STUDIES ON THE ORGANIZATION AND LOCALIZATION OF AND IN NEURONS

EDWARD R. KUCZMARSKI and JOEL L. ROSENBAUM

From the Department of Biology, Yale University, New Haven, Connecticut 06520

ABSTRACT The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg ++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the body, neurites, and microspikes ot~ both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle .

KEY WORDS neurons microspikes poisons stops the transport and causes a piling up myosin localization of material proximal to the block (44). A retro- grade transport has also been described in which Neurons exhibit a variety of motile phenomena, material moves from the axon tip toward the cell one of the most dramatic examples being nerve body (35). Some form of motility may also be outgrowth. In experiments which proved the neu- required for transmitter release at the synapse (4). ron theory, Harrison (28) first described the rapid These examples demonstrate that several nerve movement of nerve cells in tissue culture where functions involve motility. The mechanisms re- growth cones of individual nerve cells advanced in sponsible for these movements, however, are an amoeboid fashion, drawing out long processes completely unknown. In thin sections of neurons, from the stationary cell body. A second neuronal the most obvious structural components are the process characterized by motility is axoplasmic 25-nm neurotubules () and the 10- transport. Weiss (58) originally reported that mi- nm neurofilaments, and it has been suggested that tochondria and other piled up proximal these components are involved in axoplasmic to ligated regions of axons, and that upon removal transport (35, 44). Microfilaments have rarely of the ligature the bulge of axoplasm moved been seen in neurons in vivo, although biochemi- toward the axon terminal at a rate of 1 mm/d. cal evidence indicates that nerve cells contain This result has been confirmed and, in addition, substantial amounts of cytoplasmic actin (6, 22, sophisticated radio-tracer experiments have iden- 34, 42). Cytoplasmic myosin has also been iso- tified a second transport system with rates of up to lated from nerve tissue and characterized, al- 400 mm/d (35). Energy for axoplasmic transport though precise location has not been determined must be provided by each segment of the neurite, (4, 11, 34). It is likely that these contractile since local anoxia or treatment with metabolic proteins, perhaps in conjunction with microtu-

