Identification of myosin in isolated synaptic junctions

Brain Research, 225 (1981) 75-93

75

Elsevier/North-Holland Biomedical Press

I D E N T I F I C A T I O N OF MYOSIN IN ISOLATED SYNAPTIC J U N C T I O N S

ROBERT L. BEACH, PAUL T. KELLY, JOSEPH A. BABITCH and CARL W. COTMAN* (R.L.B. and C.W.C.) Department of Psychobiology, University of California, Irvine, CA 92717; (P.T.K.) Division of Biology, Kansas State University, Manhattan, KS 66502 and (J.,4.B.) Department of Chemistry, Texas Christian University, Fort Worth, TX 76129 (U.S.A.}

(Accepted March 26, 1981) Key words: myosin - - synaptic junctions - - postsynaptic densities - - gel immunoadsorption

synaptic membranes - - peptide fingerprints - - actin

SUMMARY A monospecific antibody prepared against chicken gizzard myosin reacted with only one peptide corresponding to myosin heavy chain (Mr = 200,000) in gels of synaptic plasma membranes (SPM) and synaptic j unctions (S J) prepared from several species. Preadsorption of antisera with purified brain myosin eliminated antibody reactivity to SPMs and SJs. SJs were found to contain approximately 3 times the concentration of myosin found in SPMs when assayed by an indirect immunoradiometric assay. Postsynaptic density and myelin fractions contained no myosin detectable by immunoradiometric assay, antibody binding to gels, or Coomassie blue staining. The band identified as myosin in SJ fractions yielded peptide fingerprints indistinguishable from fingerprints of purified brain myosin but distinct from fingerprints of purified smooth and skeletal muscle myosins. The distribution of exogenous [125I]myosin during subcellular fractionation indicated that myosin in isolated synaptic junctions could not have resulted from artifactual re-distribution of soluble myosin. Together these results show that a non-muscle myosin is an endogenous component of CNS asymmetric synapses.

INTRODUCTION Actin and myosin are important for motility in many non-muscle cellse°,36,4°,rt and are implicated in membrane-related events such as receptor movements2S, 57 and endocytosisTt, 7z. These proteins may also play roles in axonal growth12, sS, axonal transport 44,4~, and in synaptic functions. At the synapse, transmitter release and * To whom correspondence should be addressed. 0006-8993/81/0000-0000/$ 02.50 © Elsevier/North-Holland Biomedical Press

76 vesicle translocationg, 63, and postsynaptic receptor anchoring and spine shape changes 23 may also involve actin and myosin. One important but as yet unanswered question is whether or not myosin is actually present at the synapse and whether it is associated with the synaptic membrane. The answer to this question is a necessary prerequisite for consideration of a functional actin-myosin system at CNS synapses. Myosin, first tentatively identified in brain by Poglazov 59, has been found to be present along with actin in tissues of the central and peripheral nervous systems of various species 9,13,76. Characterization of these proteins has indicated that brain actin 19,a9 and myosin 15,41 differ from their counterparts in muscle. Actin from brain 19, neuroblastoma 5~,65 and synaptic junctions a9 has been shown to consist of many isoelectric forms which are similar to those found in a variety of non-muscle cell lines and tissues ~°. Partial proteolytic cleavage or cyanylation of the heavy chain of brain myosin yield one dimensional peptide maps which differ from those obtained from platelet, smooth muscle, cardiac muscle, and skeletal muscle myosin heavy chains 15,16. Light microscopic localization of myosin in neurons has been achieved by immunofluorescence utilizing antibodies to gizzard myosin66, 77, adrenal myosin3, 4 and brain myosin 42. Actin has also been localized in neurons by the binding of fluoresceinlabeled heavy meromyosin 67, and with antibodies to actin 75 by indirect immunofluorescence. These studies have indicated that myosin is diffusely distributed in neurons, including neurites and growth cones, and that actin is present in neurites and growth cones and may be associated with synapses and membranes. Electron microscopic evidence indicates that actin containing microfilaments are associated with membranes and synapses in neurons 17,18A6,47,52. However, complementary studies on neuronal myosin localization have not been published, and a synaptic location for myosin has not been established. Subcellular fractionation of nervous system tissues has provided well-characterized preparations which are enriched in morphologically identifiable synaptic structures21,2~,5~, 64,7~. Previous biochemical studies on these fractions have identified several proteins of chemomechanical systems2~,49,50,63. Actin, myosin, and tropomyosin have been shown to be present in brain and synaptosomal preparations9,H,62, 6s. Actin and tubulin but not myosin have been identified in synaptic plasma membrane (SPM), synaptic junction (SJ) and postsynaptic density (PSD) fractions ~°,2~,39,74,81. Actin and tubulin are major proteins in the SJ and PSD fractions ~9. In this report we have used immunological and biochemical methods to demonstrate the presence of myosin in isolated synaptic junctions. A preliminary report of some of these results has been published 6. METHODS

