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Home , Filamin, Intermediate filament

Journal of Science 102, 19-30 (1992) 19 Printed in Great Britain © The Company of Biologists Limited 1992

Identification of the -associated gyronemin as

Implications for a novel mechanism of cytoskeletal interaction

KEVIN D. BROWN* and LESTER I. BINDER! Department of , School of Medicine and Dentistry, University of Alabama at Birmingham, BHS-63l/University Station, Birmingham, AL 35294, USA *Present address: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA tAuthor for correspondence

Summary

In a previous paper, a monoclonal antibody (designated bovine gyronemin as the immunogen show this protein Ml.4) that recognized a 240 kDa polypeptide was to be associated with -containing stress fibers, characterized. This antibody stained the intermediate although our original Ml.4 antibody continued to be filaments in several cell lines, and biochemical charac- localized along filaments. Since two-dimen- teristics of the 240 kDa polypeptide led us to conclude sional electrophoretic analysis did not demonstrate a that it was a novel intermediate filament-associated difference in either relative molecular mass or iso- protein, which we termed gyronemin. Here we report electric point of this polypeptide when associated with that gyronemin is expressed in adult rat organs that either filamentous system, we conclude that filamin is a contain a substantial component. Taking bifunctional protein capable of associating with both the advantage of this observation, this protein was purified intermediate filament and actin cytoskeletal systems. from bovine uterine tissue and, by biochemical, immu- nological and sequence analysis, found to be homologous to the actin-associated protein filamin. Three novel monoclonal antibodies raised using purified Key words: intermediate filament, gyronemin, filamin.

Introduction cytoskeletal element with which they associate. How- ever, some have been shown to bind or be co- The eukaryotic is largely composed of localized with more than one type of filament (for , and intermediate fila- example, see Frappier et al., 1987; Griffith and Pollard, ments. Although they are biochemically distinct poly- 1982; Mangeat and Burridge, 1984), and therefore may meric systems, several observations indicate that they be involved, in part, in facilitating heterologous cross- are physically interconnected. For example, exposure linking of different cytoskeletal systems. Alternatively, of cells to -dismpting agents (e.g. colchi- differential functions of these proteins may be specified cine) leads not only to ablation of most of the via association with morphologically and biochemically cytoplasmic microtubule complex, but also to large distinct . morphological changes in the intermediate filament This paper is an extension of our investigation of a cytoskeleton (Goldman, 1971; Goldman and Knipe, protein that we found to be associated with the 1972), suggesting an association between these two intermediate filament cytoskeleton in several mam- cytoskeletal systems. Additionally, ultrastructural malian cell lines (Brown and Binder, 1990). We analysis of cultured cells suggests that the determined that this protein, which we termed gyrone- and intermediate filament systems are interconnected min, possessed characteristics distinct from other (Green et al., 1986, 1987). Elements that serve to previously established intermediate filament-associated stabilize, attach or bridge homologous cytoskeletal proteins (IFAPs). Here we demonstrate that gyronemin elements, the cytoskeletal-associated proteins (i.e. is expressed in tissues that contain a substantial smooth MAPs, EFAPs, actin-associated proteins), have not muscle component, and we have taken advantage of generally been found to be promiscuous in regard to the this expression pattern to purify it from bovine uterus. 20 K. D. Brown and L. I. Binder