356 J. C~LL BIOLOGY The Rockefeller University Press 0021-9525/79/02/0356-1651.00 Volume 80 February 1979 356-371 bules and neurofilaments, are responsible for the Isolation and Fluorescent Labeling of $1 motile nerve functions described above. Myosin subfragment S~, prepared from rabbit back To construct models which explain how contrac- muscle myosin by the method of Margossian and Lowey tile proteins interact to cause movements, it will (39), was labeled with fluorescein by the protected be necessary to determine the organization and method of Sanger (50) with the following modifications. location of these proteins in the nerve cell. Al- Instead of I00 pig of fluorescein isothiocyanate per nag though long, straight microfilaments have not of protein, 30 p.g/mg were used, and additional purifi- been seen in central or peripheral nerve tissue cation of labeled S~ was achieved by ammonium sulfate prepared for electron microscopy by standard fractionation (1.6-2.2 M saturated ammonium sulfate techniques, LeBeaux and Willemot (37, 38) have fraction). The purified fluorescent S~ (F-S~) was stored in 25% glycerol at -18~ To prevent protein aggrega- recently shown the presence of heavy mero- tion and to reduce nonspecific staining, bovine serum myosin (HMM)-decoratable filaments in pieces of albumin (1 mg/ml) was added to the F-S~ and the glycerinated rat caudate nucleus and substantia solution was clarified by centrifugation (35,000 g, 60 nigra. It is not known whether the glycerol-HMM min) before cell staining. treatment acted to induce the polymerization of actin or to protect existing filaments from disrup- Isolation and Labeling of tive actions of fixation and dehydration. Dorsal Myosin Antibodies root ganglion (DRG) cells grown in tissue culture Antibody was raised in rabbits as previously described are also known to contain large amounts of actin (34), and immunoglobulin G (IgG) was isolated from (22), although it has been difficult to ultrastruc- serum by ammonium sulfate fractionation (32) and turally identify actin filaments in these cells (13, diethylaminoethyl (DEAE) chromatography (57). Puri- 62). In mouse neuroblastoma cells, however, fied IgG was reacted with fluorescein isothiocyanate (10 large numbers of microfilaments have been seen /~g/mg protein) at pH 9.5 for 8 h at 4~ After labeling, in cells prepared for conventional electron micros- the preparation was desalted on Sephadex (3-25 and copy (48) and, in addition, these filaments have then chromatographed on a DEAE cellulose column. been decorated with HMM (12, 15). Myosin in Labeled IgG fractions obtained by step elution with nerve cells has not been localized directly, but, increasing salt concentrations (14) were concentrated by using cell fractionation techniques, Berl (4) has dialysis against Aquacide II (Calbiochem, San Diego, Calif.) and stored sterilely at 4~ demonstrated that a substantial portion of the brain myosin is associated with the synaptic vesicle Immunoelectrophoresis fraction. This paper describes the arrangement of actin Immunoelectrophoresis was carded out in 1% agarose containing 20 mM sodium pyrophosphate buffer, pH 8.5 filaments in neuroblastoma and DRG nerve cells (24). A crude extract was prepared by homogenizing as determined by a variety of ultrastructural tech- chick brains in an equal volume (wt/vol) of 0.6 M KC1, niques, as well as by a probe specific for actin 20 mM sodium pyrophosphate, pH 7.0, in a glass-Teflon (fluorescent $1), The localization of myosin in homogenizer. This homogenate was clarified by centrif- DRG cells was determined by use of fluorescently ugation (100,000 g for 60 min at 4~ and the supernate labeled antibody specific for brain myosin. was stored frozen in aliquots. Brain myosin was purified as previously described (34). 10 t~l of the purified brain myosin (0.5 mg/ml) or crude homogenate (9.0 mg/ml) MATERIALS AND METHODS were electrophoresed in the first dimension for 1.5 h at 15 mA. The agarose gel was then removed from the Tissue Culture apparatus, and 50/zl of antiserum was placed in a trough Mouse neuroblastoma cells were grown at 37~C in a adjacent to the electrophoresed protein and allowed to 5% COs atmosphere, according to standard procedures diffuse for 48 h at room temperature in a moist atmos- (48). Primary cultures of chicken DRG nerve cells were phere. Finally, gels were washed overnight in 20 mM prepared as described by Okun (45), and cardiac celt sodium pyrophosphate, washed briefly in water, and cultures were prepared by trypsinizing pieces of 10-d then stained for protein by the method of Fairbanks et chick embryo heart for 20 min at 37~ and using the al. (20). DRG culturing solutions. Whole-Mount Transmission Preparation Electron Microscopy (WMTEM) Myofibrils were prepared from adult chicken pectoral Electron microscope grids were prepared for tissue muscle by the method of Szent-Gyrrgyi (52). culture in the following manner: A clean glass slide was

