 IgG) altered the myosin I1 distribution in large areas of injected osteoclasts. The cell in Fig. 6A,D retained 80% of its original spread area while the osteoclast in Figure 6B,E retained 44% of its spread area. Examination of Figure 6A,B suggest that Affi M1 is able to denude actin filaments of myosin I1 because in controls Affi M1 staining appeared as intense, periodic fluorescence along actin filament bundles throughout the cytoplasm [see Warshafsky et al., 1985; Turksen et al., 19881. The denuded areas are compared at higher magnification in enlargements (Figs. 7,


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Fig. 5 . Microinjection effects on osteoclast spread area. A: The time- dependent changes in spread area are shown for 3 osteoclasts of about I X 10' km' area injected with Affi MI (V), flow through IgG (V), or MS IgG (e). B: The spread area of injected osteoclasts was normalized to the area at the time of injection (0 hours). Mean areas (mean 2 SEM) of osteoclasts injected with affinity MI (V, n = 12). MI IgG (0. n = 17), or MI IgG with tracer (0, n = 6) decreased to 50% of the original area by 20 hours. Injections with other solutions resulted in up to a 20% reduction in area by 20 hours for flow through IgC (V, n = X), nonspecific rabbit IgG ( 0 . n = I I ) , M5 IgG (e, n = 8). rhodamine goat antimouse (A. n = 1 I ) , bulfer with tracer (Dex, 0. n = 8). or buffer alone without tracer (m. n = 6) . T test analysis showed that areas of osteoclasts injected with Aifi MI P < ,0001, M I + DEX P < .0001, and MI P < .0007 were significantly less than those injected with Flow Thru antibodies.

8). Intense staining of rhodamine phalloidin was observed in all Affi M 1 -injected osteoclasts; but as seen in Figure 6D and in the enlargement (Fig. 8B), the bundles of actin filaments appeared shorter than those in controls. The actin filament distribution appeared more trabecular and an increase in vesiculation was observed near the site of injection. This suggests a role for myosin I 1 in the network organization o f actin bundles. Vesiculation was also observed in MS IgG- and control-injected osteoclasts (data not shown).

Comparison between Affi M 1 -injected osteoclasts such as in Figure 6A,B also suggested an inverse rela-

tionship between staining for myosin I1 and retention of original spread area. The arrow in Figure 6E points to an area of lamellipodia active in extensiodretraction. The absence of myosin I1 staining of this region suggests that the dynamic extensioniretraction observed does not require myosin I1 association with actin filaments in the lamellipodia. Because control osteoclasts stained with Affi M I after fixation gave staining patterns similar to previous efforts [Warshafsky et al., 1985; Turksen et al., 19881, these micrographs show that Affi MI binds to endogenous myosin I1 and is capable of depleting actin filaments of associated myosin upon microinjection. In addition, these data show that morphology and spread area are insufficient to document the full effects of myosin antibodies on osteoclasts.

For these reasons the functional significance of antibody injection was examined by observing the effects on a differentiated activity of osteoclasts, bone resorption. Osteoclasts were injected with MI IgG, M3 IgG, M5 IgG, RGAM, or buffer plus tracer by the scrape loading method of McNeil et al. [1984; McNeil and Warder 19871 and then incubated on consecutive longitudinal slices from the diaphysis of bovine femur. Con- trols with RGAM examined by fluorescence microscopy showed that 64-100% of the osteoclasts were labeled and capable of spreading on glass, plastic, or bone slices. A broad distribution of fluorescence labeling was observed as previously described; however, limited digital image processing suggested that less myosin antibody was incorporated by this technique than by needle injection, as shown by McNeil et al. [1984, McNeil and Warder 19871.

Scrape loading was also more injurious to osteoclasts than needle injection (viability 7S-100% of controls) as only 5-30% of the original osteoclasts survived loading. Nevertheless, surviving osteoclasts were able to excavate resorption lacunae that were qualitatively indistinguishable from pits made by controls (Fig. 9). Quan- titation of the area of resorption pits [Boyde et al., 1985; Jones et al., 19861 by digital image processing ot' SEM negatives (Fig. 10) showed that MI IgG significantly inhibited bone resorption with P < .01 (Student's t-test analysis). To lesser degrees, M3 IgG and M5 IgG also inhibited bone resorption ( P 5 .0.5). These data show that myosin antibodies potently inhibit resorption, suggesting that myosin I1 participates in bone resorption.

In data not shown, microinjected osteoclasts re- mained TRAP positive and responsive to calcitonin in a manner indistinguishable from controls. In addition, re- covery of M I-injected osteoclasts which did not coni- pletely retract lamellipodia was observed after 2 days suggesting that antibody effects are reversible; osteoclasts which completely retracted tended to detach. These data plus the ability to resorb bone show that in-


Fig. 6. Injection effects on osteoclast cytoplasm. Osteoclasts injected with Affi MI (A,B) were stained with fluorescein goat antirabbit (A,B) and rhodamine phalloidin (D,E) after 15 hours of incubation at 37°C. The control osteoclast in (C) was fixed before staining with Affi

M1 and rhodamine phalloidin (F). The boxes in D and F correspond to the regions enlarged in Figures 8 and 7, respectively. The osteoclasts in A and B retained 80% and 44% of their original spread area. Scale: 10 km.

jected osteoclasts retain their differentiated characteris- tics.