Molecular, biochemical and immunological criteria indicated, gels were stained with Coomassie Blue (0.1% determined that gyronemin is identical to filamin, a Coomassie brilliant blue R/50% methanol/10% acetic acid, by well-characterized actin-cross-linking protein (for re- vol.) overnight and destained (50% methanol/10% acetic view, see Weihing, 1985). These observations serve to acid, v/v) for 4-5 h. Alternatively, gels were silver stained using the Gelcode staining system (Pierce). Prestained demonstrate that, in addition to binding actin filaments, molecular weight markers were purchased from BRL (Gaith- filamin possesses the ability to associate with the ersburg, MD). intermediate filament cytoskeleton. For double-label/two-dimensional electrophoresis, CV-1 cell extract was subjected to 2-D electrophoresis as outlined above. After running the first and second electrophoretic Materials and methods dimensions, the gels were transfered to nitrocellulose sheets and probed (overnight, room temperature) with an appropri- ate dilution of Ml.4. Subsequently, the sheets were probed Sample preparation 125 with I-labeled goat anti-mouse IgM (NEN-DuPont) for 2 h Adult Sprague-Dawley rats were anesthetized by intra- at room temperature, then rinsed, dried, and subjected to peritoneal injection of a Ketamine-Rompum mixture and autoradiography for 2 h at -80°C. Following this step, the subsequently killed by intra-cardiac perfusion with ice-cold blots were "stripped" by incubation in hot (100°C) borate- buffer (100 mM Tris-HCl, pH 7.4/6 mM EDTA). Upon buffered saline (BBS) (100 mM boric acid/50 mM sodium completion of perfusion, the indicated organs were dissected, borate/150 mM NaCl, pH 7.6) containing 2% SDS/5% /3- minced with a razor blade, and transferred to a Dounce mercaptoethanol, for 20 min. The blots were then extensively homogenizer. An appropriate amount of SDS-sample buffer rinsed in BBS, reblocked in BBS/5% non-fat dried milk and (75 mM Tris-HCl, pH 6.8/1% SDS/5% /3-mercaptoethanol/ incubated in an appropriate dilution of the monoclonal 10% glycerol) was added, and the organ samples were antibody G-10. The immunoblot was subsequently developed homogenized while immersed in a boiling water bath for 5 using goat anti-mouse y-chain peroxidase-conjugated second- min. Following boiling, the samples were cleared by centrifu- ary antibody. As a control, blots were probed with anti-mouse gation (15,000 g, 5 min), the supernatant was carefully /i-chain peroxidase-conjugated secondary antibody after strip- removed, and the protein concentration of each clarified ping. This control was performed to ensure that the outlined sample was determined using the method of Lowry et al. stripping process efficiently removed all Ml.4 from the blot. (1951) after precipitation of the protein by addition of perchloric acid and phosphotungstic acid to final concen- Purification of gyronemin trations of approximately 10% and 1%, respectively. Bovine serum albumin (BSA) was used as the standard. Two hundred grams of frozen bovine uterus (—20°C) were chopped into small pieces and homogenized for 5 min in a For two-dimensional electrophoretic analysis, a segment of Waring blendor in 300 ml of cold (4°C) buffer A (100 mM rat aorta was dissected and, after removal of the adventitia, PIPES-NaOH, pH 6.9/1 mM EGTA/l mM Mg2SO4). Follow- was solubilized as described above in a minimal volume of 2% ing clarification of the homogenate by centrifugation (50,000 SDS/5% /3-mercaptoethanol. Prior to electrophoresis, the g, 1 h, 4°C), the supernatant was harvested and adjusted to sample was diluted with ten times volume of 2-D sample 20% saturation with ammonium sulfate ((NH^SO.j) while buffer (9 M urea/4% Nonidet-P 40/2% /3-mercaptoetha- stiring in an ice-bath. The homogenate was then centrifuged nol/2% pH 3-10 Ampholytes (Bio-Rad)). (50,000g, 30 min, 4°C) and the resultant supernatant adjusted African Green Monkey kidney (CV-1) cells were cultured to 35% saturation with ammonium sulfate. The insoluble in Dulbecco's modified essential medium (DMEM) sup- protein fraction from this second fractionation was harvested plemented with 10% fetal calf serum in a humidified 5% CO2 by centrifugation (same as above) and resuspended in buffer atmosphere (37°C). For electrophoretic analysis, cells were A by Dounce homogenization. This protein fraction was grown in 100 mm Petri dishes until confluent. The cells were subsequently desalted by overnight dialysis against 200 rinsed briefly with PBS prior to the addition of a minimal volumes of buffer A at 4°C. volume of hot 2% SDS/5% j5-mercaptoethanol. Sub- sequently, the cell lysate was removed from the Petri dish and Following dialysis, the fraction was centrifuged (225,000 g, 1 h, 4°C) and the supernatant applied to a cellulose phosphate immersed in a boiling water bath for 5 min. The homogenate (Pll, Whatman) column with a bed volume of approximately was then briefly sonicated, cleared by centrifugation (15,000 45 ml equilibrated in buffer A. Nonadsorbed proteins were g, 10 min) and stored at -80°C. Prior to SDS-PAGE or iso- eluted in buffer A and the column was washed with 3 column electric focusing, the extract was diluted to a proper volumes of buffer A/50 mM NaCl. Gyronemin was eluted concentration in SDS-sample buffer or 2-D sample buffer, from the column by application of buffer A/150 mM NaCl. respectively. After cellulose phosphate chromatography, gyronemin- enriched fractions were pooled and applied to a hydroxyl- Electrophoresis and immunoblotting apetite (Bio-gel HTP, Bio-Rad) column (approx. bed volume One-dimensional electrophoresis (SDS-PAGE) was per- 10 ml) equilibrated in buffer A/150 mM NaCl. The column formed as described by Laemmli (1970) on 5 cm x 8.5 cm was rinsed with 3 column volumes of buffer A/150 mM vertical gels consisting of a 4% to 8% linear acrylamide NaCl/100 mM K2HPO4, pH 6.9. Gyronemin was eluted from gradient except where noted. Two-dimensional (2-D) electro- the column with buffer A/150 mM NaCI/200 mM K2HPO4, pH phoresis was performed using a modification of the procedure 6.9. All chromatographic procedures were performed at 4°C of O'Farrell (1975) as previously outlined (Brown and Binder, at a flow rate of 20 ml/h. 1990). For immunoblot analysis, gels were electrically trans- Following hydroxylapetite chromatography, the gyro- ferred to nitrocellulose (Towbin et al., 1979), probed with an nemin-enriched samples were concentrated by ultrafiltration appropriate dilution of the indicated primary antibody, and (Cenrricon-30 microconcentrators, Amicon) at room tem- visualized using anti-mouse peroxidase-conjugated secondary perature because gyronemin was observed to form insoluble antibodies (Kirkegaard and Perry Labs, Gaithersburg, MD) macroscopic aggregates when concentrated at 4°C. However, by methods previously described (Binder et al., 1985). Where gyronemin aggregates formed during cold concentration Filamin/intermediate filament association 21 proved to be excellent immunogens and hence were utilized solid phase. Positive wells were expanded and subsequently for immunization as outlined below. screened by immunoblotting against a crude bovine uterus Filamin was partially purified from bovine uterus as extract. Hybridomas expressing immunoblot positive anti- outlined by Hock et al. (1990); however, the final HPLC bodies were subcloned three times. Antibody isotyping was fractionation was omitted. The final preparation was assayed carried out using a commercially available kit (Boehringer for filamin content by SDS-PAGE followed by Coomassie- Mannheim). Blue staining and concentrated by ultrafiltration (Centricon- 30, Amicon) at room temperature. Immunofluorescence microscopy African Green Monkey kidney (CV-1) cells were grown on Protein sequencing and amino acid analysis pre-sterilized glass coverslips as previously outlined (Brown For protein sequencing and amino acid analysis, purified and Binder, 1990). For double-label immunofluorescence, bovine gyronemin was separated by electrophoresis and cells were rinsed briefly in phosphate-buffered saline (PBS) transferee! to PVDF membranes. Briefly, 10 ng of bovine (40 mM NaH2POV40 mM Na2HPOyi50 mM NaCl, pH 7.4) gyronemin-enriched fraction was separated by SDS-PAGE, and subsequenly fixed in PBS/3% formaldehyde (Toussimi) electrically transfered to PVDF membranes (Immobilon-P, for 30 min at room temperature. Following rinses in fresh Millipore) and stained with Ponceau S (0.5% Ponceau S in 5% PBS, the cells were permeabilized by immersion in PBS/0.5% trichloroacetic acid) for approximately 15 min. The sheets Triton X-100 (5 min, room temperature). The cells were then were then briefly rinsed in de-ionized water, the bands sequentially incubated (30 min, 37°C) in the indicated primary corresponding to gyronemin cut out with a razor blade, and antibody, goat anti-mouse y-chain biotin-conjugated antibody the slices rinsed extensively in de-ionized water. All se- (Kirkegaard and Perry Labs, Gaithersburg, MD), AMCA- quencing and amino acid analysis steps were performed at the conjugated avidin (Jackson Labs, Bar Harbor, ME), and Protein and Nucleic Acid Chemistry Center at Woods Hole rhodamine-conjugated phalloidin (Molecular Probes, Oceanographic Institute, Woods Hole, MA. Eugene, OR). All antibody reagents were diluted in PBS/1% BSA; other reagents were diluted in PBS alone. Coverslips One-dimensional proteolytic peptide mapping were rinsed in PBS for 10 min and blocked in PBS/1% BSA The biochemical relationship of gyronemin and filamin was for 5 min prior to each incubation. Minimal fluorescence compared by one-dimensional limited proteolytic peptide 'bleed-through' between the adjacent AMCA and rhodamine mapping (Cleveland et al., 1977). A 5 ng sample of partially channels was noted. purified gyronemin and filamin from bovine uterus was For triple-label immunofluorescence microscopy, cells were fractionated by SDS-PAGE. The gels were then stained with fixed by immersion in cold (-20°C) methanol for 5 min. Coomassie Blue overnight and briefly de-stained. Sub- Following rinses in PBS, the cells were sequentially incubated sequently, the bands corresponding to gyronemin and filamin (37°C, 30 min) in M1.4-containing tissue culture supernatant, were excised from the gels with a razor blade and soaked for goat anti-mouse ^-chain FTTC-conjugated secondary antibody approximately 30 min in equilibration buffer (EB) (65 mM (Kirkegaard and Perry), spent G-10 tissue culture super- Tris-HCl, pH 6.8/0.1% SDS/5 mM EDTA). For this series of natant, goat anti-mouse y-chain biotin-conjugated antibody, experiments, 15% acrylamide resolving gels with a 0.5 cm AMCA-conjugated avidin, anti-vimentin (mouse monoclonal stacking gel were employed. The gel slices were then placed in IgA subclass, NEN-DuPont), and goat anti-mouse cr-chain the wells of another gel and overlaid with 10 /A of EB/20% Texas Red-conjugated antibody (Southern Biotech, Birm- glycerol. The slices were then overlaid with 20 /J of EB/10% ingham, AL). Reagents were diluted, and cells rinsed and glycerol containing 40 ng of V-8 (Boehringer blocked as outlined above. No cross-reactivity between the Mannheim). Finally, the wells were overlaid with 5 /A of mouse immunoglobulin subclass-specific fluorochrome- EB/5% glycerol that contained a minimal amount of pyronin- tagged secondary antibodies and other mouse antibody Y (tracking dye). A well with 40 ng of Staphylococcus griseus subclasses was observed. V-8 protease alone was included to evaluate its contribution to the resultant fragmentation pattern. The gels were run at 5 mA for 2 h, and then the current was raised to 30 mA for the Results remainder of the electrophoretic run. Subsequently, the gels were silver stained using the Gelcode staining system. Reactivity of the monoclonal antibody Ml. 4 in adult rat organs Monoclonal antibody production Extracts of various tissues from rat organs were Balb/c mice were initially immunized via intraperitoneal injection with a mixture containing gyronemin aggregates subjected to immunoblot analysis with the monoclonal (formed during cold concentration at 4°C) cross-linked with antibody Ml.4 (Fig. 1A). This investigation showed 1% glutaraldehyde (overnight, 4°C) and SDS-denatured that Ml.4 recognized a 380 kDa polypeptide in extracts bovine gyronemin suspended in complete Freund's adjuvant. of rat brain, corresponding to the microtubule-associ- Subsequently, the mice were boosted twice (two-week ated protein MAPI A (Brown and Binder, 1990). intervals between boosts) with a mixture of gyronemin cross- Additionally, we noted that this antibody recognized a linked to CNBr-activated Sephrose 4B (Sigma) and SDS- 240 kDa protein in extracts of rat stomach and uterus. denatured gyronemin in incomplete adjuvant administered While these two organs are functionally unrelated, they subcutaneously. Prior to fusion the mice were subcutaneously both contain visceral smooth muscle tissue. Investi- boosted a final time with SDS-denatured bovine gyronemin. gation of Ml.4 immunoreactivity in an extensive panel Splenocytes harvested from an immunized mouse were fused with SP2/0 myeloma cells using PEG 4000 by methods of extracts from other adult rat tissues showed the 240 previously described (Kohler and Milstein, 1976). Wells kDa polypeptide recognized by Ml.4 was expressed in, positive for hybridoma growth were screened by enzyme- and largely limited to, organs that possess a substantial linked immunosorbent assay (ELISA) for the presence of visceral or vascular smooth muscle component (Brown anti-gyronemin antibodies using bovine gyronemin in the and Binder, in preparation). 22 K. D. Brown and L. I. Binder

