Journal oJNeurachemislry

Raven Press, Ltd., New York Q 1992 International Society for Neurochemistry

Evidence for a New Member of the Myosin I Family from Mammalian Brain Deqin Li and Peter D. Chantler Department of Anatomy and Neurobiology, Medical College of Pennsylvania, Philadelphia, Pennsylvania, i7.S.A

Abstract: Myosin I is an actin-based motor responsible for powering a wide variety of motile activities in amebae and slime molds and has been found previously in vertebrates as the lateral bridges within intestinal epithelial cell microvilli. Although neurons exhibit extensive cellular and intracellular motility, including the production of ameboid-like growth cones during development, the proteins responsible for the motor in these processes are unknown. Here, we report the isolation of a partially purified protein fraction from bovine brain that is enriched for a 150-kDa protein; immunochemical and biochemical analyses suggest that this protein possesses a number of functional properties that have been ascribed to myosin I from various sources. These properties include an elevated K' -EDTA ATPase, a modest

actin-activated Me-ATPase, the ability to bind calmodulin, and a ready association with phospholipid vesicles made from phosphatidylserine, but not from phosphatidylcholine. The combination of these properties, together with a molecular mass of 150 kDa (most myosin I molecules found to date have molecular masses in the range 1 10-130 kDa) yet recognition by an anti-myosin I antibody, suggests the presence of a new member of the myosin I family within mammalian brain. Key Words: Myosin I-Brain myosinActin-binding motor-Lipid vesicle binding. Li D. and Chantler P. D. Evidence for a new member of the myosin I family from mammalian brain. J. Neurochem. 59, 13441351 (1992).

Myosin I is the name given to members of a growing family of globular molecules, best understood in Acantharnoeba and Dictyostelium, whose singleheaded structure possesses substantial sequence and functional homology with the head region of conventional, two-headed myosin I1 (Pollard and Korn, 1973; Cote et al., 1985; Hammer et al., 1986; Korn and Hammer, 1988; Mooseker, 1989; Spudich, 1989; Titus et al., 1989). The powering of ameboid locomotion (Fukui et al., 1989) and propulsion of intracellular organelles (Adams and Pollard, 1986) appear to be functions of myosin I, which is presumably responsible for the continued motility of amebae upon functional removal of myosin I1 by genetic manipulation (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987). Our knowledge of myosin I in higher organisms has been limited, so far, to the gene product of the Drosophila ninaC locus (Monte11 and Rubin,

1988) and the 1 10-kDa calmodulin complex which comprises the lateral bridges in vertebrate intestinal epithelial cell microvilli (Collins and Borysenko, 1984; Coluccio and Bretscher, 1987; Conzelman and Mooseker, 1987; Hoshimaru et a!., 1989; Collins et al., 1990). However, the phenomenological similanties between the extension and retraction of pseudopodia during ameboid motion and of filopodia during neuronal cell growth cone motility originally suggested to us that myosin I may also be present in vertebrate neurons, a possibility we confirmed recently by immunocytochemistry (Miller et al., 1992). Here, we present both immunochemical and functional evidence that a 150-kDa protein, isolated from bovine brain, represents a new member of the myosin I protein family. This work has been presented in preliminary form (Li and Chantler, 1991). While this work was in progress, two reports appeared in abstract form

Received October 9, 1991 ; revised manuscript received March 30, 1992; accepted March 30, 1992. Address correspondence and reprint requests to Dr. P. D. Chantler at Department of Anatomy and Neurobiology, Medical College of Pennsylvania, 3200 Henry Ave., Philadelphia, PA 19 129, U.S.A.

Abbreviations used: DTT,dithiothreitol; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.

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BRAIN MYOSIN I which also suggest, on the basis of immunoreactivity, that myosin I-like molecules are present in brain (Bridgman and Kordyban, 1989; Bahler, 1990).

