Proc. NatL Acad. Sci. USAVol. 80, pp. 1738-1742, March 1983Neurobiology

Immunocytochemical localization of actin and microtubule-associated protein MAP2 in dendritic spines

(immunocytochemistry/cellular motility/neuronal plasticity/cytoskeleton/synaptic modulation)

ALFREDO CACERES*, MICHAEL R. PAYNEt, LESTER I. BINDERt, AND OSWALD STEWARD*§Departments of *Neurosurgery and tBiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908; and tDepartment of Anatomy, Basic ScienceBuilding, New York Medical College, Valhalla, New York 10595

Communicated by Francis Crick, December 13, 1982

ABSTRACT To determine whether dendritic spines containactin, we evaluated the immunocytochemical localization of actinin the hippocampal formation and cerebral cortex of the rat.Monoclonal hybridoma antibodies were prepared against adult quailbreast muscle actin. The culture supernatant of two cell lines (QABIand QAB2) was examined. Both antibodies bound only actin in crudebrain homogenates, and neither exhibited species specificity.Electron microscopic analyses of sections reacted with QABI re-vealed staining of postsynaptic densities and dendritic microtu-bules but little staining of the cytoplasmic compartment of spines.However, sections reacted with QAB2 exhibited staining at the cy-toplasmic compartment of spines as well as the sites stained byQABI. We also evaluated the immunocytochemical distribution of(3-tubulin and high molecular weight microtubule-associated pro-tein (MAP2) utilizing monoclonal antibodies. MAP2 was found inthe dendritic spine as well as in the parent dendrite. However, 1-tubulin was found only in the postsynaptic density and in the mi-crotubules of the parent dendrite. The combined results indicatethat actin is present in the spine along with MAP2 and that thereis a difference in the actin (or the state of actin) in the spine in com-parison with other neuronal compartments.

Dendritic spines are wineglass- or mushroom-shaped protru-sions from dendrites that represent the principal site of ter-mination of excitatory afferents on many vertebrate neurons,particularly in cortical regions. Although there have been nu-merous speculations about the functional significance of spines(1), a recurring theme is that spines are involved in modifyingsynaptic efficacy and thus perhaps in the storage of experientialinformation (2). This hypothesis derives from the observationsthat spines are sensitive to both the integrity and functional ac-tivity of their accompanying synapse (3, 4). It has long been rec-ognized that spines in various brain regions can be modified byexperience, presumably in response to variations in the patternsof activity over the presynaptic fibers (2-4). Furthermore, briefperiods of electrical activity that induce changes in synaptic ef-ficacy (long-term potentiation) also result in dramatic changesin spine shape (5, 6). Because the size and shape of a spine wouldbe expected to result in severe attenuation of current flow be-tween the synapse and the parent dendrite during a transientdepolarization (7), a change in spine shape could bring aboutchanges in synaptic efficacy as a consequence of changes in cur-rent flow during synaptic activation.The above-mentioned studies and many others have con-

tributed to the emerging hypothesis that spines are dynamicstructures that are capable of very rapid structural modifica-tions, which, in turn, alter synaptic efficacy (2, 4-9). How theseshape changes are brought about is of considerable interest. Onesuggestion has been that spines contain a contractile apparatus

similar to that which operates in other nonmuscle cells to reg-ulate cell form, specifically a "cytomusculature" comprised ofproteins similar or identical to actin, myosin, etc. (2). A meansto test this hypothesis has become available in the form of mono-clonal antibodies to contractile proteins which can be localizedimmunocytochemically. The present study describes our initialexploration of the potential cytomusculature of the dendriticspine with antibodies to actin with additional observations onmicrotubule-associated protein MAP2 and f3-tubulin. Some ofthese results have been reported earlier (10).