KUCZMARSrd AND ROSENBAU~ Localizationof Nerve Actin and Myosin ~7 dipped into a 0.5% solution of Formvar in ethylene Cells prepared by one of the above-described methods dichloride and allowed to air dry. With a clean razor were stained with the fluorescent probe for 30 min at blade, the film surface was scored to give three 22-mm room temperature in a humidified chamber. Unbound squares which were then gently floated onto a clean reagent was removed by washing in three changes of water surface. Several acetone-cleaned 200-mesh stain- PBS over a 30-min period. The cells were rinsed briefly less steel grids were placed on each square, and the in distilled H20, and then mounted in 50% glycerol combination was picked up from above with a glass containing 10 mM K+-carbonate buffer, pH 8.5, and cover slip. A fairly heavy layer of carbon was then examined with a Zeiss microscope equipped with epiflu- evaporated onto the Formvar-covered grids, and, after orescence or darkfield fluorescence optics. Unfixed my- glow discharge treatment to render the grids hydrophilic, ofibrils were stained by suspension in the fluorescent they were placed in a polylysine solution (1 mg/ml in reagent and washed by low-speed centrifugations. Pho- H20). After 3-4 h, the coverslip with the grids attached tomicrographs were taken on Tri-X film (Kodak) and was washed in several changes of distilled water, trans- development was carried out for 11 min in Acu-1 ferred to a 35 x 10 mm plastic petri dish, and sterilized developer (Acufine, Inc., Chicago, Ill.). for 30 min under an ultraviolet light source (Sylvania To demonstrate specificity of fluorescent staining, germicidal no. G15T8, Sylvania Chemical Co., Mill- several control experiments were performed. Staining of burn, N. J.), at a distance of 25 cm. Just before plating cells treated with F-St could be reversed by washing the cells, the petri dishes were rinsed once with sterile cells with buffer containing 5 mM ATP, which served to culture media. dissociate the acto-F-Sl complex. Reversal of staining The cells, which were growing on electron microscope would also have been obtained, however, if the ATP grids, were prepared for WMTEM as described by solubilized the cellular actin. This was not the case since Buckley and Porter (10). Cells were fLxed for 10 min at cells pretreated with 5 mM ATP retained their phase- room temperature in 2 % glutaraldehyde made up in 0.1 dense stress fibers and stained normally with F-S1 after M KCI, l mM CaC12, 2 mM MgC12, 10 mM piperazine- removal of ATP. N-N'-bis[2-ethane sulfonic acid] (PIPES) buffer, pH 6.9 The following controls established specificity of the (PMCK buffer). The cells were rinsed in buffer, post- fluorescent antibody staining: (a) Cells treated with fixed in 1% osmium tetroxide (in PMCK buffer) for 10 fluorescently labeled nonimmune serum did not stain. rain, stained with uranyl acetate, dehydrated, and critical (b) Cells which were pre-incubated with unlabeled im- point dried exactly as described by Buckley and Porter mune serum did not stain, while pre-incubation with (10). After coating with a layer of carbon,, specimens unlabeled pre-immune serum had no effect on the were examined in a Philips 201 electron microscope staining. For these studies the cells were incubated in operated at 80 or 100 KV. unlabeled pre-immune or immune serum (diluted 1:2 with PBS) for 30 rain, washed in buffer for 5 min, and Studies on Detergent-Treated Cells then reacted with the fluorescein-labeled immune IgG (the labeled IgG contained either immune or pre-im- Cells grown on stainless steel, electron microscope mune serum in a ratio of one part serum to three parts grids as described above were gently opened by the method of Clarke et al. (16). The electron microscope labeled IgG). After a 30-min incubation, the preparation was washed and mounted as described above. (c) Anti- grid was removed from the glass coverslip and held in a body which had been adsorbed with antigen failed to pair of fine forceps while a stream of PMCK buffer stain the cells. Brain or breast myosin (0.2 mg) in 0.6 M containing 0.01% Triton X-100 was aimed at the cells KCL was added to the fluorescently labeled antiserum through a drawn-out Pasteur pipet. These detergent- (0.1 mg) and then dialyzed overnight against PBS. The treated cells were then either fixed and prepared for myosin aggregated under these conditions and could be WMTEM as described above or immediately negatively sedimented by centrifugation, thereby removing the stained with 1% aqueous uranyl acetate. Cells to be specific antibody. The supernate was then tested for its stained with fluorescent antibody were rinsed once with ability to stain cells or myofibrils. warm phosphate-buffered saline (PBS, 0.15 M NaC1, 10 mM phosphate buffer, pH 7.0) and then fixed for 10 rain at room temperature in PBS containing 4 % formalin RESULTS and 0.15 M sucrose. The sucrose was required to prevent blebbing of the nerve cells during fLxation. Fixed cells Actin Localization were rinsed once in PBS, placed into 100% acetone at WMTEM ANALYSIS OF 0~ for 10 min, and then air dried. Cells prepared in this ORGANIZATION manner could be successfully stored desiccated at -18~ for several days. Cells to be reacted with F-St were Fibroblasts examined by the whole-mount either treated with 0~ acetone for 1 min and air dried, method contained mitochondria, 25-nm microtu- or exposed to 0.01% Triton X-100 in PMCK buffer for bules and 6- to 7-nm microfilaments which paral- 30-60 s and then rinsed in detergent-free buffer. leled the long axis of the cell, as reported by