MATERIALS AND METHODS Myosin preparation Myosin I was prepared by methods based on published procedures (Pollard and Korn, 1973; M a m a et al., 1979; Collins and Borysenko, 1984; Lynch et al., 1986; Conzelman and Mooseker, 1987; Collins et al., 1990). Bovine brain (800- 1,200 g) was homogenized in an equal volume of 10 mM imidazole, pH 7.3,75 m M KCl, 1 mM EGTA, 1 mM EDTA, I mM dithiothreitol (DTT), and a protease inhibitor cocktail comprising the following: 3 mM NaN,, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/L trypsin inhibitor, 10 mg/L tosyl L-arginyl methyl ester, 10 mg/L benzyl L-arginyl methyl ester, 2.5 mg/L aprotinin, 2.5 mg/L leupeptin, and 2.5 mg/L pepstatin A. AAer being spun at 10,000 rpm for 30 min, the supernatant was brought to 5 m M ATP and then spun at 100,000 g for 1 h. The precipitate resulting from ammonium sulfate fractionation in the range 2 0 4 0 % was resuspended in a buffer containing 10 mMimidazole, pH 7.3, 0.6 M KCI, 1 mMATP, I mM EGTA, 1 mM MgCI,, 5% sucrose, and the above inhibitor cocktail, and then dialyzed overnight against the same buffer. The clarified supernatant was loaded onto a Sepharose CL4B column, and fractions immunopositive for myosin I were passed successively through hydroxyapatite and Sepharose Q columns. For details of the individual column chromatography stcps, see the legends of Figs. 1-3.

Antibodies An antibody [rabbit immunoglobulin G (IgG)] against chicken brush border myosin I was a generous gift from Drs. P. Matsudaira and K. Collins (Whitehead Institute, Cambridge, MA, U.S.A.). This antibody has been characterized (Collins et al., 1990) and recognizes the 110-kDa heavy chain of brush border myosin I, but not myosin 11. An antibody (rabbit IgG) against mouse neuroblastoma (Neuro2A) myosin 11, developed and characterized in this laboratory (Mil!er e! al., 1992), recognizes the heavy chaic of neuroblastoma and brain myosin 11, but does not cross-react with brain myosin I. Horseradish peroxidase conjugated goat anti-rabbit IgG was obtained from Cappel.

Immunoprecipitation Aliquots from the pcak of anti-myosin I immunoreactivity eluting from the Sepharose Q column, anti-intestinal brush border myosin I IgG, and anti-neuroblastoma myosin I1 IgG were dialyzed separately against 10 mM imidazole, pH 7.5, 0.5 MKCl, 1 mMDTT, 0.2 mMPMSF, 0.3 mM NaN,. The specific K+-EDTA ATPase rate of the sample was measured (see below). One-milliliter samples were then incubated with 4O-pl aliquots of either anti-myosin I or antimyosin I1 at 4°C with gentle inversion. After 1 h, 0.3 ml of protein A-agarose was added to this mixture and equilibrated with the sample for 15 min. Protein A-agarose was spun down at 2,000 rpm for 10 min and the specific K+EDTA ATPase rate of the supernatant remeasured (correcting for changes in volumc). Pellets were washed three times in the above buffer containing 1 mg/ml bovine serum albumin, prior to sodium dodecyl sulfate (SDS>acrylamide gel electrophoresis and western blot analysis.

134.5

Calmodulin overlay Fractions enriched for myosin I were run on 7.5% SDSacrylamide gels (Matsudaira and Burgess, 1978) and transferred to nitrocellulose (Towbin et al., 1979), whereupon the blot was placed in a blocking buffer for 1 h (5% nonfat dry milk, 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl,). The blot was incubated with biotinylated calmodulin (a generous gift of M. Billingsley, Milton S. Hershey Medical Center, Hershey, PA, U.S.A.), at a concentration of 25 pl of 2 mg/ml biotinylated ealmodulin for each 5 ml of fresh blocker (Billingsley et al., 1985), to which EGTA had been added to a concentration of I . 1 mM. The blot was then washed in this same buffer, with the omission of milk powder, prior to incubation for 1 h in fresh blocker, to which had been added 50 pl of a 1 mg/ml solution of avidinperoxidase conjugate (Sigma). Excess reagent was removed by washing, prior to color development (Hawkes et al., 1982).

ATPase measurements KC-EDTAATPase assays (Collins and Borysenko, 1984) were made after separate dialysis of all column fractions against 10 mMimidazole, pH 7.5,0.5 MKC1, 1 mMDTT, 0.2 mM PMSF, 3 mM NaN,. The final reaction solution contained 0.1 ml of sample, 0.4 ml of the above sample buffer, plus EDTA to 0.2 mMand 5 pl of 0.1 M [y3'P]ATP ( 1 pCQ2.5 PI). Typically, a 45-min assay time interval was used. Ca2+-ATPasemeasurements werc made in 10 mM imidazole,pH 7.5,0.5MKCl, lOmMCaCl,, 1 mM[y-32P]ATP. Rates were measured by estimation of "Pi release by standard procedures (Collins et al., 1990). Mg2+-ATPaserates in the presence and absence of actin were made after separate dialysis of column fractions against 10 mM imidazole, pH 7.5, 0.5 M KCl, 10 mM MgCl, and were assayed in the same buffer plus 0.5 mM EGTA, 0.5 mM CaCI,, 1 mM [Y-~~PIATP, conditions which optimize the rates for brush border myosin I (Collins et al., 1990). Actin-activated rates were measured in the presence of 0.5 mg/ml rabbit F-actin.