MATERIALS AND METHODSPreparation of Antiactin Hybridoma Antibodies. Actin was

purified from adult Japanese quail breast muscle by using themethod of Spudich and Watt (11) and antibodies were preparedas described (12). Each antiactin hybridoma cell line was re-cloned four times by limited dilution over a 2- to 3-month pe-riod. Large quantities of antibody from each hybridoma cell linewere obtained as culture supernatant and were purified by re-peated centrifugation to remove cellular debris, two cycles ofammonium sulfate fractionation (0-50%), and then protein A-Sepharose (Pharmacia) affinity chromatography (13).The binding specificity of two antiactin hybridoma antibod-

ies (QAB1 and QAB2, both of which are IgG) was initially ex-amined by the solid-phase enzyme-linked immunoassay (12) asbeing positive for quail actin and having no crossreactivity topurified quail myosin, quail muscle tropomvosin, and bovinebrain tubulin. Crossreactivity to other proteins was examinedby reacting the antiactin with electrophoretic blots (14) of bo-vine cerebellum (see legend to Fig. 1).

Immunocytochemistry. Adult male Sprague-Dawley-de-rived rats were perfused with 4% paraformaldehyde/0. 5% glu-taraldehyde/0.01% tannic acid (Mallinckrodt) in 0.1 M phos-phate buffer (pH 6.8). Tannic acid was included because it appearsto stabilize actin against destruction by osmium (15). Vibratomesections of the hippocampal formation and the cerebral cortexwere collected in phosphate-buffered saline and stained ac-cording to the three-layered method of Sternberger (16). Briefly,the sections were preincubated in 1% normal rabbit serum andthen were incubated in the primary antibody for 24 hr at a finalprimary antibody concentration of 40, 20, or 10 ,ug/ml in 1%normal rabbit serum. After the 24-hr incubation, the sectionswere washed for 3 hr in phosphate-buffered saline (pH 7.2) andthen were incubated for 3 hr in rabbit anti-mouse IgG (Stern-berger-Meyer, Jarrettsville, MD) at a dilution of 1:40. The sec-tions then were washed in phosphate-buffered saline (3 hr) andincubated for 1.5 hr in mouse peroxidase-antiperoxidase diluted

Abbreviations: PSD, postsynaptic density; MAP, microtubule-associ-ated protein; QAB, quail breast muscle actin antibody.§ To whom reprint requests and other correspondence should be ad-dressed.

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

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1:40. The sections then were washed again for 1 hr and incu-bated in 3,3'-diaminobenzidine and H202 as described (17).Stained sections were osmicated and embedded in Epon-Ar-aldite according to standard procedures, and ultrathin sectionswere observed without additional staining on a Hitachi HU12electron microscope.We also evaluated the distribution of two other cytoskeletal

proteins (,-tubulin and the high molecular weight MAP2) usingmonoclonal hybridoma antibodies for those proteins (12). All ofthe hybridoma antibodies were cell culture supernatants thatwere purified by protein A-Sepharose affinity chromatographyand used in immunocytochemical preparations at dilutions of1:80, 1:200, and 1:300.

Controls for all of the immunocytochemical procedures in-volved absorption of the antibodies with a molar excess of thespecific antigen or incubation with nonimmune serum. The im-munocytochemical preparations were compared with conven-tional electron microscopic preparations and with preparationsfrom animals perfused transcardially with 2% paraformalde-hyde/2% glutaraldehyde/0.2% tannic acid in 0.1 M phosphatebuffer.

RESULTSAs illustrated in Fig. 1, of the large numbers of proteins thatcould be visualized on 5-20% gradient NaDodSO4 electropho-retic gels of bovine cerebellum (Fig. 1, lane'B), only the actinband, as determined by migration comparison with purified ac-tin, was recognized by either antiactin hybridoma antibody (Fig.1, lanes C and D).

The ultrastructural appearance of dendritic spines has beendescribed in numerous previous works (see ref. 18), and ex-amples of typical spines from the hippocampus are illustratedin Fig. 2 A and B. Typically, spines extend at right angles fromthe main dendrite, and each spine is contacted by a presynapticelement (usually only one per spine). The region of synaptic

A B C D

FIG. 1. Specific binding of the antiactin hybridoma antibodiesQAB1 and QAB2 to the proteins of bovine brain. Lane A, purified quailbreast muscle actin electrophoresed on a 5-20% gradient NaDodSO4/polyacrylamide gel, Lane B, fresh bovine brain solubilized-by homog-enization in 1*a NaDodS04/0.1% 2-mercaptoethanol and. heating inboiling water for 3 min. The preparation was centrifuged to remove in-soluble material. The supernatant showsa large numberof protein bandswith electrophoresis on 5-20% gradient gels, as revealed by Coomassieblue staining. Lanes C and D, unstained gradient gels were electro-phoretically blotted onto nitrocellulose, incubated with either hybri-.doma antibody QAB1 (lane C) or QAB2 (lane D),.and then incubatedwith-horseradish peroxidase-conjugated goat F(ab')2 anti-mouse IgG(Tago, Burlingame, CA) by using o-dianisidine HCl as the substrate(14).