DISCUSSION

The abundant, highly organized actin filament network in the cytoplasm of osteoclasts [King and Holtrop, 1975; Zambonin-Zallone et al., 19831 suggested to us that actin-binding proteins may be important in modulating osteoclast function. However, because mammalian osteoclasts are available in insufficient quantities for biochemical experimentation, single cell experiments were conducted with immunological and micromanipulation

techniques to ascertain protein interactions important in the mechanism of bone resorption. Toward this goal antibodies to cytoplasmic myosin I1 from perfused rat liver were raised based on the speculation that osteoclasts may have a similar myosin isoenzyme. This approach was necessary because while there are well-characterized myosin I1 antibodies [Claviez et al., 1982; Kiehart et al., 1984; Citi and Kendrick-Jones, 19871, obtainable antibodies [Kiehart et al., 19841 were found to be non-specific based on blots and immunofluorescence of rat osteoclasts. For this reason, functional polyclonal antibodies were raised and chosen based on the ability to inhibit the actin-activated Mg-ATPase because this ac-


Fig. 7. Myosin and actin staining of control. In control osteoclasts Affi M 1 (A) staining colocalized with rhodamine phalloidin staining (B), although the reverse was not true. Both actin filament bundles and actin filament dots which colocalize with podosomes were observed.

Periodic myosin I1 staining along actin bundles was observed with some but not complete colocalization with actin filament dots [Marchi- sio et al., 1984; Warshafsky et al., 1985; Turksen et al., 19881. Scale: 10 pm.


Fig. 8. Myosin and actin staining after microinjection. Figure 6A and D were enlarged near the site of Affi M I injection. Large areas of the cytoplasm were devoid of staining for myosin I1 and shorter bundles of actin filaments were observed. The actin filament network appeared more trabecular and vesiculation of the cytoplasm was observed. Scale: 10 Fm.

260 Sato and Grasser

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Fig. 9. Resorption pits on bone slices. Scanning electron microscopy (SEM) allowed the comparison of resorption lacunae in slices of bovine femur produced by osteoclasts injected with various solutions (A-D). Resorption lacunae were made by osteoclasts scrape loaded with buffer plus fluorescent tracer (A), RGAM (B), M3 IgG (C), or MI IgG (D). Scale: 100 pm.

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Fig. 10. Quantitation of resorbed area. The area of resorption pits was quantitated by digital image processing of SEM negatives from the various samples of scrape-loaded osteoclasts on bone slices. Mean values (mean ? SEM) corresponding to the resorbed areaislice ( 6 X

6 x 0.1 mm) were plotted for 3 sets of experiments comparing MI (n = 2 ) to RGAM (n = 2) and M3 (n = 2) to RGAM (n = 2) and buffer with IOK tetramethyl rhodamine dextran (Dex, n = 2) and M5 (n =

2) t o RGAM (n = 2). Because of the variability in osteoclnst number the three sets were not directly compared. Howevcr each showed that myosin antibodies (including M5) injected into osteoclasts inhibit the area resorbed. For each set. osteoclasts wcrc isolated from baby rats of the same litter.

- tivity correlates with the mechanochemical transduction of myosin [Warrick and Spudich, 19871. In the absence of previous biochemical or sequence data concerning osteoclast myosins, this approach appeared most able to

elucidate the functional significance of myosin I1 in osteoclastic activity.

Northern analysis with cDNA to a myosin heavy chain from chicken intestinal epithelial cells showed that all non-muscle tissues examined express a similar mes- sage, including kidney, spleen, brain, and liver [Shohet et al., 19891. Blots, immunofluorescence, and microinjection experiments suggest that osteoclasts share a myosin I1 isoenzyme similar to liver myosin and the effects of MI (and M3) Ab or IgG injection on osteoclast morphology suggests that myosin I1 participates in the main- tenance of spread lamellipodia. Compared to injected controls, these antibodies promoted the extensioniretraction of lamellipodia but inhibited the continued mainte- nance of spread lamellipodia. The former may correspond to the enhanced lamellipodia motility noted by Honer et al. [ 19881 upon antibody injection of tenth but not earlier passage chicken embryo fibroblasts. How- ever, unlike chicken embryo fibroblasts, injected osteoclasts were not observed to translocate more than 1 cell diameteri20 hr and actually decreased the spread area at the plane of the substrate with time. This may indicate that myosin I1 participates in anchoring osteoclasts to substrate.