Gel filtration assay for myosin I binding to lipid vesicles The gcl filtration a s s q for myosin 1binding to lipid vesicles was performed according to the procedures of Adams and Pollard (1989). A Sepharose CL-4B (LKB-Pharmacia) column (0.7 x 16 cm) was equilibrated in 60 Mp o t a s s i u m glutamate, 10 Mimidazole-HC1, pH 6.4,2 M E G T A , 2 mM MgCl,, and 1 mM ATP. Phosphatidylserine or phosphatidylcholine (0.1 ml of 5 mg/ml in chloroform) was dried under vacuum, then resuspended in 0.5 ml of the above buffer prior to sonication. Protein sample (0.35 ml) and 0.15 ml of sonicated lipid vesicles were mixed together by stimng at 4°C for at least 30 min before use. A 0.5-ml sample was loaded onto the column, followed by I ml of column buffer. The sample was then followed directly with the same buffer plus 0.3 A4 NaC1, as described (Adams and Pollard, 1989). Fractions of 0.4 ml were collected.

Other SDS-acrylamide gel electrophoresis (Matsudaira and Burgess, 1978)and electrophoretic protein transfer onto nitrocellulose membranes (Towbin et al., 1979) were performed according to standard procedures. Myosin I, immobilized on nitrocellulose membrane, was detected by indirect immunocytochemistry using horseradish peroxidase J. Neurocltem., Vol. 59, No. 4, 1992

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conjugated goat anti-rabbit IgG (Cappel). Color was developed with 0.01% H,02 and khloro-1-naphthol as sub strates (Hawkes et al., 1982).

Myosin II

Myosin I

RESULTS AND DISCUSSION By using procedures based on earlier myosin I preparations from protozoa and chicken brush border (Pollard and Korn, 1973; Maruta et al., 1979; Collins and Borysenko, 1984; Lynch et al., 1986; Conzelman and Mooseker, 1987; Collins et al., 1990), we were able to isolate a fraction from bovine brain enriched for myosin I. Our primary criteria for myosin I identification were as follows: (a) antigenicity towards a well characterized antibody against myosin I from chicken brush border (Collins et al., 1990); (b) possession of a K+-EDTA ATPase (Collins and Borysenko, 1984; Conzelman and Mooseker, 1987), which is often considered a property that distinguishes myosins from other ATPascs; (c) a polypeptide chain size significantlj: lower than that of the myosin 11 heavy chain, which can be separated chromatographically from myosin 11; and (d) possession of a MgZ+-ATPasethat can be elevated in the presence of F-actin. Secondary criteria, not necessarily exhibited by all members of the myosin I family, included calmodulin binding and association with lipid vesicles. The profiles of the eluates from all three columns used in the preparation are seen in Figs. 1-3. Myosin I and myosin I1 are both present in the initial stages of

Myosin II Myosin I

,,.., O . ~ . " ' , ' ' ' ' '' I " ' ! ' I ' , I 15 20 25 30 35 40 46 60 55 80 65 I

FkW&dl

FIG. 1. Elution profile of material passing through the Sepharose CL-4B column. Sepharose CL-4B (LKB/Pharmacia) was the first column employed in the brain myosin I preparation. The precipitate resulting from ammonium sulfate fractionation in the range 20-40% was resuspended in and dialyzed against 10 mM imidazde,pH 7.3.0.6M KCI, 1 mM ATP. 1mM EGTA, 1 mM MgCl2.5% sucrose, plus the protease inhibitor cocktail (see Materials and Methods). The clarified supernatant (30 ml at -9 mg/ml) was loaded onto a 500-ml Sepharose CL-4B column which had been preequilibrated in the same buffer. Every third fraction eluting from the column was run on two sister 10% SDS-acrylamidegels, which were transferred to nitrocelluloseand visualized by indirect immunochemistry (Materialsand Methods)using primary antibodies to either neuroblastoma myosin II (upper strip) or chicken brush border myosin I(lower strip). In this figure, the immunoblots are positioned exactly over their appropriate fractions within the elution profile. The elution profile (0)was obtained by monitoring absorbance at 280 nm. A partial separation of myosin Ifrom myosin I1was obtained.