358 THE JOURNAL OF " VOLUME 80, 1979 Buckley and Porter (10). The neurites and micro- microtubules on the basis of size and shape. spikes of mouse neuroblastoma cells contained an Bundles of 6- to 7-nm microfilaments were seen abundance of filamentous structures. A low mag- to emerge from this -containing cen- nification view of a neurite with many microspikes tral area (arrows, Fig. 1 a). At higher magnifica- is shown in Fig. 1 a. The darkly staining central tion, microfilaments in the fine neuroblastoma portion of the neurite contained structures which microspikes were wavy and appeared to form a at higher magnification could be identified as meshwork (Fig. 1 b-d). Microfilaments were also

Fmu~ 1 Micrographs of mouse neuroblastoma cells prepared for WMTEM. (a) Neurite containing many microspikes (s). Microfilament bundles (mfb) extend from the center of the neurite out to the tips of individual microspikes. (b) Microfilament bundles extend from microtubules (mt) at the center of the neurite. (c and d) additional examples of microspikes. Note the wavy appearance of the microfilm-nents. Bars, 0.5 tan. (a) x 10,600. (b) x 25,000. (c) x 90,000. (d) x 52,000.

Kue~ AND ROS~h'a^UM Localization of Nerve Actin and Myosin 359 observed in whole-mount preparations of chicken increase resolution while at the same time reduc- DRG nerve cells. The highly spread growth cone ing the number of manipulations, detergent- contained many criss-crossed microfilaments (Fig. treated cells were immediately negatively stained 2), and groups of microfilaments were seen in the with uranyl acetate and examined with the elec- microspikes (insert, Fig. 2). tron microscope. Detergent-treated, negatively stained chicken fibroblasts contained meshworks OBSERVATIONS OF DETERGENT-TREATED, of criss-crossing "stress fibers" and individual mi- ATTACHED CELLS crofilaments (Fig. 4a). In microspike regions, CRITICAL-POINT DRIED PREPARATIONS. microfilaments joined to form bundles, and these An accelerating voltage of 100 KV was insuffi- bundles in turn fused to form a rodlike structure cient to examine thicker regions of intact critical- in the microspike (Fig. 4 b). point dried cells. To better visualize these internal Fig. 5A shows part of a detergent-treated, structures, therefore, the upper of negatively stained neuroblastoma cell. At low attached cells was gently removed with a stream magnification, it was possible to see filamentous of detergent-containing buffer before fixation and material in the interior of the neurite as well as critical-point drying. Fig. 3a illustrates a neuro- darkly staining microspikes at the periphery. blastoma cell treated in this manner, revealing When examined at higher magnification, individ- large cables composed of microfilaments. As in ual microfilaments could be recognized in these intact cells, the bundles emanated from the central microspikes. Fig. 5 B illustrates a fine microspike part of the neurite and fused to form a tight rod with its membrane partially splayed open, reveal- which filled the microspike. It was also possible to ing a rod composed of tightly packed microfila- discern the individual microfilaments which joined ments. Figs. 5 C-E depict microspikes with com- to form the bundles (Fig. 3 b). pletely removed membranes. Bundles of straight, NEGATIVELY STAINED PREPARATIONS. TO aligned microfilaments which sometimes appeared

FIGURE 2 WMTEM preparation of DRG growth cone. Note meshwork of filaments in the growth cone which extend into the microspikes. Proximal portion of the growth cone is printed lighter to reveal intracellular organelles. (inset) High magnification of a microspike which contains microfilaments. Bars, 1.0 p,m. x 4,800. (inset) x 12,500.

360 THE JOURNAL OF CELL BIOLOGY 9VOLUME 80, 1979 FIGURE 3 Electron micrographs of detergent-treated, critical point dried neuroblastoma cells. (a) Neurite with many microspikes. Microfilament bundles (mfb) fuse and insert into microspikes. (b) Higher magnification of microspikes. Note individual microfilaments (mf). Bars, 0.5 p.m. (a) x 11,000. (b) x 19,000. to be in register were apparent. following criteria: (a) The F-S1 attached to puri- PARACRYSTAL FORMATION. The fact that fied muscle f-actin filaments to form characteristic filaments within a microspike were occasionally in arrowheads (Fig. 7a). Formation of these struc- register suggested the possibility of inducing para- tures was prevented by washing the electron mi- crystals in situ. Addition of 25 mM MgC12 to the croscope grid with buffer containing 1 mM Mg- detergent buffer enhanced the paracrystalline or- ATP. (b) Addition of F-S1 to muscle myofibrils der of the filament bundles (Fig. 6). The observed resulted in a distinctive staining pattern (Fig. 7 b); periodicity was -35 nm, the same as the cross- the actin-containing "I" bands appeared as bright over repeat for purified actin paracrystals (34). doublets intersected by the unstained "Z" bands, and there was no staining of the "A" bands which ACTIN LOCALIZATION WITH F-S1 contained myosin. This characteristic staining pat- CHARACTERIZATION OF F-SI. The fluo- tern was prevented when the incubation was car- rescein-labeled myosin subfragment S~ (F-S~) was ried out in either 5 mM Mg-ATP or 5 mM Na- shown to be a specific probe for actin by the pyrophosphate. (c) The F-S1 also labeled the