Preparation of contractile proteins Steer and rat brain myosin were purified essentially according to the method derived for platelet myosin by Pollard et al. 6°. Brains were cleaned free of meninges prior to homogenization. Phenylmethylsulfonylfluoride (PMSF, 0.1 mM) was added to all solutions to reduce proteolysis. For some preparations, 0.6 M KCI was used instead of 0.6 M K1, to reduce denaturation during column chromatography in

77 Sepharose 4B. When necessary, brain myosin was concentrated by ultrafiltration using an Amicon XM-100 membrane. Smooth muscle myosin was purified from chicken gizzards according to the method of Wang s°. A final column of Whatman DE-52 was employed to purify monomeric myosin. Skeletal myosin was extracted from the leg and back muscles of mice and rabbits in 0.6 M KCI, 25 mM Na4P207 pH 7.0, 1.0 mM EDTA, 0.1 mM dithiothreitol (DTT). Myosin preparations were stored at --20 °C in 50 ~ glycerol. Actin was prepared from chicken gizzards, mouse skeletal muscle, and rat brain essentially by the method of Spudich and Watt 7°.

Preparation of antisera to myosin The chicken gizzard myosin, which was used as the immunogen, was purified by ion-exchange chromatography on a Whatman DE-52 column and was 98 ~ pure as judged by densitometric scans of polyacrylamide gels. Gizzard myosin (100/~g) in 5 ml of 0.6 M KC1 was emulsified in an equal volume of complete Freunds adjuvant. Approximately 0.1 ml of the emulsion was injected at each of 40 intradermal sites along the backs of 4-5 pound male New Zealand white rabbits, as described by Vaitukaitus et al. 78. Two weeks later 40 adjacent sites were injected in the same manner. Fourteen days later and at intervals of 2-3 weeks the rabbits were boosted with 0.05 mg myosin in incomplete Freunds adjuvant subcutaneously in available sites along the back. Bleedings were by cardiac puncture 7-10 days following each boost. Antibodies were detected by immunofluorescent and immunoperoxidase staining of myosin in stress fibers in a clone of mammary tumor cells (MTF 7) 56 in the second and subsequent bleedings. All antisera used here were obtained from one rabbit (H2) which gave detectable filament staining at titers up to 1:300 by immunofluorescence and 1:1200 by immunoperoxidase. For some experiments the myosin antibodies were partially purified by (NH4)zSO4 fractionation. Immunofluorescence staining The distribution of myosin was observed in cultures of neuroblastoma C-1300 clone N-18, and MTF 7 cell lines as well as in primary cultures derived from neonatal rat hippocampus as described elsewhere (Beach et al., submitted). Cells grown on coverslips were fixed and permeabilized in acid alcohol (95 ~ ethanol/5 ~o acetic acid) or 100 ~ acetone, at --10 °C. Similar results were obtained with cultures fixed in 3.7 formalin in phosphate-buffered saline (PBS) (136.9 mM NaC1, 2.7 mM KC1, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) after permeabilization with 0.3 ~ Triton X-100 (TX-100) and treatment with 0.1 M glycine in PBS. Optimal staining was obtained using antimyosin sera diluted between 1:60 and 1:100 in PBS and rhodamine or fluoresceinconjugated IgG fraction of goat antirabbit IgG (Cappel) diluted 1:20 in PBS. Antibodies were incubated for 45 min at room temperature, followed by thorough rinsing in PBS. Coverslips were mounted inverted on slides with Gelvatol (Monsanto) and observed with episcopic illumination on a Nikon Fluorophot. In primary cultures, astrocytes were labeled with antibodies to glial fibrillary acidic protein and neurons were identified by labeling with tetanus toxin, and goat antibodies to tetanus toxoid, in a double label procedure as described elsewhere (Beach et al., submitted).