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Fraction

FIG. 2. Elution profile of material passing through the hydroxyap atite column, showing correspondence of myosin I immunoreactivity and K+-EDTAATPase activity. The peak of myosin Iimmunoreactivity obtained by passage through the Sepharose CL-4B column was pooled and dialyzed against the starting buffer for the hydroxyapatitecolumn [ l o mM imidazole, pH 7.5, 0.2 M KCI, 1 mM K2HP04,1 mM ATP, 1 mM MgQ, 1 mM EGTA, plus the inhibitor cocktail (see Materials and Methods)]. The clarified, dialyzed material (80 ml at -2 mg/m!) was diluted with an equal volume of the above buffer, then applied to a 100-mlhydroxyapatite column (Hoechst), and washed in the above buffer. Material was eluted from the column with a linear 0.001-1 M K2HP04 gradient (8-mi fractions, 32 ml/h). Every third fraction eluting from the column was run on two sister 10% SDS-acrylamide gels, which were transferred to nitrocelluloseand visualized by indirect immunochemistty (Materialsand Methods) using primary antibod ies to either neuroblastoma myosin I1 (upper strip) or chicken brush border myosin I (lower strip). In addition, fractions were collected separately and individually dialyzed prior to K+-EDTA ATPase determination (M; see Materials and Methods). The a b sorbance at 280 nm (0)was used to determinethe elution profile. lmmunoblots are positioned exactly over their appropriate fractions within the elution profile. This column separated myosin II from myosin 1.

the preparation and are partially separated on the Sepharose CL-4B column (Fig. I). The single band that is immunoreactive towards anti-myosin I corresponds to a molecular mass of 150 kDa. Myosin I is separated away from the remaining myosin I1 by passage through a hydroxyapatite column (Fig. 2); a peak of K+-EDTAATPase activity coincides with the peak of myosin I immunoreactivity. Further purification is achieved upon passage through a Sepharose Q column (Fig. 3), where myosin I is separated away from a large amount of material absorbing in the aromatic region of the ultraviolet spectrum. An enhanced Ca2+-ATPaseis associated with all myosin peaks (data not shown). Although the 150-kDa immunopositive band has a higher chain weight than that of other myosin I molecules observed to date, it is unlikely that the peptide we are detecting is simply a breakdown product of myosin 11. First, the anti-myosin I antiserum used does not detect brain myosin 11 prepared from the same starting material, yet readily detects the 110kDa peptide from chicken brush border and the 150kDa peptide from bovine brain (Fig. 4B, E, and H). Secondly, our affinity-purified pol yclonal antibody against neuroblastoma myosin I1 (Miller et al., 1992) readily detects the bovine brain myosin I1 heavy

BRAIN MYOSIN I Myosin

I

m c

1 I

n

a

Fractkn

FIG. 3. Further purificationof brain myosin I upon elution through Semarose Q. The peak of myosin I immunoreactivity from the hydroxyapatite column was pooled and dialyzed against 20 mM imidazole, pH 7.5, 75 mM KCI, 0.1 mM MgCI,, 0.1 mM EGTA, 1 mM ATP, plus inhibitor cocktail (see Materials and Methods). After clarification, 120 ml of this material (-0.3 mg/ml) were loaded onto a 40-ml Sepharose Q column (LKB/Pharmacia) and eluted with a 0-1 .OM KCI gradient. Every second fraction eluting from the column was run on 10% SDS-acrylamide gels, which were transferred to nitrocelluloseand visualized by indirect immunochemistry(Materials and Methods) using a primary antibody to chicken brush border myosin I. lrnrnunoblots are positioned exactly over their appropriate fractions within the elution profile, which was determined through observation of the absorbance at 280 nrn (0).In addition, fractions were collected separately and individually dialyzed prior to K’-EDTA ATPase determination (M; see Materials and Methods). This column separated myosin I from a large amount of other material absorbing at 280 nm.

chain, but is not cross-reactive against either the 110kDa peptide from chicken brush border or the 150kDa peptide from bovine brain (Fig. 4C, F, and I). Thirdly, the anti-myosin I antibody only detects a single 150-kDa band in crude brain homogenates (Fig. 4K), whereas anti-myosin I1 detects only higher molecular mass material at 2 10 kDa (Fig. 4L). Finally, no bands immunopositive for myosin I are found when purified brain myosin I1 is digested by chymotrypsin (Fig. 5), demonstrating that it is unlikely that a new immunopositive peptide can be obtained by generation of a novel conformation as a result of proteolysis. The considerably lower molecular mass of the 150-