KUCZMARSKI AND ROSENBAUM Localization of Nerve Actin and Myosin 361 Fmu~ a Electron micrographs of detergent-opened, negatively stained chicken fibroblasts. (a) Low magnification reveals large bundles of microfilaments at edge of the opened fibroblast. Note microspikes (s). Bar, 1.0 /~m. (a) x 10,000. (b) Higher magnification showing individual double helical actin filaments in a microspike. Bar, 0.1 p.m. (b) • 78,000. actin-containing stress fibers (26) of chick fibro- satellite cells stained specifically with the F-S1 (see blasts (Fig. 7c). Again, this staining pattern could Fig. 7c). DRG nerve cells, on the other hand, be reversed by the addition of ATP. revealed no discrete fluorescent localization. In- ACTIN LOCALIZATION IN NERVE CELLS. stead, the neurites and growth cone regions were Mouse neuroblastoma cells reacted with F-S~ ex- stained uniformly (Fig. 8 C and D). Localization hibited a diffuse staining throughout the perinu- in these structures was specific, however, because clear region and neurites. In addition, actin-con- this staining pattern was not seen in the control taining microspikes stained intensely (Fig. 8 A and which was washed with ATP-containing buffer. B). If the reaction was carried out with ATP, the microspikes no longer stained, and fluorescence in Myosin Localization the cell body was greatly reduced. The distribution of actin was also investigated CHARACTERIZATION OF MYOSIN ANTIBODIES in chicken DRG nerve cultures. In addition to Antibodies to brain or breast myosin were nerve cells, these cultures usually contained a tested against antigen by the Ouchterlony double- small number of fibroblast-like satellite cells. As diffusion technique. The antibody to chicken brain expected, the actin-containing stress fibers in these myosin reacted strongly with purified chicken

362 THE JOURNAL OF CELL BIOLOGY 9VOLUME 80, 1979 brain myosin but did not react with myosin from with chicken brain or gizzard myosin, or with chicken gizzard or breast, or rabbit back muscle. rabbit striated muscle myosin (see Fig. 13 of Antibody to chicken breast muscle myosin gave a reference 34). The antibody to brain myosin was reaction with chicken striated muscle myosin only. further characterized by immunoelectrophoresis. No reaction of chicken breast antimyosin was seen A single precipitin arc was obtained for the pure

Fmu~ 5 Electron micrographs of detergent-opened, negatively stained neuroblastoma ceil. (A) Large neurite with several microspikes (s). Bar, 1.0 ttm. (B-E) Higher magnification of A, showing detail of microspikes. (B) microspike with membrane partially removed, exposing a microfilament bundle. (C, D, and E ) microspikes with membranes completely removed to reveal bundles of actin filaments. Bars, 0.1 ttm. (A) x12,000. (B) x 51,000. (C) x 40,000. (D) x 110,000. (E) x 95,000.