12 3 4 5 6

B PH 8.2 78 7.4 68 224-

Mr

224' 109-

72- 107-

72-

46-

28-

Fig. 1. Immunoreactivity of Ml.4 in adult rat organs. (A) Extracts of indicated organs were fractionated by SDS-PAGE (4% to 10% linear polyacrylamide gradient superimposed on a 1 M to 8 M urea gradient) followed by immunoblot analysis with the monoclonal antibody Ml.4. All extracts were loaded at 25 ^g (total protein)/lane. Lane 1, uterus; lane 2, brain; lane 3, stomach; lane 4, cardiac muscle; lane 5, ; lane 6, liver. Note Ml.4 immunoreactivity with the MAPIA present in brain extract (arrow) as well as a 240 kDa polypeptide in extracts of stomach and uterus (arrowhead). (B) Two-dimensional electrophoretic analysis of adult rat aorta extract. A 5 f.ig sample of extract from rat aorta was exposed to iso-electric focusing followed by SDS-PAGE, and subsequent immunoblot analysis with the monoclonal antibody Ml.4. The 240 kDa protein recognized by Ml.4 in this organ (arrow) possessed an iso-electric point of 3 approximately 7.5. Mr denotes relative molecular mass (xlO~ ).

Two-dimensional electrophoresis of an extract of rat kDa protein that was immunoreactive with Ml.4 was aorta, an organ enriched in vascular smooth muscle, observed (Fig. 2A, lane 5). Typically, 200 g of bovine followed by immunoblot analysis with Ml.4, showed uterine tissue yielded 4-7 mg of protein that was greater that the 240 kDa polypeptide exibited an iso-electric than 50% 240 kDa polypeptide by weight as determined point of approximately 7.5 (Fig. IB). This closely by densitometric gel scanning (data not shown). agrees with the reported pi (7.7) of human HeLa cell Two-dimensional electrophoretic analysis deter- gyronemin (Brown and Binder, 1990), indicating that mined that the Ml.4 immunoreactive 240 kDa polypep- this previously indentified mammalian IFAP is also tide possessed an iso-electric point of 7.3-7.7 (Fig. 2C), present in certain rat organs. further indicating that the high relative molecular mass polypeptide present in the final enriched fraction was gyronemin. Additionally, silver stains of 2-D gels failed Purification of gyronemin and subsequent to detect the presence of any other protein species identification as the actin-binding protein filamin unreactive with Ml.4 at 240 kDa (Fig. 2B). Using bovine uterus as our starting material, we have The final gyronemin fraction also contained an Ml.4 obtained a protein fraction that is highly enriched for immunoreactive polypeptide present at 215 kDa (Fig. gyronemin. The purification scheme that was designed 2A, lane 5). We presume this to be a proteolytic utilizes ammonium sulfate precipitation of the protein fragment of gyronemin generated during purification, from a soluble tissue extract followed by cellulose since: (1) the quantity of this species (215 kDa) varied in phosphate and hydroxylapetite chromatography (see proportion to gyronemin (240 kDa) from preparation to Materials and methods). The protein composition of preparation (data not shown); and (2) it is not detected fractions collected during gyronemin purification were in whole organ extracts when precautions are taken to analyzed by SDS-PAGE followed by Coomassie Blue limit proteolysis (see Fig. 1). staining (Fig. 2A, lanes 1-4). An enrichment of a 240 As a first step toward characterization of gyronemin Filaminjintermediate filament association 23 A i 3 4 pH 7.3 7.7 B V Mr 224. 224-

109" 72-. 109-

72-

224s 46-

109- 28" 72- dye.

Fig. 2. Purification of gyronemin from bovine uterus. Bovine uterus was fractionated as outlined in Materials and methods. (A) One-dimensional electrophoretic analysis of protein fractions collected during the purification protocol. Lanes 1-4, Coomassie-Blue stain; lane 5, immunoblot with Ml.4. Lane 1, initial bovine uterus homogenate; lane 2, protein fraction insoluble at 20-35% ammonium sulfate saturation; lane 3, gyronemin-enriched fraction following cellulose phosphate chromatography; lanes 4 and 5, gyronemin-enriched fraction following hydroxylapetite chromatography. All lanes were loaded with 10 ^g protein/lane except lane 5, which contains 250 ng protein. Note the clear enrichment of a Ml.4 reactive 240 kDa polypeptide (+) through this protocol as well as the presence of an Ml.4 immunoreactive 215 kDa polypeptide (++) in the final fraction. (B and C) 500 ng of protein from the final fraction in the outlined purification protocol was subjected to two-dimensional electrophoresis followed by silver staining (B) or immunoblot analysis with Ml.4 (C). Shown is the high relative molecular mass region of two gels run in parallel. Note that the electrophoretic mobility of the 240 kDa polypeptide detected by silver staining coincides precisely with the Ml.4 immunoblot signal (arrowheads). MT denotes relative molecular mass (xlO~3). we obtained amino acid sequence on electrophoreti- phoretic mobilities (Fig 4A). One-dimensional limited cally purified gyronemin. Since the amino terminus was proteolysis peptide mapping with V-8 protease showed found to be blocked, we obtained internal peptide that the fragmentation patterns generated from filamin sequence from three electrophoretically distinct cyano- and gyronemin coincided exactly (Fig. 4B). gen bromide-cleaved polypeptides (see Materials and We have also compared gyronemin and filamin, on methods). Each of the obtained sequences (Fig. 3) the basis of immunological criteria (Fig. 5). We found showed extensive homology with the deduced sequence that an anti-filamin monoclonal antibody, designated of actin-binding protein (ABP) (Gorlin et al., 1990), the Mabl (supplied by Drs. J. Gorlin and J. H. Hartwig, supposed non-muscle form of filamin (Wallach et al., Harvard Medical School, Boston, MA), reacted with 1978). The only exception was a threonine to alanine both purified filamin and gyronemin (Fig. 5, panel 1). substitution at ABP residue 1228. Likewise, an affinity purified anti-filamin polyclonal As a further basis of comparison we determined the antibody (supplied by Dr. D. W. Speicher, Wistar amino acid composition of bovine gyronemin. This Institute, Philadelphia, PA) was also reactive with both analysis showed that the composition of gyronemin is polypeptides (Fig. 5, panel 2). highly comparable to filamin polypeptides obtained As a further means of studying this protein we have from both mammalian and avian sources (Table 1). selected a panel of anti-gyronemin monoclonal anti- Weihing (1985) noted that among mammalian bodies using our bovine gyronemin preparation as the the Lys/Arg ratio is nearly 2.0; in close agreement with immunogen. Each of these antibodies, designated G-2 this observation is the obtained Lys/Arg ratio (1.7) of (subclass IgGO, G-3 (IgM), G-10 (IgGj), G-ll (IgGj) bovine gyronemin. and G-12 (IgGi), as well as Ml.4, were reactive with To investigate further the relationship of filamin and bovine filamin (Fig. 5, panels 3-8). Subsequent epitope gyronemin we have partially purified filamin from mapping studies have determined that each of these bovine uterus utilizing the protocol of Hock et al. monoclonal antibodies recognizes distinct gyronemin (1990). Comparison of bovine filamin and gyronemin by epitopes (data not shown). SDS-PAGE followed by Coomassie Blue staining On the basis of molecular (amino acid sequence), showed that these two proteins have identical electro- biochemical (peptide mapping), and immunological 24 K. D. Brown and L. I. Binder