78

lmmunoradiometric assay for myosin To quantitate the amounts of immunoreactive myosin in particulate subcellular fractions, an assay was developed which is based on the binding of [125I]protein A to antimyosin antibodies which were previously bound to subcellular fractions (SPM, SJ, myelin and PSD). Immune and preimmune sera were preadsorbed with calf brain microtubules and intact rat red blood cells and fractionated with (NH4)2SO4, and were diluted 1:100 in 0 . 5 ~ ovalbumin in PBS. Each subcellular fraction (50 /zg) was pelleted at 8730 g for 12 min in a Beckman Microfuge B, washed in 1.0 ml of the ovalbumin buffer and then incubated for 1 h at 4 °C in 0.1 ml of the antibody dilution. Buffer was then added (0.9 ml) and the samples were pelleted as before and washed 3 times with 1 ml buffer. [125I]protein A (2 × 107 cpm, 1-2/~g) in 0.1 ml buffer was added to each sample. The samples were incubated at 4 °C for 30 min, then diluted and washed 3 times as before. The radioactivity of the pellets was determined in a Beckman 9000 gamma counter (counting efficiency for 1251 was 85 ~). Protein A iodination Protein A (Pharmacia) was iodinated by the method of Dorval et al. z6. Specific activity obtained was 1-2 × 107 cpm/#g. After Sephadex G-25 chromatography and dialysis, the [125I]protein A was diluted in 5 ~ ovalbumin in PBS to 2 × 107 cpm/ml and stored in 1 ml aliquots at --20 °C. Myosin iodination Rat brain myosin (0.4 mg in 0.2 ml of 50 ~ glycerol/50 ~ column eluate) was dialyzed against 50 mM Na4PeOT, 1 mM ethylenediaminetetraacetate (EDTA) and 1.43 mM fl-mercaptoethanol (buffer B) for 4 h and then dialyzed for 2 h versus 50 mM Na4PzOT, 1 mM EDTA, 0.1 mM fl-mercaptoethanol (buffer A). To 0.1 ml (0.2 mg) of this myosin, the following were added with mixing: 0.31 U lactoperoxidase (Sigma, 61 U/mg) in 0.1 ml of buffer A, 0.02 ml (2 mCi or 2 nmol) of Na[l~5l] (Amersham), 11.6 #1 of 2 mM H202. After 1 min an additional 10/A of 2 mM H202 was mixed in and after 3 min, the reaction was terminated by addition of 0.02 ml of buffer B. The iodinated sample was immediately dialyzed against buffer B containing 0.1 mg/ml NaI, 3 × 100 ml/h each, and then overnight against 3 changes of buffer B. The [125I]myosin (spec. act. = 3.0 × l0 s cpm/#g) was centrifuged 20 min in a Beckman airfuge (136,000 g) immediately prior to use. The [125I]myosin bound to filamentous actin and was pelleted with the actin filaments (data not shown). Gel electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 0.5-0.9 mm thick slab gels using a discontinuous buffer system described by Laemmli and Favre 43 in chambers according to the design of Studier 7a. Exponential-linear gels a8 of 7-15.5 ~ or 8.5-15.5 ~o were used. Gels were fixed and stained in 25 ~ isopropanol, 10 ~ acetic acid with 0.2 ~o Coomassie brilliant blue R. Destaining was carried out in a solution of 25 ~ isopropanol/10 ~ acetic acid followed by 10 ~o acetic acid.

79

Localization of myosin in polyacrylamide gels Myosin immunoreactivity was localized in polyacrylamide gels by a modification of the [125I]protein A binding method of Adair et al. 1. Following complete destaining, the gels were incubated 30 min in 10 ~ acetate followed by 8-10 changes over a 10 h period in Tris-buffered saline (TBS) (50 mM Tris, 150 mM NaCI, 0.01 ~o merthiolate, pH 7.8). The gels were immersed in antisera diluted in TBS and were incubated 8 h to overnight. The gels were rinsed with 8-10 changes of TBS and were incubated 4 h to overnight submerged in 2 × 107 cpm of [125I]protein A in TBS. After 8-10 rinses in TBS the gels were restained, dried and exposed to Kodak XR-5 film, usually for 1-2 days.

Subcellularfractionation Synaptic plasma membrane (SPM), synaptic junction (SJ) and postsynaptic density (PSD) fractions were prepared as described by Cotman et al. zz. Fractions enriched in mitochondria, microsomes and soluble proteins were also obtained by this method. Purified myelin was prepared according to Norton and Poduslo 58.

Redistribution of [125I]myosin during subcellularfractionation To test for possible adventitious adsorption of soluble myosin to various components during subcellular fractionation, [125I]myosin was added at two different stages during the preparation of subcellular fractions. In one experiment, 8 × 107 cpm lzsIlabeled rat brain myosin (27/zg) was added during the initial homogenization. In another experiment 1.3 × 107 cpm (4.3/~g) of [125I]myosin was added at the osmotic lysis stage of the crude mitochondrial-synaptosomal pellet (P2). Samples were taken from all stages during subcellular fractionation to determine protein (as previously describedag), and [125I]myosin radioactivity (in a Beckman 9000 gamma counter) and for SDS-PAGE. SPM fractions were divided after harvesting; 6 0 ~ were used to prepare PSDs, the remainder to prepare SJs.

Tryptic fingerprint analysis The method of peptide mapping of radiolabeled proteins (actin and myosin) in single polyacrylamide gel slices described by Elder et al. 29 was used with minor modifications3L RESULTS

Characterization of antisera to myosin Antibodies to chicken gizzard myosin were shown to decorate myosin containing stress fibers in fibroblastic cells in primary cultures of rat hippocampus using an indirect immunofluorescence procedure (Fig. 1A). A periodic pattern of myosin staining in cytoplasmic stress fibers was evident in some well-spread fibroblasts (Fig. 1A), although not all fibroblastic cells showed striations. This intermittent staining has been noted previously35,s2, as. Hippocampal neurons have been identified in these cultures by reaction with tetanus toxin and their morphology (Beach et al., submitted).