KUCZMARSKI AND ROSENBAUM Localization of Nerve Actin and Myosin 363 FIGURE 6 Micrographs of detergent-opened, negatively stained neuroblastoma cells. Paracrystalline arrays of actin filaments in microspikes induced by MgCI2 (25 mM). Note alignment of helical crossover points. Bar, 0.1 /~m. x 130,000. myosin (Fig. 9a). In the reaction with the crude cells (Fig. 11A and B). Often, the staining oc- homogenate, a single precipitin line was also curred in discrete patches along the length of a obtained (Fig. 9b), indicating that the antibody stress fiber, giving rise to regular periodicities. was monospecific. The antibody to chick brain myosin did not stain Fluorescently labeled antibodies were also stress fibers of mouse neuroblastoma, A-9, or tested for specificity by staining isolated breast primary fibroblast cells. Likewise, human macro- muscle myofibrils. Fluorescently labeled antibody phages, HeLa, or MRC-5 cells did not stain. to brain myosin did not stain myofibrils (Fig. 10a Stress fibers in baby hamster kidney (BHK) cells and b). The antibody to breast muscle myosin, did, however, exhibit distinct staining with the however, did stain the myosin-containing "'A" chicken antibody. The significance of these inter- bands of the myofibrils (Fig. 10c and d). This specific differences is not obvious. staining pattern was abolished when the immune To further demonstrate specificity of the brain serum was pre-adsorbed with chicken striated myosin antibody, various control experiments muscle myosin. Staining was not affected, how- were carried out, with the characteristic staining ever, by pre-adsorhing the serum with chicken of fibroblast stress fibers as a test system. Large gizzard or brain myosin (data not shown). numbers of fibroblasts, as well as some myoblasts, were obtained from 10-d-old chick embryo hearts. MYOSIN LOCALIZATION WITH In one study, cardiac cell cultures were pre-incu- FLUORESCENTLY LABELED MYOSIN bated with either pre-immune or immune serum ANTIBODY before staining with fluorescently labeled anti- Fluorescently labeled antibody to nerve myosin brain myosin antibody. The unlabeled immune (obtained from the chicken brain) was used to serum prevented staining of fibroblast stress fibers stain cultured nerve cells of the chicken dorsal (Fig. 12a and b), while the pre-immune serum did root ganglion. Staining was diffuse and not local- not affect the staining (Fig. 12c and d). It was ized to any particular region of the neuron. Flu- also possible to prevent staining of fibroblast stress orescent staining occurred in the growth cone, fibers by adsorbing the antibody with antigen. neurite, and cell body but not in the nucleus (Fig. Thus, the anti-brain myosin anti-body which had 11 C-F). This diffuse staining of the nerve cells been adsorbed with the brain myosin antigen could be prevented by blocking the reaction with failed to stain the stress fibers in the fibroblasts unlabeled immune serum. Satellite cells which (Fig. 12g and h). However, the brain myosin were present in the nerve cultures also stained antibody which had been adsorbed with breast with the antibody to brain myosin. Fluorescent myosin did stain the stress fibers (Fig. 12 e and f), staining was localized in the stress fibers of these indicating that the antibody was specific for brain

364 THE JOURNAL OF CELL BIOLOGY 9VOLUME 80, 1979 by the fact that myofibrils within the cardiac cells never exhibited staining of the myosin-containing "A" bands. This characteristic type of staining could, however, be obtained by reacting the car- diac cells with fluorescently labeled antibody to striated muscle myosin (from chicken breast). This antibody stained the cardiac muscle cells brightly, and the characteristic banding pattern resulting from staining of myofibril "A" bands could be seen (Fig. 13 C and D). In contrast, the cardiac fibroblasts present in this preparation were not stained by the antibody to striated muscle myosin.

F/GUIt~ 7 Characterization of the fluorescent-S~ re- agent. (a) Electron micrograph of negatively stained actin filaments reacted with fluorescent-S1. Note "arrow- heads" distributed along the length of the actin fila- ments. Bar, 0.1 /~m. x 40,000. (b) Light micrograph showing the interaction of F-S~ with chicken myofibril. Actin in the "I" bands stained as a bright doublet intersected by the unstained "Z" lines. The "A" bands which contain myosin did not stain, x 4,800. (c) Light micrograph showing staining of actin-containing stress fibers in a confluent layer of chicken fibroblasts, x 3,200. myosin and, further, that removal of antibody by brain myosin was not caused by nonspecific bind- ing to myosin protein. Not only fibroblasts, but also myoblasts of the cardiac cell cultures stained with anti-brain myosin antibody. It was not always possible to morpho- logically identify myoblasts, but occasionally my- ofibrils within these cells could be recognized with phase contrast optics. Staining of such cells with fluorescent anti-brain myosin antibody was often diffuse, although stress fibers similar to those seen in fibroblasts were sometimes seen (Fig. 13A and B). The fact that the cardiac muscle cells in culture stained with anti-brain myosin suggested that Fmu~ 8 Light micrographs of nerve cells stained either the antibody cross-reacted with cardiac with F-S1. (A and B) Mouse neuroblastoma cells. Note muscle myosin or, more likely, that the cardiac intense staining of microspikes. A, • 3,000; B, x 5,400. muscle cells contained both cytoplasmic and mus- ( C and D) Cultured chicken DRG nerve cells showing cle . This latter possibility was supported uniform staining of neurite. • 1,300.