contact is marked by a prominent membrane specialization, thepostsynaptic density (PSD). Unlike the parent. dendrites thatcontain longitudinally oriented arrays of microtubules, numer-ous mitochondria, and vacuoles and cisterns of various types,the spine contains few organelles except for the so-called spineapparatus, a membranous structure found frequently in the spineneck (18). In addition, it is sometimes possible to discern fila-mentous structures in the spine of approximately the size of mi-crofilaments (see Fig. 2A). -In some preparations the filamentsappear especially prominent within the spine neck, where they.assume a parallel orientation. Spines also contain a flocculentmaterial which makes their cytoplasm distinct from that of theparent dendrite (18). The difference between the cytoplasm ofthe spine and the parent dendrite is emphasizedin material pre-pared for electron microscopy when tannic acid has been in-cluded in the perfusion solution (see Fig. 2B).

As illustrated in Fig. 2 C-E, immunocytochemical prepara-tions of central nervous system material with antibodies to actinclearly reveal that the molecule is present within the spine, aswell as in other neuronal compartments. In the case of QAB2,strong immunoreactivity was found within the spine neck andhead, and the staining in the PSD was particularly prominent(Fig. 2 E andF). Reaction product also was found in associationwith the plasma. membrane and with cisternal specializationswithin the spine neck, which probably represent spine apparati(Fig. 3A). Interestingly, although the other antibody to actin(QAB1) stained the dendritic microtubules extensively along withthe PSI), little immunoreactivity was found within the spinecytoplasm (Fig. 3 B and C).

Because actin is prominent in the spine and because the cy-*toplasm of the spine is relatively simple in comparison to themain dendrite, the question arose whether actin might be theprincipal cytoskeletal protein within the spine. To evaluate thisquestion, immunocytochemical studies were carried out withmonoclonal.antibodies to ,(3tubulin and MAP2. As illustrated inFig. 3D, immunoreactivity for MAP2 was present within thecytoplasm of the dendritic spine as well as in association withdendritic microtubules. In addition, the PSD was stained prom-inently. However, in the case of /3-tubulin the spine head andneck contained little reaction product, whereas dendritic mi-crotubules and PSDs were heavily stained (Fig. 3E). Thus, al-though actin is not the only cytoskeletal protein contained withinthe spine, there is a differential distribution of cytoskeletal pro-teins in the spine and the parent dendrite.As illustrated in Fig. 3 F and G, immunoabsorption of the

monoclonal antibodies against actin with a molar excess of thespecific antigen or incubation with nonimmune serum yieldedimmunocytochemical preparations that exhibited essentially nostaining.

DISCUSSIONThe present study demonstrates actin within the cytoplasmiccompartment of the dendritic spine as.well as in dendritic shaftsand in the PSD. The presence-of actin in dendrites is not sur-prising in view of previous biochemical evidence -that actin is amajor component of the so-called slow component b of axo-plasmic transport (19). Furthermore, LeBeux and Willemot (20)have described microfilaments in neuronal dendrites which canbe decorated with heavy meromyosin as well as within elementswhich presumably represent dendritic spines. The concentra-tion of actin immunoreactivity around dendritic microtubuleswould not necessarily have been predicted from previous stud-ies, but it is not surprising in view of the emerging evidence ofvery close associations between actin and the so-called MAPs(21-23). Furthermore, the presence of actin in the PSD also isin. keeping with biochemical evidence (24-26).