Fig. 1. Photomicrographs of cultured cells after immunocytochemical staining for myosin. A: fibroblastic cells in a primary culture of rat hippocampus which have been stained with rabbit antibody to myosin (1:80) and rhodamine-coupled IgG fraction of goat anti-rabbit lgG (1:20). Note the periodic staining on the filament bundles; 16 days in vitro. Bar = 10/tin. B: hippocampal neuron growing on glial cells in a 6 days in vitro culture of rat hippocampus, which was stained as in A. Note the diffuse staining throughout the soma and processes, as well as the fainter staining of the underlying astrocytes. Bar = 10/~m. C: phase contrast of field in B. Bar ~ 10/~m.

81 In these neurons a diffuse but intense fluorescence was seen in cell bodies and throughout neuritic processes (Fig. 1B). The phase contrast image of this field is shown in Fig. 1C. The underlying astrocytes (which have been identified in other cultures with antibody to glial fibrillary acidic protein) display a less intense, and occasionally almost filamentous fluorescence. Similar neuronal staining was seen in neuroblastoma cultures (data not shown). Reaction of antibodies directed against smooth muscle myosin with neuronal myosin has also been observed by similar immunofluorescence studies in cultures 66 and in tissue sections 77. Myosin antibodies from another rabbit T1 showed less reactivity with neuronal myosin than the antibodies used in this paper (rabbit H2). The antibodies to myosin were further characterized by their binding to proteins from the penultimate stage of the antigen preparation (before chromatography on DE 52). The proteins of gizzard myosin were separated by SDS-PAGE, and the immunoreactive species were identified by incubation with antisera and subsequent binding of [125I]protein A and autoradiography. As shown in Fig. 2b, lane 4, the antimyosin sera reacted strongly with the heavy chain of the gizzard myosin (Mr = 200,000). No detectable reaction was apparent in the molecular weight region of the light chains. When preparations of brain myosin were resolved by SDS-PAGE, only the band at Mr = 200,000 was observed to react with the antibody (Fig. 2d, lane 1). Ouchteriony plates and immunoprecipitates also demonstrated a single component which reacts with the antibodies (data not shown). The antibodies used in this work appear to satisfy adequate criteria to define them as monospecific for the myosin heavy chain.

Myosin localization in subcellular fractions on polyacrylamide gels The locations of myosin and actin in gels of rat and bovine SJs are shown in Fig. 2a, lanes 1 and 3, respectively. Immunoreactive myosin was found in SPM and SJ preparations but not in the PSD fraction (Fig. 2b, lanes 1, 2 and 3, respectively). In Coomassie blue stained gels of PSDs no band corresponding to myosin is seen (not shown). One autoradiographic band (Mr ~ 200,000) was observed in each of 9 independent preparations of SPMs and SJs, and myosin immunoreactivity was not detected in the 6 different PSD preparations tested. Other brain fractions where myosin immunoreactivity was observed (homogenate, P2, synaptosomes, data not shown) only one band at Mr = 200,000 was also found. The foregoing observations were true for material of rat, mouse or steer origin. Skeletal muscle myosin showed very little reaction with the antibodies prepared against gizzard myosin (compare Fig. 2c, lane 3 with Fig. 2d, lane 3). The reaction of antimyosin sera with steer synaptic junctional myosin and partially purified rat brain myosin are also shown in Fig. 2. The myosin heavy chains in these fractions did not bind as much [12~I]protein A as did gizzard myosin (cf. Fig. 2b, lane 4 with Fig. 2d, lanes 1 and 2). Note that 0.5 /~g of gizzard myosin (lane 4) gives a very strong autoradiographic spot with the antibody relative to the SJ and SPM myosins (about 0.1-0.2 /tg myosin present). This is due to the much stronger reaction with the immunogen. Brain myosins showed considerably more reaction with antigizzard myosin than was observed with skeletal muscle myosin (Fig. 2d, lane 3, compare

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Fig. 2. Identification of myosin in gels of subcellular fractions by immunoadsorption. A: Coomassie blue staining profiles of 8.5-17.5 ~ gel of rat SJ (55/~g, lane 1), purified rat brain myosin (2.5/~g, lane 2) and bovine SJs (55/~g, lane 3). The band at Mr -- 200,000 which corresponds to myosin heavy chain is indicated by the arrow. The myosin bands in this figure do not perfectly comigrate since the sample composition and loads vary and since lane 3 was not adjacent to lane 2 on this gel. B: autoradiograph of a 8.5-17.5 ~ gel of rat brain fractions and chicken gizzard myosin alter the gel was incubated with antibody to myosin (diluted 1:50) and [125I]protein A (see Methods). Lanes: 1, rat synaptic plasma membranes (50 pg) ; 2, rat synaptic junctions (50/~g); 3, rat postsynaptic densities (50 pg); 4, chicken gizzard myosin (0.5/~g). Lanes were exposed for two days. Note that the arrow in lane 4 is based on the band seen after only 4 h of exposure. The myosin bands do not perfectly comigrate due to differences in sample composition and loading. C: 8.5-17.5 ~ gel and the corresponding autoradiograph (D) of brain fractions, after the gel was incubated with antibody to myosin diluted 1:100, and [x25I]protein A. Lanes: 1, crude bovine brain myosin (2.5 pg); 2, bovine SJs (70 g); 3, mouse skeletal muscle myosin (I0 g); 4, mouse CNS myelin (60/~g). Gel was exposed for two days. E, F: autoradiograph of gels which demonstrate specific immunoadsorption of SJ and SPM myosin reactivity. Gels were run and incubated with antimyosin as in B and C. In (F) the antibody was adsorbed with brain myosin prior to incubation with the gel. Antimyosin was used at 1:100 dilution. Gels were 7 - 1 7 . 5 ~ acrylamide. Lanes: 1, gizzard myosin (0.5 pg); 2, rat SPM (50/~g); 3, rat SJ (50 /~g); 4, rat PSD (50 #g). E and F were exposed for two days.