Previously, in fibroblasts, stress fibers were shown to apply tension and anchor cells through adhesion plaques to the substrate [ Isenberg et al., 1976; Kreis and Birchmeir, 1980J. While osteoclasts are devoid of stress fibers and have podosomes instead of adhesion plaques [Zambonin-Zallone et al., 1983. 1989; Turksen et al.,


nipulation experiments have limitations that should be recognized. Microinjection is injurious to cells as shown by Sinard and Pollard [ 19891. Evidence for this was the injection-induced 20% decrease in the spread area of controls with time and the vesiculation of cytoplasm observed in even buffer-injected osteoclasts. Extensive efforts to improve the empirically derived injection buffer or microinjection technique by either criteria have not been successful, to date. The observations made on injected osteoclasts were assumed to be generally valid, but the possibility exists that because of anchorage selection in the culture methods, viability selection, and size selection (5 X 103-7. 1 X lo4 pm2) the conclusions may be relevant to a subpopulation of osteoclasts. Nev- ertheless, we argue that injected osteoclasts are reason- able models of isolated osteoclasts on the basis of TRAP staining, calcitonin responsiveness, antibody staining (data not shown), and ability to resorb lacunae in bone slices [Vaes, 1988; Sat0 and Rodan, 19891.

These data show that the indirect approach of using functional antibodies to probe the antigen in possibly related cells has promise in elucidating protein interactions important in osteoclastic activity. However, the utility of this approach is limited by the biochemical characterization of the antigen and characterization of the antibodies. Myosin activity is regulated by the confor- mational state which in turn is affected by the phosphor- ylation state of light chains, heavy chains, polymeriza- tion, and possibly by the binding of proteins (such as antibodies) to specific sites along the molecule [Wamck and Spudich, 1987; Korn and Hammer, 19881. To ascertain which domain of myosin I1 is important in bone resorption will require further analysis.

19881, periodic (sarcomeric) staining of myosin and other components (alpha actinin) of contractile fibers along actin filaments have been observed in proximity to the podosomes [Marchisio et al., 1984; Warshafsky et al., 1985; Turksen et al., 19881 (see Fig. 3). Therefore, we speculate that myosin I1 is part of the mechanism by which the lamellipodia is stabilized via vitronectin-like receptor complexes attached to substrates including bone [Zambonin-Zallone et al., 1989; Davies et al., 1989; Sat0 et al., in press].

The fact that M1-injected osteoclasts continue to extendhetract lamellipodia may be explained by incom- plete depletion of myosin I1 by the antibodies or by the existence of alternative myosins in osteoclasts. Similar lack of inhibition of motility in other cells [Knecht and Loomis, 1987; DeLozanne and Spudich, 1987; Honer et al., 1988; Sinard and Pollard, 19891 may be explained by the existence of myosin I [Pollard and Korn, 19731 which was shown to bind to membranes and move ves- icles along actin filament bundles [Adams and Pollard, 19891. The identification of similar proteins in vertebrate cells [Collins and Borysenko, 1984; Hoshimaru and Na- kanishi, 19871 suggests the possibility of a myosin I in osteoclasts.

The rounded, compact morphology produced by M1 injection occurred more slowly but was not dissim- ilar to calcitonin (0.1 nM) effects on osteoclasts [Cham- bers et al., 1984; Arnett and Dempster, 19871. There- fore, the inhibition of myosin I1 activity may be part of the mechanism by which calcitonin-receptor binding in- duces retraction of the lamellipodia and the cessation of bone resorption [Chambers et al., 19841. Other support- ive evidence for this kind of mechanism includes the finding that calcitonin elevates cytoplasmic CAMP [Nicholson et al., 1986, 19871 which in other systems activates CAMP-dependent protein kinases to inhibit actomyosin ATPase activity and force transduction [Sellers and Adelstein, 19871.

The finding that M1, M3, and M5 antibodies all inhibited the excavation of bone is evidence for the importance of myosin I1 in the processes of bone resorption. Because the amount of antibody injected by scrape loading was less than by microinjection as seen in fluorescence microscopy and because these cells spread on glass, plastic, and bone, myosin I1 activity appears to be more important in resorption than in modulating osteoclast morphology. These data also show the importance of examining a differentiated cell function over cell morphology in evaluating the effect of antibodies because while M 1 potently inhibited ATPase activity, spread area, and resorption, M5 also significantly inhibited resorption but had little effect in the 2 former assays.

While our data suggest that myosin I1 is a regulatory protein in osteoclast shape and function, microma-

ACKNOWLEDGMENTS

The authors thank R. Murrills and D. Dempster (Helen Hayes Hospital) for instructions in osteoclast isolation from rat, J. Ianacone (Merck) for excellent tech- nical assistance, and W. Pfender (Merck) for help with the JEOL JSM 840. We also thank J. Murray (Univ. of PA), G. Rodan (Merck), and J. Sellers (NIH) for criti- cally evaluating the manuscript. We would also like to thank D. MacDonald for help in assembling the manuscript. Finally, we would like to dedicate this effort to Robert D. Allen (1927-1986) who encouraged me to try the “impossible.”

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