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kDa immunopositive polypeptide chain as compared with that of the myosin I1 heavy chain, together with the lack of immunogenic cross-reactivity between these isoforms and coupled with the possession of myosin I-like properties by the 150-kDa protein, as described below, makes it unlikely that this molecule is a low molecular mass isoform of myosin 11. Currently, at least three myosin I1 heavy-chain isoforms have been detected in brain (Kawamoto and Adelstein, 1991; Murakami et al., 1991; Sun and Chantler, 1991), I-X3ne of which shows a heavy-chain molecular mass below 196 m a . In order to demonstrate that an association of K+EDTA ATPase activity with the 150-kDa immunopositive band is not coincidental, we measured the K+-EDTAATPase rates before and after immunoprecipitation with anti-brush border myosin I. The results are shown in Table 1. Over 70%of the K+-EDTA ATPase activity is removed from the peak of activity eluting from the Sepharose Q column through immunoprecipitation with anti-myosin I. By contrast, a control immunoprecipitation using anti-myosin I1 caused the K+-EDTAATPase to decline by only 1770, suggesting that the loss of ATPase brought about by myosin I immunoprecipitation was specific and was not brought about by denaturation of the sample during treatment or by nonspecific absorption. SDSacrylamide gel electrophoresisand western transfer of these samples, before and after immunoprecipitation, are seen in Fig. 6. The 150-kDa peptide is specifically removed from the starting material by the anti-myosin I antibody. These data, together with the association of K+-EDTAATPase activity with 150-kDa peptide elution in all column chromatography elution profiles (Figs. 1-3), provide strong evidence that the 150-kDa protein is responsible for the K+-EDTA ATPase activity. The final specific activity of 150-kDa proteinenriched material eluting from the Sepharose Q column, divided by the initial specific activity of the ho-

FIG. 4. Demonstration of the specific210 kD ity of antibodies used. Chicken brush border myosin I(lanes A-C; a generous 150 kD gift from P. Matsudaira and K. Collins, 110 w ) Whitehead Institute, Cambridge, MA, U.S.A.). bovine brain myosin I1 (lanes D-F), a bovine brain fraction that had passedthrough all three columns of our purification procedure and was enriched for the 150-kDa protein (and completely separated from myosin 11; lanes G-I), or a freshly prepared bovine brain homoQenate [lanes J-1) was run A B C D E F G H I J K L on 7.5% SDS-acryiamide gels (Matsudaira and Burgess, 1978) and transferred to nitrOcelluloSeffowbin et al.. 1979). Blots were Drobed usina either Dolvclonal antibodies directed aaainst chicken brush border myosin I(6, E, H, K) or neuroblastomamyosin II (C, F, I,L). Lanes A, DyandG are-gels stained with Coomassi~Blue;laneJ is a blot stained with amido black. Rainbow standards (Amersham) were used as molecular weight markers for each of the separate runs (not shown). Gel loadings were as follows: 1pg brush border myosin I; 3.5pg brain myosin 11; 10 pg of total protein (brain myosin I); 15 pg of total protein (brain homogenate).

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210.

Myosln I-

95-

A

45. ABCD

EFGH

I J K L

FIG. 5. Demonstration that proteolysis of brain myosin 1 I does not produce fragments irnmunopositivefor anti-myosin 1. Myosin II, purified from bovine brain, was solubilized in 0.6 M NaCI, 25 mM Tris, pH 7.5, 0.1 mM DTT at 0.63 mg/ml and digested with chymotrypsin (12.5 @g/ml)for 30 min. Aliquots were removed at 0-, 5-,15, and 30-min intervals, and proteolysis terminated by the addition of PMSF to 1 mM, prior to preparation for western blotting. Lanes A-D: Coomassie Blue stained gel. Lanes E-H: Sister gel that was transferred to nitrocellulose and stained by indirect immunochemistry using a primary antibody against brush border myosin 1. Lanes I-L Sister gel that was transferred to nitrocellulose and stained by indirect immunochemistry using a primary antibody against neuronal myosin II.