83 autoradiograph Fig. 2d with Coomassie blue stain Fig. 2c). Gels of mouse brain myelin did not bind antimyosin (Fig. 2d, lane 4), nor did they contain a Coomassie blue stained band at 200,000 daltons (Fig. 2c, lane 4). Purified rat brain myelin did not react with antimyosin by this technique or by immunoradiometric procedures (see below). To examine the specificity of the reaction of the antiserum with myosin, the antiserum to gizzard myosin was preabsorbed with purified rat brain myosin prior to incubations with the gels. As shown in Fig. 2e and f, this treatment abolished the reactivity of the serum against SJ and SPM fractions and reduced, but did not eliminate the binding to gizzard myosin. Quantitative analysis o f myosin in subcellular fractions An immunoradiometric assay was developed to quantify relative amounts of myosin immunoreactivity in several particulate fractions of rat brain. In Table I, the difference between the binding of immune and preimmune sera was employed to determine the relative quantities of myosin in myelin, SPM, SJ and PSD fractions. Myelin and PSD displayed little or no specific binding. By this method, SJ fractions were shown to contain about 3 times more immunoreactive myosin than was observed in SPMs. These results confirmed the enrichment of myosin in SJs relative to SPMs seen in gel binding experiments by visual observations (Fig. 2) and liquid scintillation counting of [125I]antibodies bound to myosin in bands cut from gels (data not shown).

TABLE I Immunoradiometric assay of myosin in subcellular fractions of rat brain The indicated fractions were treated as described for the immunoradiometric assay for myosin in the Methods section. SPM and SJ fractions were from single preparation from rat brain and the PSD fractions were isolated from steer brain according to Cotman et al. 2~. Myelin used was purified according to Norton and Poduslo58. (NH4)aSO4 fractions of immune and preimmune sera were both adsorbed with microtubules and rat red blood cells (see Methods). Values are averages of duplicate determinations. CPM bound specifically equals the counts bound when antisera raised against myosin were used minus the controls. The controls were tubes carried through in the same manner with the same dilution of preimmune sera replacing the antisera to myosin. Subcelhdar fraction

Primary antibody dilution

CPM bound specifically

Binding relative to SPM

SPM

1 : 1 :

100 200 1 : 100 1 : 200 1 : 100 1 : 200 1 : 100 1 • 200

11,218 6,589 31,790 16,613 1,307 1,495 --596 --1,238

1.0

SJ PSD Myelin

2.9 0.2 --

84 TABLE II Distribution of exogenous [125I]myosin during subcellular fractionation

Distribution of exogenous [~25I]myosin. Fractions were prepared and assayed as described in the methods. H, original homogenate; P1, 3000g pellet; S1, 3000g supernatant; $2 and P2, supernatant and pellet, respectively, of 14,000 g centrifugation; LS, supernatant after osmotic lysis and centrifugation; myelin, 0.8-1.0 M sucrose interface in SPM gradient. Values in experiment A are for an experiment where 8.0 × 107 cpm (27/~g) [~2~I]myosin was added during homogenization. Values in experiment B are for an experiment where 1.3 × 107 cpm (4.3/~g) [~25I]myosinwas added at the stage of osmotic lysis. Subcellular fraction

mg protein/fraction

CPM {1251]myosin~ fraction ( × 103)

CPM [1251]myosin/#g total protein (%) *

Experiment ,4 : 8 × 107 CPM [1251]myosin addedat homogenization H 397 79,800 P1 120 3,600 SI 272 73,400 $2 122 63,400 P2 92 5,610 LS 31 3,530 Myelin 20 339 SPM 5.8 57 SJ** 0.96 8.1 PSD** 0.10 4.2

201 30 270 520 61 114 17 10 8.3 41

(100) (15) (135) (255) (31) (57) (8.6) (5.0) (4.2) (20)

Experiment B: 1.3 × 107 CPM [1251]myosin added at osmotic lysis P2 80 13,000 LS 25 12,500 Myelin 13 39.0 SPM 5.5 17 SJ** 0.92 2.1 PSD** 0.15 0.62