KUCZMAaSK1 AND ROSENBAUM Localization of Nerve Actin and Myosin 365 been seen in thin sections of cultured DRG cells (references 13, 62, and our unpublished observa- tions). Instead, microfilaments are organized in a meshwork or lattice arrangement similar to that seen in the ruffling membrane region of fibroblasts (62). Likewise, stress fibers were not seen in DRG cells examined by WMTEM, although indi- vidual microfilaments were observed in the thin, FIGURE 9 Immunoelectrophoresis using antibody against native brain myosin. Antigen was placed in the well-spread growth cones (Fig. 2). Microfilaments circular wells, electrophoresed, and then reacted with which entered microspikes present at the periph- antiserum placed in the center trough. (a) Reaction with ery of the growth cone did, however, associate purified brain myosin to give a single line. (b) Crude into loose bundles (Fig. 2, inset). In addition, brain homogenate also gave a single precipitin line. microfilaments which were randomly distributed throughout the growth cones converged at, and DISCUSSION appeared to continue into, the large axon which connected the growth cones to the cell body. Actin Localization However, the organization of flaments in the LOCALIZATION WITH WMTEM. Thin sec- compact neurite could not be determined by this tioning of individual nerve cells is a tedious proc- technique because of the thickness of the struc- ess and provides only limited information on ture. filaments which course in and out of the plane of Burton and Kirkland (12) first showed that section. To study the localization and organization mouse neuroblastoma cells contained microfila- of actin in cultured nerve cells, therefore, new ments which could be decorated by HMM and, approaches which avoided some of these problems with a modified glycerination procedure, Chang were used. One such method was that developed and Goldman (15) subsequently demonstrated by Buckley and Porter in which whole cells were that HMM-decorated filaments were present in all studied in the transmission electron microscope regions of the neuroblastoma cell, including the after positive staining and critical-point drying cell body, neurites, and microspikes. In a detailed (10). Because the cells retained their three-dimen- ultrastructural study Ross et al. (48) described the sional structure, it was possible to follow individ- organization of these microfilaments in neuroblas- ual filaments over long distances, and to trace toma cells. Particularly interesting was the report their course by use of stereo pairs of electron that microspikes contained large numbers of mi- micrographs. With this technique, the filament organization in thinner regions of cultured cells was investigated with a standard transmission elec- tron microscope operated at 100 KV. Thin-sectioned fibroblasts contain arrays of lin- early arranged microfilaments (known as stress fibers [9], sheath [59] or microfilament bundles [25]) which are in close association with the basal membrane, and serial sectioning reveals that sim- ilar structures can sometimes be found near the upper membrane (26, 59). Microfilaments in these bundles bind HMM (26), indicating that they are composed of actin. In ruffling membrane regions of fibroblasts, microfilaments do not form FIGURE 10 Specificity of antimyosin antibodies. stress fibers but rather are seen as a loose mesh- Chicken breast muscle myofibrils were reacted with work or lattice (59). Both stress fibers and three- antibody and then photographed in the fluorescence dimensional lattice formations were maintained in microscope. The myofibril seen by phase microscopy (a) chicken fibroblasts observed by the whole-mount failed to react with fluorescently labeled antibody to method described above. brain myosin (b). The myofibril shown in c, however, The highly organized microfilament bundles reacted with fluorescently labeled antibody specific for (stress fibers) characteristic of fibroblasts have not breast muscle myosin (d). x 3,430.