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However, biochemical studies could not detect actin withinthe cytoplasmic compartment of the spine itself, because iso-lated synaptosomes or other isolated synaptic constituents, suchas synaptic plasma membrane fractions, synaptic junctions, andPSDs, do not include the cytoplasmic components of the post-synaptic member of the synapse. Thus, the present results pro-

vide information that can be derived only from immunocyto-

FIG. 2. (A and B) Electron micro-A- . m- I-graphs from the ventral leaf of the

dentate gyrus showing filamentousmaterial (arrows) in spines from con-ventionally prepared tissue (A) and thesharp demarcation between the shaftand the spine in material perfused withtannic acid in the fixative (B). (Cali-bration bar = 0.25 /Am.) (C-F) Elec-tron micrographs from a section ofdentate gymrs reacted with QAB2 (20jg/ml). (C) Note the staining in thespine neck (arrows), head, PSD, and themain shaft. (Calibration bar = 0.25pn.) (D) Note the intense staining of

T the PSD and the fibrillar appearanceofthe reaction product in the spine neck(arrows). (Calibration bar = 0.1 pm.)(E) Electron micrograph showing theappearance of the reaction product in

I uI_ the spine head and PSD. (Calibrationbar = 0.1 ,Am ) (F) Same as in E. (Cal-

;^_.r_^ ibration bar = 0.1 pum.) T, presynapticterminal; S, spine; Den, dendrite.

chemistry and demonstrate that at least one of the molecularcomponents that might comprise a cytomusculature is presentin the dendritic spine itself. This conclusion is consistent withother studies that have appeared since the present work was un-dertaken, which include the demonstration of actin filamentswithin dendritic spines by decoration with heavy meromyosin(27) and by ferritin-labeled antibody studies and heavy mero-

Proc. Nad Acad. Sci. USA 80 (1983)

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FIG. 3. (A) Electron micrographfroma section of rat cerebral cortex re-acted with QAB2 (10 pg/ml) showingthe reaction product in association withthe spine apparatus (sa). (Calibrationbar = 0.25 pm.) (B and C) Electron mi-crographs from the dentate gyrus re-acted with QAB1 (40 jig/ml). Note thelight staining of the spine cytoplasmand the presence of reaction productin the PSD. (Calibration bar = 0.25pm.) (D) Electron micrograph from asection reacted with a monoclonal an-tibody against MAP2 (AP9). Note theintense staining of the shaft, spineneck, and head. (Calibration bar = 0.25jsm.) (E) A section reacted with amonoclonal antibody against P-tubu-lin. Note the intense staining of thedendritic shaft but the absence ofreaction product in the spine cyto-plasm(s). However, the PSD is stained.(Calibrationbar = 0.25 pm.) (Fand G)Electron micrographs from two sec-tionsreacted with QAB2 (F) and QAB1(G) previously absorbed with a 5M ex-cess of pure actin. (Calibration bar =0.25 pum.) T, presynaptic terminal; Den,dendrite; S, spine.

myosin labeling of actin in the spine-like appendages on red nu-cleus neurons (28).The presence of actin in spines assumes special importance

when considered in the light of the evidence regarding the cel-lular localization of the other two cytoskeletal proteins that wereevaluated (tubulin and MAP2) and the differential staining bythe two different actin antibodies. The demonstration of actinimmunoreactivity in the spine would be trivial if the cytoplasmand cytoskeleton of the spine were simply an extension of thecytoplasm and cytoskeleton of the parent dendrite. It is highlyunlikely that the entire neuronal dendrite is a motile structureand, if all of the proteins that would constitute a cytomuscu-lature were uniformly distributed, this would imply that evi-dence regarding the localization of these proteins is not likelyto imply function. However, the differences in the distributionof cytoskeletal proteins between the spine and the parent den-drite suggest that localization of certain proteins may indicate

a microspecialization of the spine cytoskeleton or cytomuscu-lature, or both.A particularly interesting, but currently enigmatic, clue is the

differential staining for the two actin antibodies. Both antibod-ies recognize only actin in brain homogenates but yield com-pletely different staining patterns in situ. The differences are

probably not related to a higher titer of QAB2, because even athigh concentrations the staining of the spines with QAB1 was

not prominent, although staining of the dendritic microtubuleswas quite dramatic. It is conceivable that the epitope for QAB1is more sensitive to fixation, histological preparation, etc., insome locations, whereas in other neuronal compartments theepitope is protected as a consequence of some interaction withother neuronal components. For example, perhaps actin inter-action with MAPs on microtubules, which has been described(21-23), protects the QAB1 epitope, whereas in the spine itself,the presence of MAP2 without microtubules does not protect

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1742 Neurobiology: Caceres et al.

the epitope. Alternatively, it may be that QAB1 recognizes anepitope that can be concealed in some neuronal compartments.In this case, one would predict that the molecular arrangementor configuration of actin within the shaft or in the PSD is dif-ferent from that in the spine cytoplasm. Another possibility isthat these two hybridoma antibodies are distinguishing betweentwo different actins (isoforms) present in the spine. Though thedifferential staining for the two antibodies is likely to eventuallyprovide clues about the supramolecular organization of actin indifferent sites, pertinent conclusions can be drawn only whenmore is known about where these two antibodies react on theactin molecule.