mogenate, gives rise to a minimal purification factor, which was typically in the range of 15-20-fold. However, the total ATPase within the homogenate, and within material applied to both the Sepharose CL4B and hydroxyapatite columns, contains a substantial component due to myosin 11. One method to estimate the relative proportions of myosin I and I1 originally TABLE 1. Demonstration by immunoprecipitation that the 150-kDaprotein is the source of the Kf-EDTAATPase K+-EDTA ATPase (nmol of Pi/min/ml) Treatment

Anti-myosin I

Anti-myosin I1

Before immunoprecipitation After immunoprecipitation

317 83

426 352

K+-EDTAATPase activity of the myosin I-enriched peak eluting from the Sepharose Q column was determined before and after immunoprecipitation with anti-brush border myosin I IgG or antineuroblastomamyosin I1 IgG. After removing an aliquot to test for K+-EDTA ATPase activity, the remainder of the myosin I peak from the Sepharose Q column was incubated at 4°C with antibody in 10 mM imidazole, 0.5 M KCI, 1 mMDTT', 0.2 mMPMSF, 0.3 mM NaN,, pH 7.5. After 30 min, an excess of protein A-agarose (equilibratedin the same buffer) was added, equilibrated, and then spun down. Aliquots from the supernatant were removed to test for K+-EDTA ATPase activity (see Materials and Methods). Results were obtained from two separate preparations. These data show that 74%ofK+-EDTA ATPase activity was removed from the activity peak of the Sepharose Q column through immunoprecipitation with anti-myosin I, whereas the activity was only diminished by 17%upon immunoprecipitationwith anti-myosin 11, used here as a control, all myosin I1 having been removed at an earlier stage in the preparation. These results suggest that the 150-kDa protein is responsible for the associated K+-EDTA ATPase activity. J. Neurochem., VoI. 59. No. 4, 1992

B

C

D

E

F

FIG. 6. Demonstrationthat the 150-kDa protein is removed specifically upon immunoprecipitation with anti-myosin 1. Fractions containingthe 150-kDa protein eluting from the Sepharose Q column were immunoprecipitated with either anti-myosin I (lanes AC) or anti-myosin ll (lanes D-F), as described in Materials and Methods and in the legend to Table 1. Material was examined on Coomassie Blue stained 12.5% SDS-acrylamide gels both before (lanes A and D) and after (lanes B and E) immunoprecipitation. Sister lanes of the immunoprecipitatedmaterial were blottedonto nitrocellulosepaper and examinedby indirect immunocytochemistry using primary antibodies against either myosin I (lane C) or myosin II (lane F). The 150-kDa band corresponding to myosin I immunoreactivity is indicated in the figure. The IgG employed in the irnmunoprecipitationsalso appears on the gels (arrowheadsin lanes B and E) and is recognizedthrough application of the appropriate antibody, during western blot development (arrowheads in lanes C and F). The heavy band seen in the Coomassie Blue stained gels of the immunoprecipitates (lanes B and E) located above the IgG heavy chain is bovine Serum albumin, used as a carrier in the final washes of the immunoprecipitations.

present is to measure the areas under their respective ATPase peak profiles upon elution from the Sepharose CL4B column (data not shown). Myosin I1 was found to contribute at least 75% of the ATPase activity. Following the example of others (Swanljung-Collins and Collins, 199I), one can use this value as an estimate of the contribution of myosin 11to the ATPase within the homogenate (it is not possible to assess the relative contributions of the myosin isoforms within the homogenate directly). Taking this into account, the purification factor achieved here is in excess of 60-fold. Densitometry of Coomassie-Blue stained gels, such as those seen in Fig. 4G and 7A, suggests that myosin I represents 5-10% of the partially purified fraction eluting from the Sepharose Q column. Now, if myosin I and myosin I1 both have similar specific activities, a lower boundary for the myosin I1 to myosin I ratio within the homogenate would be threefold. However, taking into account the measured ATPase rates seen in Table 1 and the degree of purity of the sample, which suggest that the specific K+-EDTAATPase rate of the 150-kDaprotein is substantially higher than that of myosin 11, then this ratio could be 10-fold higher. We, therefore, suggest that the ratio of myosin I1 to myosin I found in the starting material is in the range three- to 30-fold, and is probably closer to the higher ratio asjudged by inspection of Coomassie-Blue stained gels and western blots from early stages of the preparation, making the purification factor achieved correspondingly higher.