162 500 3.0 3.1 2.3 4.1

(100) (309) (1.9) (1.9) (1.4) (2.5)

* ~ is 100 time the ratio of the CPM [125I]myosin//~gtotal protein in the fraction under consideration to the CPM [12q]myosin/#g total protein on the homogenate (experiment A) or P2 (experiment B) * * SJ and PSD values were corrected to account for the fact that 6 0 ~ of the SPM were used to make PSD and 40 ~ to make SJ. Values are averages of duplicate determinations. Disposition o f exogenous [125I]myosin during subcellular fractionation

H a v i n g clearly shown the presence o f m y o s i n in synaptic fractions, a d s o r p t i o n experiments were p e r f o r m e d to d e t e r m i n e whether this myosin c o u l d represent myosin which artifactually redistributed during f r a c t i o n a t i o n a n d adventitiously b o u n d to S P M s a n d SJs. The results in Table II are f r o m two experiments where m y o s i n which h a d been purified f r o m rat brains a n d i o d i n a t e d was a d d e d either (experiment A ) during h o m o g e n i z a t i o n or (experiment B) during o s m o t i c lysis o f the crude s y n a p t o s o m a l - m i t o c h o n d r i a l (P2) pellet. The a d d e d m y o s i n p a r t i t i o n e d p r i m a r i l y as a soluble p r o t e i n in these experiments, being enriched in s u p e r n a t a n t fractions (S1, $2, a n d LS) is b o t h cases. This result is consistent with a previous r e p o r t b y H a r t w i g a n d Stosse134, w h o f o u n d t h a t non-muscle ( m a c r o p h a g e ) m y o s i n was soluble in isotonic sucrose solutions. F r o m the h o m o g e n a t e to the S P M fractions, a r e d u c t i o n in a p p a r e n t specific activity ( C P M [125I]myosin/#g total protein) o f a b o u t 20-fold occurred when the m y o s i n was a d d e d during the h o m o g e n i z a t i o n (experiment A). M o r e t h a n 50-fold

85 reduction in apparent specific activity was found comparing the lysed P2 and SPM fractions when [125I]myosin was added during osmotic lysis (experiment B). The apparent specific activity of [125I]myosin in both experiments was lower in SJ fractions than in SPM fractions and higher in PSD fractions than in SJ or SPM fractions. The specific activity of [125I]myosin in the myelin fractions was comparable to their respective SPM fractions in both experiments. The ratio of apparent specific activities in the fraction under consideration to that of the homogenate is shown as a per cent in the final column of Table II. Previous studies21 have used this ratio as a measure of contamination during fractionation. Thus 4.2~o of the myosin in SJs may be a contaminant from soluble myosin in the homogenate. Likewise 1.4 ~ may come from soluble myosin present during osmotic lysis. An alternative approach to calculating contamination yields similar values. Thus if the [125I]myosinadded during homogenization equilibrated with the myosin in brain (0.3 ~15), then the myosin recovered in SJs as a result of adsorption from this pool is calculated from the data in Table II to be 120 ng myosin/952/~g SJ*. Densitometry of Fig. 3a, lanes 1 and 3, gives about 150 ng endogenous myosin/55/~g SJ. Thus, at the most, 4.6~ of junctional myosin could be due to artifactual adsorption during fractionation. Since some of the myosin is not released from compartments where it is occluded or bound, the actual specific activity of myosin accessible to the membranes is higher than this figure. Therefore, the actual adsorption of myosin is probably considerably lower than 4.6 ~ of the total myosin found in SJs. A comparison of the results of adsorption experiments with those obtained from myosin localization in gels (Fig. 2) or by immunoradiometric assay (Table I) clearly shows that the quantities of exogenous [125I]myosin recovered in the various fractions is unrelated to the amounts of endogenous immunoreactive myosin in the same subcellular fractions. For example, while PSDs or myelin display relatively high [125I]myosin apparent specific activity in adsorption experiments compared to SJ or SPM, endogenous myosin is not immunologically detectable in either fraction. Very similar results were obtained when myosin, which had been more mildly iodinated by Enzymobeads (Biorad) in the presence of 0.6 M KCI, was used in the same experiment (data not shown). These results show that there is no selective o~ adventitious adsorption of myosin to synaptic fractions during their isolation.

Tryptic peptide maps To compare molecular proteins of the various types of myosins, tryptic fingerprints were obtained for heavy chains of myosins which had been run on polyacrylamide gels. As seen in Fig. 3b, the apparent molecular weight of the different myosin standards on 8.5-17.5 % gels was quite similar to that of the myosin band in gels of SJ fractions. However, brain (lane 4) and SPM or SJ associated myosin (lanes 2 and 3, respectively) migrated slightly behind the gizzard (lane 5) and skeletal muscle (lane 1)

*

8.1 × 10s CPM mg myosin x 0.003 7.98 × 107 CPM mg protein × 397 mg protein = 120 ng myosin.