Gyronemin Fragment A >• X-P-G-T-Y-T-V-T-I-K-Y-G-G

C-P-G-A-Y-T-V-T-I-K-Y-G-G •* Human ABP 1225 1237 (residueno)

Gyronemin Fragment B >- V-K-K-R-A-E-F-T-V-E-T-R-S-A-G-Q-G-E-V-L-X-Y-V-E-D V-K-K-R-A-E-F-T-V-E-T-R-S-A-G-Q-G-E-V-L-X-Y-V-E-D •* Hunan ABP 298 322 (residue no)

Gyronemin Fragment C: X-L-F-A-D-Q-A-T-P-T V-L-F-A-D-Q-A-T-P-T < Hunan ABP 850 859 (residue no)

Fig. 3. Amino acid sequence analysis of bovine gyronemin. Gyronemin was subjected to amino acid analysis as outlined in Materials and methods. We determined that the amino terminus of this polypeptide was blocked, thus CNBr fragmentation to obtain internal sequence was required. Sequence analysis was obtained on three electrophoretically distinct peptides. The obtained sequences for gyronemin fragment A (15 kDa), fragment B (16 kDa), and fragment C (65 kDa) are shown. Note that each fragment possessed absolute identity with portions of the deduced amino acid sequence of human ABP (nonmuscle filamin) (Gorlin et al., 1990). The only exception was found at residue no. 4 of fragment A, which was determined to be a threonine to alanine substitution at residue 1228 of the ABP sequence.

Table 1. Amino acid composition of bovine gyronemin and other filamin polypeptides Human smooth Guinea pig vas Avian smooth Amino acid Bovine gyronemin muscle filamin" deferens filaminb muscle filamin0 Asp 8.1 8.5 9.2 7.6 Thr 6.3 6.6 6.4 5.7 Ser 7.6 8.0 6.8 6.4 Glu 9.7 11.1 10.8 9.4 Pro 6.8 8.0 8.7 8.4 Gly 13.0 11.9 12.0 12.9 Ala 7.4 7.3 7.7 9.2 Val 9.4 10.1 9.8 11.5 Met 1.1 1.0 1.1 1.0 He 5.0 3.9 4.1 3.1 Leu 5.8 5.4 6.0 5.7 Tyr 3.1 3.2 2.7 1.9 Phe 3.0 2.8 2.2 2.7 His 2.7 2.4 2.1 2.2 Lys 6.9 6.2 6.4 5.2 Arg 4.1 3.6 3.5 5.5 'Hock et al., 1990. "Wallach et al., 1978. 'Shizuta et al., 1976.

(antibody cross-reactivity) criteria, we conclude that 6A-C). Furthermore, the observed co-localization of the IFAP we identified as gyronemin is identical to these antibodies with fluorescently labelled phalloidin filamin. (Fig. 6a-c) indicated that they stained the actin filament containing stress fibers in this cell line. Immunoblot Localization of filamin in cultured mammalian cells analysis of CV-1 cell extracts has shown each of these Since gyronemin was originally identified as an IFAP antibodies to be monospecific for filamin in this cell and filamin is known to associate with actin filaments, line. The monoclonal antibodies G-2 and G-3 were we studied the localization of filamin in a cultured relatively non-reactive in indirect immunofluorescence mammalian cell line with our newly derived panel of assays (data not shown). anti-gyronemin/filamin monoclonal antibodies. Indirect Although G-10, G-ll and G-12 exhibited co-localiz- immunofluorescence microscopy of cultured African ation of filamin with stress fibers, we had previously Green Monkey kidney (CV-1) cells with the mono- shown that Ml.4 localized this protein on intermediate clonal antibodies G-10, G-ll and G-12 displayed filaments. Consequently, we wished to determine the localization on fibrous structures within these cells (Fig. localization of filamin using G-10 and Ml.4 within the Filamin/intermediate filament association 25 G F E GFGF GF GF — — ** Mr 72- 224-