BRAIN MYOSIN I FIG. 7. Demonstration of calmodulin binding to the 150-kDa pro210. tein by means of the calrnodulin overlay technique. Fractions containing the 150-kDa protein eluting s. from the sepharose Q column were run on 7.5% SDS-acrylam68 ide gels (lane A stained by Coomassie Blue) and transferred to nitrocellulose. One strip was devel45. oped using anti-brush border A B C myosin I as primary antibody (lane B).A sister strip (lane C)was incubated with biotinylated calmodulin. Incubation conditions chosen provided a [Ca"],, of 0.5 X 10-6 M, established using an iterative Ca'' binding program (Chantler and Szent-Gyorgyi, 1980). Following binding, the blot was washed, then incubated with horseradish peroxidase-conjugated avidin. Color was developed using 0.01% H,02 and 4chloro-1-naphthol as substrates. See Materials and Methods for details.

Material eluting from the Sepharosc Q column at the combined peak of both myosin I immunoreactivity and K'-EDTA ATPase activity showed a threefold enhancement of Mg2+-ATPaseactivity upon addition of a large molar excess of F-actin. Under the conditions described in Materials and Methods, which were optimal for assessing the actin-activated ATPase rate of brush border myosin I (Collins et al., 1990), the myosin I peak fraction had a Mg2+-ATPase activity of 6-8 nmol/min/mg in the absence of actin, and a Mg*+-ATPaseactivity of 18-20 nmol/min/mg in the presence of actin. The addition of calmodulin to 20 pM (Collins ct al., 1990) had no effect on these rates. This enhancement is consistent with that found by others for brush border myosin I (Conzelman and Mooseker, 1987). Recently, Collins et al. (1990) have noted up to a 40-fold actin activation for brush border myosin I and attributed earlier lower enhancements to incomplete purification. Although the hydroxyapatite column completely separates brain myosin I away from myosin I1 (Fig. 2), the material from the Sepharose Q column is still impure, as noted above; the presence of other proteins in the preparation may be responsible for attenuating the extent of the measured actin-activated ATPase. We have made exhaustive attempts to purify brain myosin I completely, including passage through ADP-agarose, phosphocellulose, DEAE-cellulose, CM-Sepharose, and calmodulin affinity columns. In our studies, application of all protocols successful for brush border myosin I (Collins et al., 1990)does not enhance further the purity of brain myosin I as compared with the fraction we have already obtained. Similarly, with protocols that have proven successful for the purification of protozoan myosin I, we were also unable to achieve a higher degree of purity than presented here. Therefore, in order to effect a functional characterization of brain myosin I using this material, we had to choose techniques that would lead to specific answers even when employed on protein mixtures containing myosin I.

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The calmodulin overlay technique and the lipid vesicle binding assay, described below, fulfill these requirements. The 1 10-kDa heavy chain of chicken brush border myosin I is associated with two to four subunits of calmodulin (Matsudaira and Burgess, 1979;Collins et al., 1990), rather than a single, specialized light chain, as in the case of Acanthamoeha myosin I (Albanesi et a]., 1984). We investigated whether the 150-kDa protein from bovine brain would associate with calmodulin by means of a sensitive overlay method using biotinylated calmodulin (Billingsleyet al., 1985)(Fig. 7). We chose conditions to give micromolar levels of free Ca2+,so as to maximize calmodulin binding (Collins et al., 1990). Calmodulin was found to associate with the 150-kDa band (Fig. 7C), suggestive of an interaction similar to that seen in brush border myosin I. A characteristic feature of a number of forms of myosin I is the ability to bind to membrane lipids (Adams and Pollard, 1989; Miyata et al., 1989) and pure phospholipid vesicles (Adams and Pollard, 1989; Hayden et al., 1990). We followed the gel filtration assay of Adams and Pollard (1989) to examine the ability of the 150-kDa protein from bovine brain to bind to anionic lipid vesicles made from phosphatidylserine. Figure 8 (lanes B and D) demonstrates that the 150-kDa protein associates with phosphatidylserine vesicles and appears in the void volume upon passage through a Sepharose CL-4B column. In the absence of phosphatidylserine (Fig. 8, lanes A and C), or after incubation with neutral lipid vesicles made from phosphatidylcholine (Fig. 8, lane E), the 150-kDa protein moves much more slowly through the column, eluting close to the salt volume. These results are similar to those described earlier for Acanthamoeba myosin I (Adams and Pollard, 1989) and chicken brush border myosin I (Hayden et al., 1990), and serve to illustrate that the 150-kDa protein from bovine brain is analogous to myosin I from other sources in that it can interact with vesicles made from anionic phospholipids, but not with vesicles made from neutral phospholipids. Several authors have commented on the variety of intracellular tasks that may be accomplished by a family of myosin I molecules (Pollard and Korn, 1973; Korn and Hammer, 1988; Adams and Pollard, 1989; Miyata et al., 1989; Mooseker, 1989; Spudich, 1989). Some (Albanesi et al., 1984; Lynch et al., 1986), but not all (Collins et al., 1990), myosin I molecules possess a second actin binding site at the C-terminal end of the molecule, which is ATP-independent and allows for cyclic interaction with actin at one end while binding tightly to another actin filament at the other. At present, we are unable to assess the ability of the 150-kDa protein to bind to actin in an unambiguous manner; this will become possible once this enzyme has been purified to homogeneity. The ability to interact with the plasma membrane (Adams and Pollard, 1989; Miyata et al., 1989; Hayden et al.. 1990) J. Neurochem..