86 myosins. On 7-17.5 ~ gels (Fig. 3d) myosin migrated farther and was separated more from a major stained band at Mr = 260,00 in synaptic junctions. Comparisons of the tryptic fingerprints of these different myosins revealed a clearer picture of differences in their respective molecular structures. Fig. 4 shows the tryptic fingerprints of the different myosins. Myosin bands from either rat (Fig. 3a or 3c, lane 1) or steer (Fig. 3a, lane 3) SJ fractions produced very similar fingerprints (Fig. 4a and 4b, respectively). Both of these fingerprints displayed a high degree of similarity to the fingerprint of purified brain myosin (band from Fig. 3a or 3c, lane 2, fingerprint shown in Fig. 4c). In fact, when equal amounts (based on CPMs) of radioiodinated peptides of rat SJ and brain myosin were mapped simultaneously, six of their major autoradiographic spots comigrated (Fig. 4d). Further comparisons show that SJ myosin contains no major autoradiographic spots that are not also present in the fingerprint of brain myosin. This observation suggests that the myosin-containing bands in gels of SJ fractions contain predominantly brain myosin polypeptides. Also shown in Fig. 4 are the fingerprints from gizzard (Fig. 4e) and skeletal muscle (Fig. 4t")myosin. Comparisons of these two muscle myosins and brain myosin show that significant differences exist among them with respect to their molecular structures. Moreover, the fingerprints of each myosin type is highly distinct from the fingerprint of the other two.

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-..~myosin 123 Fig. 3. Coomassie blue staining profiles of purified myosin standards, synaptic plasma membrane (SPM) and synaptic junction (SJ) fractions, a: 8.5-17.5 ~ exponential gradient gel. Lane 1, rat SJ (55 pg); lane 2, rat brain purified myosin (2.5/~g); lane 3, bovine SJ (55 Fg). b: 8.5-17.5 ~ exponential gradient gel. Lane 1, skeletal myosin (2 ~g) ÷ gizzard filamin (0.5/~g); lane 2, bovine SPMs (55/~g), lane 3, bovine SJs (55 Fg); lane 4, rat brain myosin (3 Fg); lane 5, gizzard myosin (2 #g) ÷ gizzard filamin (0.5/~g). Small unlabeled arrows in lanes 2 and 3 indicate myosin heavy chain, c: expanded view of the high molecular weight region (160-320 kdaltons) of lanes 1 and 2 of a. d : expanded view of high molecular weight region of a 7-17.5 ~ exponential gradient gel; lanes 1 and 2 each contain 55/~g and are from two independent preparations of bovine SJs.

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Fig. 4. Two-dimensional tryptic fingerprints of radioiodinated myosin heavy chain peptides, a: SJ myosin (rat preparation 164, Fig. 3a and 3c, lane 1). b: SJ myosin (bovine preparation 12, Fig. 3a, lane 3). c: purified rat brain myosin (Fig. 3a and 3c, lane 2). d: a mixture, using equivalent amounts of radioactivity, of tryptic peptides from samples a and c. e: purified gizzard myosin, f: purified skeletal myosin (from preparation shown in Fig. 3b).

Tryptic fingerprint analyses were also carried out with SJ associated actin and actin standards purified from rat brain, skeletal muscle and chicken gizzard. The fingerprints of various actin species show that SJ associated actin (Fig. 5c) is indistinguishable from cytoplasmic actin purified from rat brain (Fig. 5a and see mixing experiment in Fig. 5b). The peptide fingerprints of cytoplasmic actin purified from chick brain was indistinguishable from the fingerprint of either rat brain actin (data not shown). Furthermore, in the fingerprints of various actin species, there exist discrete autoradiographic spots that allow brain, gizzard (Fig. 5d) and skeletal muscle (Fig. 5e) actins to be easily distinguished from each other (see spots designated with asterisks and arrows in Fig. 5).

88

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b

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B Fig. 5. Two-dimensional tryptic fingerprints of radiolabelled actin peptides, a: purified rat brain actin. b: a mixture, using equivalent amounts of radioactivity, of tryptic peptides from samples a and c. c: SJ actin (rat preparation 164, Fig. 3a and 3c, lane 1). d: purified gizzard actin, e: purified skeletal actin. Asterisks in respective fingerprints (panels a and d) denote peptides which appeared distinct between rat brain and gizzard actins following mixing experiments. Likewise arrows (panels b and e) denote peptides which appeared distinct between rat brain and skeletal actins following mixing experiments. Actins from chicken gizzard and mouse skeletal muscle were purified by the method of Spudich and Watt r°.