46- 224- 109- 72- 28- IT

109- *

15- G F 72- I 46 •

Fig. 4. Electrophoretic comparison of bovine gyronemin and filamin. Filamin was purified from bovine uterus as outlined in Materials and methods. (A) A 15 fig sample of gyronemin (G) and filamin (F) enriched fractions was fractionated by SDS-PAGE followed by Commassie Blue staining. Note that these two proteins possess identical electrophoretic mobilities (arrowhead). (B) Bovine gyronemin (G) and filamin (F) were compared by one- dimensional limited proteolyis peptide mapping (see Fig. 5. Immunological comparison of gyronemin and Materials and methods). An adjacent well was loaded with filamin. A 500 ng sample of purified bovine gyronemin (G) enzyme alone (V-8 protease) (E) to determine its and filamin (F) was fractionated by SDS-PAGE and contribution to the observed fragmentation patterns. subsequently immunoblotted with a panel of anti-filamin or Shown is a silver stain of the peptide mapping gel. Note anti-gyronemin antibodies. The anti-filamin monoclonal that the resultant peptide maps of gyronemin and filamin antibody Mab 1 (panel 1) (Gorlin et al., 1990) as well as 3 are identical. Mr denotes relative molecular mass (xlO~ ). an affinity-purified anti-filamin rabbit polyclonal antibody (panel 2) (a gift from Dr. D. W. Speicher) showed reactivity with both filamin and gyronemin (arrowhead). same cell. Triple-label immunofluorescence microscopy Several novel anti-gyronemin antibodies (G-2 (panel 4), G- with G-10, Ml.4 and an anti-vimentin antibody is 3 (panel 5), G-10 (panel 6), G-ll (panel 7), and G-12 shown in Fig. 7. This investigation confirmed our (panel 8)), as well as Ml.4 (panel 3), were also reactive previous findings demonstrating that, while G-10 with both filamin and gyronemin. MT denotes relative localizes filamin on stress fibers (Fig. 7C), Ml.4 (Fig. molecular mass (xlO ). 7B) colocalizes with the vimentin filaments (Fig. 7A). No obvious overlap between the observed Ml.4 and G- spots on 2-D gels in extracts of rat aorta, CV-1 cells and 10 localization was noted. Immunoblot analysis, how- other cultured cell lines (Brown and Binder, unpub- ever, showed that Ml.4 and G-10 were both mono- lished observations). It is likely that Ml.4 did not specific for filamin in CV-1 cells (Fig. 8A). recognize multiple isoelectric variants in this exper- The observation that filamin is associated with both iment due to the lack of sensitivity of the 125I-labeled microfilament and intermediate filament secondary antibody employed. Under any circum- prompted us to determine if Ml.4 detects a specific stances, these results clearly indicate that Ml.4 and G- isoform of filamin present within these cells. Therefore, 10 recognize bona fide forms of filamin associated with a CV-1 cell extract was subjected to two-dimensional both actin-containing stress fibers and vimentin-con- electrophoresis, followed by a double-label immuno- taining intermediate filaments. blotting protocol (see Materials and methods). This analysis showed that Ml.4, the antibody that localizes along intermediate filaments recognizes a single spot at Discussion 240 kDa that exibits a pi of approximately 7.5. G-10 recognized two 240 kDa spots, the more basic of which Previously, we showed that the monoclonal antibody was superimposable with the observed Ml.4 reactivity Ml.4 was specific for the microtubule-associated pro- (Fig. 8B,B' and C,C). In other experiments, we have tein MAP1A in brain but that it also recognized a 240 observed that Ml.4 also recognizes multiple 240 kDa kDa polypeptide in HeLa and other cultured mam- 26 K. D. Brown and L. I. Binder

Fig. 6. Double-label immunofluorescence localization of filamin along stress fibers in cultured African Green Monkey kidney (CV-1) cells. Cells were subjected to indirect immunofluorescence microscopy with the antibodies G-10 (A), G-ll (B), or G-12 (C), followed by incubation in rhodamine-conjugated phalloidin (a, b, c). Note the clear co-localization of these antibodies along microfilament stress fibers. Bar, 5 jim. malian cells (Brown and Binder, 1990). Indirect these polypeptides constitute a functionally and bio- immunofluorescence microscopy showed this polypep- chemically related family of proteins. The term filamin tide to be associated with both and vimentin is applied to the protein expressed in muscle tissues filaments in HeLa cells and with the vimentin filament while the acronym ABP is applied to the protein cytoskeleton in other cell lines that did not express present in non-muscle cells. Since its discovery, filamin MAP1A. Additionally, immunoelectron microscopy (or ABP) has been found in avian skeletal and cardiac revealed that the 240 kDa polypeptide localized muscle (Bechtel, 1979; Koteliansky et al., 1981), chick periodically along bundled intermediate filaments neuroglia (Lemmon, 1986), platelets (Rosenberg et al., (tonofilaments) in HeLa cells. Further characterization 1981), neutrophils (Valerius et al., 1981), Dictyostelium of this polypeptide led us to conclude that it was a (Hock and Condeelis, 1986), and a number of avian and unique intermediate filament-associated protein mammalian cultured cells (for review, see Weihing, (LFAP), which we designated gyronemin. Here we 1985). report that this polypeptide is expressed in rat organs Filamin has been shown by indirect immunofluor- that contain a substantial smooth muscle component. escence microscopy (Wang et al., 1975) and immuno- Amino acid sequence analysis and exhaustive biochemi- electron microscopy (Langanger et al., 1984) to be cal and immunochemical analyses demonstrated that localized along bundled actin filaments (stress fibers) in the polypeptide we originally identified as the IFAP several cultured cell lines. Additionally, Mittal et al. gyronemin is identical to the actin binding protein (1987) found that fluorescently labeled filamin, when filamin (Wang et al., 1975); hence, Ml.4 is an anti- microinjected into various cultured cells, associated filamin antibody. with microfilaments. These studies support several in The microfilament-associated protein filamin was vitro studies that established that filamin (or ABP) initially isolated from chicken gizzard (Wang et al., associates with F-actin (Shizuta et al., 1976; Brotschi et 1975; Shizuta et al., 1976; Wang, 1977). Subsequently, al., 1976; Wang and Singer, 1977; Rosenberg and Wallach et al. (1978) isolated a protein from guinea pig Stracher, 1982). Electron microscopy of ABP/actin vas deferens that possessed several filamin-like proper- complexes demonstrated that ABP can induce forma- ties. These investigators also found that polyclonal tion of rigid orthoganal arrays of actin filaments in vitro antibodies raised against the guinea pig protein were (Hartwig et al., 1980; Rosenberg and Stracher, 1982). cross-reactive with both chicken filamin and ABP, a Recently, platelet ABP has been shown to bind high relative molecular mass actin binding protein specifically the membrane glycoprotein Ib-IX (Ezzell et identified in extracts of rabbit aveolar macrophages al., 1988; Andrews and Fox, 1991), suggesting that a (Hartwig and Stossel, 1975). It is currently thought that subset of platelet ABP may anchor the cortical Fig. 7. Triple-label immunofluorescence localization of vimentin filaments and filamin using the monoclonal antibodies G- 10 and Ml.4. Cells were fixed as outlined in Materials and methods and stained for vimentin (A), and filamin using the antibodies Ml.4 (B), and G-10 (C). Note that while G-10 localizes filamin on stress fibers, Ml.4 clearly shows filamin co-localization with vimentin type filaments in this cell. Bar, 2 j«n.