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FIG. 8. The 1 SO-kDa protein binds to phospholipidvesicles made from phosphatidylserine, but not from phosphatidylcholine. The myosin I enriched fraction eluting from the Sepharose Q column was incubated with lipid vesicles using conditions identical to those described by Adarns and Pollard (1989). After incubation, material was applied to a Sepharose CL-46 column (see Materials and Methods for details). Material eluting from the Sepharose CL-46 column was subjected to 7.5% SDS-acrylamide gel electrophoresis (Matsudaira and Burgess, 1978) and western blotting (Towbin et al., 1979) using anti-brush border myosin I as primary antibody. One experiment is shown in lanes A and B. Another set of data, obtained from a different preparation of my* sin I, is shown in lanes C-E. Lanes A and C: Control runs which show that myosin I, in the absence of lipd vesicles, elutes near the salt volume, as demonstrated for Acanthamoeba (Adams and Pollard, 1989) and brush border (Hayden et al., 1990) myosin 1. Lanes B and D: in the presence of lipid vesicles composed of phosphatidylserine,myosin I elutes at the void volume. The locations of other proteins present in this still impure myosin l fraction remain mattered upon elution, irrespective of prior incubation with phosphatidylserinelipid vesicles (data not shown). Lane E: In the presence of lipid vesicles composed of phosphatidylcholine, the elution of myosin 1 remains in a position similar to that attained in the complete absence of phospholipids.

allows for movement of the plasma membrane relative to polarized actin filaments. The Drosophila ninaC gene encodes a product with a protein kinase moiety contiguous with the myosin head (Monte11 and Rubin, 1988). All these properties suggest a heterogeneous group of molecules whose common theme is a single, functionally conserved myosin head structure. Indeed, evidencc (Titus et al., 1989) from Dictyostelium molecular genetics suggests a number of types of myosin I may be present within a single cell, each possessing a unique set of attributes (e.g., the presence or absence of a second actin binding site, membrane binding domain, additional enzymatic function, and so on). The versatility of this family of actin-based motors makes it unlikely that their presence is restricted either to amebae and slime molds, on the one hand, or solely to structural functions in cells of higher organisms, on the other. The ameboidlike motions exhibited by neuronal growth cones during development (Smith, 1988) and the complexities of organelle transport within mature neurons (Lasek et al., 1984) make the mammalian nervous system an

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ideal place to search for myosin I. Here, we have shown that a preparative method applied to bovine brain, devised to purify myosin I, produces a fraction enriched in a 150-kDa protein which is immunoreactive with an antibody against chicken brush border myosin I, possesses K+-EDTA ATPase, Ca2+-ATPase, and actin-activated ATPase activity, binds calmodulin, and associates with anionic phospholipid vesicles; all these properties are characteristic of vertebrate myosin I. Based on these data, we suggest that a membrane-anchored myosin I functions in brain cells by mechanisms analogous to those already proposed for myosin I molecules from other sources (Pollard and Korn, 1973; Maruta et al., 1979; Korn and Hammer, 1988; Adams and Pollard, 1989; Fukui et al., 1989; Miyata et al., 1989; Spudich, 1989) and that it may be a key player involved in neural circuit formation and mature brain function. Acknowledgment; We are indebted to Drs. Paui Matsudaira and Kathy Collins for advice and gifts of anti-brush border myosin I and its antigen, and to Mark Miller and Drs. Yulai Wang, Weidong Sun, and Rhea Levine for help and discussion. This work,was supportcd by grants from the American Heart Association (870688) and the N.I.A.M.S.D.(AR 32858).

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