DISCUSSION

This work provides the first clear evidence for the presence of myosin in synaptic junctions. SJs are enriched in immunoreactive myosin relative to synaptic plasma membranes, and immunoreactive myosin is not found in postsynaptic densities. The association of myosin with synaptic junctions is unlikely to be a result of artifactual redistribution of soluble myosin as shown by studies on recovery of exogenous [125I]myosin. Brain and SJ myosin heavy chains yield peptide maps which are indistinguishable from each other, but which clearly differ from peptide maps of smooth and skeletal muscle myosin heavy chains. In addition, the peptide maps of SJ actin are different from those of smooth or skeletal muscle actins. Thus synaptic junctions have actin and myosin, and these proteins differ from their counterparts in muscle. The antibody to chicken gizzard myosin used here reacts with brain and SJ myosin but shows only very weak reaction with skeletal muscle myosin. Other authors have noted a lack of cross-reaction between striated and smooth muscle myosins 7,s,a3. Also, other authors have produced antibodies to chicken gizzard myosin which react with non-muscle myosins3a,54, a3, including neuronal myosin 86,77. While smooth

89 muscle and non-muscle myosins are antigenically related, the peptide maps of these myosins and skeletal muscle myosins show that all three myosins possess some clear differences in structure. Burridge and Bray15 have also shown distinct differences in the one dimensional electrophoretic pattern of peptides obtained after partial proteolytic cleavage or cyanylation of smooth, skeletal and brain myosins. SJ and SPM fractions isolated from rat, bovine, or chick brains contain bands which approximately comigrate with the heavy chains of skeletal muscle, gizzard and brain myosins, and react specifically with antibodies against gizzard myosin heavy chains. The peptide fingerprints of the immunoreactive band in SJ fractions and the fingerprints of purified brain myosin heavy chain are indistinguishable. However, the peptide fingerprints of purified brain or SJ myosin heavy chain are considerably different from those of smooth and skeletal muscle myosins. Thus, we conclude that synaptic junctions isolated from several species contain a distinct type of myosin which is closely similar or identical to the predominant myosin which is isolated from brain. Peptide maps of cytoplasmic actin isolated from brain and actin from SJ are indistinguishable. The present peptide maps of these actins distinguish them not only from the a-actin of skeletal muscle but also from the y-actin of smooth muscle. These results can be explained by recent sequence data on mammalian actinsTL Non-muscle actins differ by at least 25 amino acid replacements from skeletal muscle a-actin and the y-forms of smooth muscle actin. Our results indicate that at this level of resolution, SJ actin is quite indistinguishable from cytoplasmic actin isolated from brain. The enrichment of myosin in SJ relative to SPM suggests a preferential localization of myosin at the synaptic region. Since PSDs do not contain detectable amounts of myosin heavy chain, it may be present in structures which are partially or totally solubilized by sarcosinate, e.g. the synaptic junctional membranes or pre- or postsynaptic paramembraneous speeializationse2, 51. Thus, a precise subsynaptic localization of synaptic myosin remains to be demonstrated. Nonetheless, our data show that myosin in mature CNS neurons is associated with synapses. In this location, myosin could effect structural changes such as spine shape changes or movement of material to the synapse. Myosin-mediated shape changes have been shown in intestinal microvilli where the terminal web myosin acts to retract microvillar actin filaments shortening the microvillila,27,sL Synaptic myosin could also effect vesicle translocation by interacting with membrane and vesicle associated actin5. The localization of myosin at CNS synapses supports hypotheses for an active role for a mechanochemical system in synaptic functions. It should be noted that in many motile cells myosin is present in much smaller amounts than actin, and the ratio of myosin to actin in SJs is similar to that in motile cells61. Thus the amount of myosin in SJs is consistent with its possible functional roles at the synapse. The regulation of myosin activity in neurons must be considered in the light of current information on regulation of non-muscle myosins. Smooth muscle and cytoplasmic actin activated myosin ATPase activities are regulated by the state of phosphorylation of the P-light chain2, 25. The state of phosphorylation (and ATPase activity) is controlled in a calcium dependent manner 69, mediated by calmodulin and

90 myosin light chain kinase24,s6. While myosin light chains were not detected in these experiments they may be present as they are not recognized by this antibody. Myosin light chain kinase has been isolated from brain 24. In addition, the calcium dependent regulator protein calmodulin has been identified previously in fractions enriched in PSDs a2,4a and demonstrated by immunocytochemical methods to be associated with the postsynaptie densities at synapses 48,a4. Thus the components required for calcium dependent actomyosin activity appear to be available at the synapse. ABBREVIATIONS SJ SPM PSD SDS PAGE

synaptic junction synaptic plasma membrane postsynaptic density sodium dodecyl sulfate polyacrylamide gel electrophoresis

D1V EDTA TBS PBS

day in vitro ethylenediamine tetraacetate tris-buffered saline phosphate-buffered saline

ACKNOWLEDGEMENTS We would like to t h a n k Susanne Bathgate for her excellent help with the cultures a n d staining, Paul M o n t g o m e r y for peptide mapping, a n d Susanne Bathgate a n d D e b o r a h F r a n k s for their help preparing the manuscript. This work was supported by N I H G r a n t s NS08597 (C.W.C.), NS15554 (P.T.K.), NS 12485 (J.A.B.), a n d a N I H postdoctoral fellowship (R.L.B.). J.A.B. is a recipient of a n R C D A (NS00233).

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