Filamin/intermediate filament association 27

Fig. 8. Electrophoretic analysis of Ml.4 and G-10 reactivity microfilament cytoskeleton to the plasma membrane in in CV-1 cell extracts. (A) A 5 fig sample of total CV-1 cell this cell type. extract was subjected to immunoblot analysis with Ml.4 While the data presented in this paper represent the (lane 1) or G-10 (lane 2). Note that these two antibodies display monospecificity for filamin in this cell line first account of an association between filamin and (arrowhead). (B and C) Double-label/two-dimensional intermediate filaments, other investigators have ob- electrophoretic analysis of CV-1 cell extract. A 5 fig sample served that filamin co-localized with intermediate of CV-1 extract was subjected to two-dimensional filament-rich areas within muscle cells. For instance, electrophoresis followed by transfer to nitrocellulose several laboratories have noted that filamin is located at sheets. The blot was first probed with Ml.4 followed by the Z-lines of avian skeletal and cardiac muscle incubation in 125I-labeled goat anti-mouse IgM (B). After (Bechtel, 1979; Gomer and Lazarides, 1981; the antibody stripping process Koteliansky et al., 1986), which are known to contain ( described in Materials and intermediate filaments (Granger and Lazarides, 1979; I 1 methods the blot was probed with Gard and Lazarides, 1980). Furthermore, in smooth G-10 followed by rabbit anti- muscle cells, Small et al. (1986) found that filamin was mouse y-chain-specific peroxidase- located in the same area of the cell as ; however, Af, conjugated secondary antibody these investigators were unable to speculate on a • -« (C)' Note that M1-4 and G'10 possible filamin/intermediate filament association. 224- immunoreactivity co-localize on A recent biochemical study has also suggested that a this immunoblot at a 240 kDa spot that has a pi of connection exists between filamin and intermediate approximately 7.5 (arrowheads). filaments. Malmqvist et al. (1991) studied the changes Higher magnification views of in filamin, , actin and desmin in hypertrophied Ml.4 and G-10 immunoreactivity urinary bladder smooth muscle and found that while the 109 (B' and C) show that while the quantity of actin remained unchanged in hypertrophied bulk of G-10 reactivity co- smooth muscle, there was a net increase in desmin. localizes with the observed Ml.4 Additionally, the filamin/actin ratio was found to reactivity (arrowheads) we increase coordinately with the desmin/actin ratio, observed G-10 to be reactive with an acidic isoelectric variant of leading these investigators to suggest that a connection filamin (arrow) that showed no exists between increased filamin and desmin expression apparent Ml.4 reactivity. MT in the absence of any correlative structural analysis. denotes relative molecular mass Moreover, perhaps related to these findings are the (xlO~3). studies of Berner et al. (1981), which showed, micro- scopically, that smooth muscle hypertrophy resulted in a net increase of intermediate filaments in this cell type. basic acidic Why does Ml.4 localize filamin on intermediate filaments? M Of all of our anti-filamin monoclonal antibodies only Ml.4 co-localizes with intermediate filaments. The reason for this is unknown. We have previously shown 224 that Ml.4 fails to bind to either vimentin or on immunoblots (Brown and Binder, 1990). Neither does this antibody bind to native isolated vimentin filaments, as judged by solid-phase binding assays (data not shown). Therefore, the only reasonable explanation for the localization pattern exibited by this antibody is that Ml.4 is binding to a protein associated with the intermediate filament polymers. The most straightfor- ward interpretation, based on the data presented here, is that this protein is filamin. The two-dimensional electrophoresis/double-label immunoblot experiments suggest that the form of filamin recognized by Ml.4 is also detected by G-10, a monoclonal antibody that co-localizes with actin fila- 224 ments. However, we have demonstrated the existence of multiple filamin isoelectric variants and, moreover, filamin isoforms are known to exist (Gomer and Lazarides, 1983a,b; Mangeat and Burridge, 1983; 109 Pavalko et al., 1989). Thus, the possibility that there is a difference between the form of filamin associated with 72 1 intermediate filaments versus the microfilaments can- I not be ruled out. It is also possible that, when filamin is 28 K. D. Brown and L. I. Binder