It has been suggested that if the cytoplasm of the spine-isenriched in actin in comparison to the parent dendrite, then thisdistribution would add a new dimension to our view of dendriticspines (2). Our results did not indicate a preferential localizationof actin in the spine but indicated that actin was present in bothspines and the parent dendrite. However, our results did dem-onstrate that, although actin is in both the spine and the parentdendrite, one type of tubulin is not. We, also have evaluated thedistribution of tubulin using animal antisera and three mono-clonal antibodies against 8-tubulin, and none stained the spinecytoplasm (unpublished data). However, all did stain the PSD,which is consistent with previous evidence (24; 29). It is con-ceivable that our preparative procedure is not adequate to pre-serve tubulin that is not incorporated in microtubules. Alter-natively, the absence of tubulin in the spine proper may be realand, in fact, may be what makes the cytoplasm of the spine dis-tinct from the cytoplasm of the parent dendrite. Specifically,actin is found in nonmuscle cells in various forms, and the reg-ulation of the state of actin may be carried out byother proteins.Some of these proteins are gelation factors- because they col-laborate in gelating actin in solution in a calcium-dependentfashion. It is interesting to note that MAPs also are capable ofinteracting with actin to form gels (21-23). Thus, one hypothesisis that MAP2 in the dendritic spine regulates the gel-sol tran-sitions of the actin that is present there. These interactions areconceivable because the spine contains low concentrations of atleast one type of tubulin (,&tubulin), which has a much higheraffinity for MAP2 than actin, and thus would interfere with MAP-actin interaction. Because actin networks are isotropic gels, theycould well be responsible for the characteristic shape of thedendritic spine. It is probable that any such gel-sol transitionswould be highly sensitive to calcium concentrations within thespine, which, in turn, would be regulated by functional activity.

All of these speculations are, of course, very preliminary atthe present stage of our knowledge, and further developmentof the concept of the spine as a structure speoialized for motilitywill require studies of-the localization of other contractile pro-teins and detailed studies of the supramolecular organization ofthese proteins within the spine.Note Added in Proof. Since the present study was.accepted for pub-lication, a study has appeared which also localizes actin in dendritic spinesimmunocytochemically (30). Although the study by Matus et aL and thepresent study agree that actin is present in the spine, other aspects ofthe results differ. Matus et al report little staining of the dendrite proper,whereas our immunocytochemical preparations with both antibodiesyield heavier staining of the dendrite than of the spine cytoplasm. In-deed, the staining pattern described by Matus et aL is virtually a reverseimage of the pattern produced in the present study by QAB2, exceptthat the PSD is stained by all of the antibodies. It seems likely that actin

is present as several isoforms or states, which are differentially localizedwithin the neuron and which yield quite different immunochemicalstaining patterns depending on the antibodies utilized. Therefore, spe-cial caution is required for interpreting a lack of staining in particularcellular compartments.

The hybridoma antibodies used in these experiments were preparedby M.R.P. while a fellow of the Muscular Dystrophy Association. Wethank Dr. Irwin Konigsberg of the Department of Biology, Universityof Virginia, for his hospitality and acknowledge support from the Na-tional Institutes of Health and Muscular Dystrophy Association grantsto him; F. Lee Snavely for his excellent technical assistance with theelectron microscopic immunocytochemistry, photography, and figurepreparation; and Sharon Vinsant and Patricia Palmer for their technicalassistance with the conventional electron microscopy and tannic acidprocedures, respectively. Special thanks also are given to William B.Levy for his very helpful advice in the course of these studies. This re-search was supported by National Institutes of Health Grant NS12333(to O. S.) and Fogarty International Fellowship F5TW 02910A (to A. C.).O.S. is the recipient of Research Career Development Award NS00325.

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