asssociated with intermediate filaments, the binding al., 1983) and microtubules (Zagon et al., 1986). sites of other anti-filamin antibodies are blocked, due to Consequently, is thought to play a role as a the association of filamin with other proteins and/or cytoskeletal cross-linker (Goodman et al., 1988; conformational changes within the filamin polypeptide. Coleman et al., 1990). The unique Ml.4 staining pattern remains puzzling. was first identified as an intermediate fila- However, unlike our other antibodies (G-10, G-ll, G- ment-associated protein on the basis of its co-isolation 12), Ml.4 is also an anti-MAPI A antibody, originally and co-localization with intermediate filaments from raised against this bovine brain microtubule-associated several cultured cell lines (Wiche et al., 1982). In protein (Brown and Binder, 1990). Furthermore, we addition to its ability to bind intermediate filaments, found that when a polyclonal anti-filamin reaction was solid-phase binding assays have shown plectin to be elicited in five different mice immunized with purified capable of binding MAPI, MAP2 and spectrin (Herr- filamin, cross-reactivity with MAP1A was never ob- mann and Wiche, 1983). These observation have led to served (data not shown). This suggests that perhaps the the view that this protein may serve a cytoplasmic cross- Ml.4 site on filamin is normally "immunogenically linking function (for review, see Wiche, 1990). silent" and that others have not obtained an antibody An attractive hypothesis suggested by the obser- that displays a localization pattern similar to Ml.4 vations presented in this paper is that filamin serves to because their immunogen was filamin rather than interconnect the microfilament and intermediate fila- MAPI A. Identification of the precise location of the ment cytoskeletal systems. A putative heterologous Ml.4 epitope along filamin may greatly aid in our cytoskeletal cross-linking function for filamin may have understanding of which portions of the molecule are the greatest significance in smooth muscle, since this available for antibody binding when the protein is cell type has both extensive microfilament and inter- bound to F-actin versus intermediate filaments, and mediate filament cytoskeletons. While ABP, the non- may also facilitate the production of other anti-filamin muscle filamin homolog, has been shown to be antibodies that display a localization pattern similar to expressed in platelets, it is unlikely that ABP would M1.4. play a structural role involving intermediate filaments in this cell type, since platelets possesses few, if any, of What is the significance offilamin/'intermediate these filamentous elements (Tuszynski, 1987). On the filament association? other hand, our findings do not rule out the possibility Classically, cytoskeletal-associated proteins are classi- that filamin, when in association with intermediate fied by their association with formed polymers in vitro filaments, plays a role that is independent of the and/or by co-localization with filaments in situ. Further- microfilament cytoskeleton. more, most members of this group of proteins are In conclusion, we have found that gyronemin, a thought to be monospecific for one type of formed protein that we previously identified as an intermediate . However, several cytoskeletal-associated pro- filament-associated protein, is the microfilament-as- teins have been shown to be capable of associating with sociated protein filamin. These findings indicate that biochemically distinct polymers. For instance, some filamin plays a broader role within the cytoskeleton microtubule-associated proteins (MAPs) have also than was previously believed. been shown to bind intermediate filaments in vitro (Aamodt and Williams, 1984; Heimann et al., 1985); The authors thank Drs. J. Gorlin, J. Hartwig and D. and Bloom and Vallee (1983) showed that MAP2 co- Speicher for generously supplying antibodies used in this localized with the intermediate filament cytoskeleton in study. We gratefully acknowledge the contribution of Dr. R. cultured brain cells, by immunofluorescence mi- Ridge of the Woods Hole Oceanographic Institute for croscopy. Additionally, Pollard and co-workers have gyronemin sequence and amino acid composition analysis. This work was supported by NIH grant AG 06969 to L.I.B.; shown that MAP2 possesses the ability to bind actin K.D.B. was supported by NIH predoctoral traineeship no. filaments (Griffith and Pollard, 1982; Selden and 5T32HL07553. Pollard, 1983; Pollard et al., 1984). Furthermore, Morales and Fifkova (1989) also noted that MAP2, a protein known to localize along dendritic microtubules References in situ (Caceres et al., 1984), co-localized with actin filaments in dendritic spines. These studies suggest that Aamodt, E. J. and Williams, R. C. (1984). Microtubule-associated some microtubule-associated proteins may, in part, proteins connect microtubules and in vitro. serve as cross-bridges between microtubules and other 23, 6023-6031. Andrews, R. K. and Fox, J. E. B. (1991). Interaction of purified actin- non-microtubule cytoskeletal systems. binding protein with platelet membrane glycoprotein Ib-IX Multi-functional cytoskeletal associations are not complex. J. Biol. Chem. 266, 7144-7147. Bechtel, P. J. (1979). Identification of a high molecular weight actin- limited to MAPs. The actin-cross-linking protein spec- binding protein in skeletal muscle. J. Biol. Chem. 254, 1755-1758. trin has been shown to bind both microtubules and Berner, P. F., Somlyo, A. V. and Somlyo, A. P. (1981). Hypertrophy intermediate filaments in vitro (Ishikawa et al., 1983; induced increase in intermediate filaments of vascular muscle. /. Fach et al., 1984; Langley and Cohen, 1986, 1987; Cell Biol. 88, 96-101. Frappier et al, 1987). Various spectrin isoforms have Binder, L. I., Frankfurter, A. and Rebhun, L. I. (1985). The distribution of tau in the mammalian central nervous system. /. Cell also been shown to co-localize with intermediate Biol. 101, 1371-1378. filaments (Mangeat and Burridge, 1984; Hirokawa et Bloom, G. S. and Vallee, R. B. (1983). Association of microtubule- Filaminjintermediate filament association 29

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