366 THE JOURNAL OF CELL BIOLOGY" VOLUME 80, 1979 (Fig. 1). Because of the depth of field available with this method, it was possible to discern the overall organization of microfilaments in a manner not possible with thin-sectioning techniques. In the whole-mount preparation, microfilaments which extended from the microtubule region were organized into bundles which joined with similar bundles present at the cell margin, forming a single structure which inserted into the microspike (Fig. 1). It was possible to obtain a clearer view of this microfilament organization by gently remov- ing the plasma membrane before processing for WMTEM (Fig. 3). In contrast to preparations of intact neuroblastoma cells, microfilament bundles in the detergent-treated cells seemed to be more intact and to stain more darkly. While this appear- ance may have resulted from partial collapse of the bundles as a result of detergent treatment, it is likely that the absence of a membrane permeabil- ity barrier allowed better fixation as well as a less turbulent CO2 substitution during the critical- point drying process, resulting in the observed morphology. The fact that the microfilament bun- dles were not removed by the detergent treatment suggested a firm attachment to the lower cell membrane. ACTl~ IN MICROSP~KES. At the tip of a growing nerve axon is a flattened region known as the "growth cone" from which extend long, rigid microspikes (28) having a constant diameter of between 0.1 and 0.2 p.m (8). These dynamic structures, which can grow outwards as far as 25 /~m in 1 min, have been seen to wave about in the medium or along the solid substratum (59). Mi- crospikes are also found in fibroblasts and other cell types (2), and are believed to be involved in cell-cell interactions (23), membrane spreading (61), exploration of the cellular environment, and cell adhesion (1). Ultrastructural studies have FIGURE 11 Cellscultured from chick dorsal root gan- shown the microspike to be composed of an glia stained with fluorescently labeled anti-brain myosin elongated network of microfilaments (2, 48, 59). antibody. (A and B) Stress fibers in fibroblasts stained Microtubules and 10-nm filaments are not present specifically with the antibody. Periodic staining can in the microspikes (2, 48, 59), and recent immu- occasionally be seen along the length of a fiber (arrow). nofluorescent localization studies indicate that • 3,100. (C-F) Neurites and growth cones of DRG myosin is also absent from the microspikes (24). nerve cells stained uniformly with the anti-brain myosin Microspikes of detergent-opened, negatively- antibody. • 1,440. stained neuroblastoma cells contained straight mi- crofilaments aligned in parallel arrays (Fig. 5), crofilaments which emanated from the main neu- and a similar organization of microfilaments was rite and sometimes appeared to be spatially asso- seen in fibroblast microspikes (Fig. 4). Similar ciated with the neurite microtubules (see Fig. 20 results have been reported for Dictyostelium and of reference 48). Similar results were obtained in sea urchin cells (16, 19). The microfilament bun- the present study with the whole-mount procedure dles did not just fill the microspike, but extended

KUCZMARSKI AND ROSENBAUM Localization of Nerve Actin and Myosin 367 back into the cell for a considerable distance, and branched to form smaller bundles (Figs. 1 and 3). These bundles appeared to be firmly attached to the lower cell membrane. This type of bundle arrangement is likely to be responsible for both the rigidity and motility of the micpospike. Indeed, a model has been proposed in which movement results from the interaction of myosin with actin filaments in the microvilli of intestinal brush-bor- der cells (41). The straight, intact microfilaments revealed in negatively stained neuroblastoma cells differed from the wavy, network image obtained by either thin section (15, 48) or WMTEM (Fig. 1). This suggests that processes involved in preparing the cells for electron microscopy by the latter proce- dures may have disrupted filament organization. Indeed, Maupin-Szamier and Pollard (40) have shown that purified, in vitro assembled actin, when fixed in osmium tetroxide, exhibits reduced viscosity and appears broken and wavy when examined in the electron microscope. Further- more, these authors have also shown that tow concentrations of osmium tetroxide rapidly cleave the actin molecule to produce lower molecular weight species. LOCALIZATION OF ACTIN USING F-S 1. To rapidly localize actin and study its distribution within an entire cell, the fluorescent $1 probe for actin was used with both neuroblastoma and DRG nerve cells. As already noted, microfilaments were present in the neuroblastoma cell body, neurites, and microspikes (references 15, 48 and Figs. 1, 3, and 5). These same regions reacted specifically with F-S1, and the most intense stain was seen in the microspikes which contained high concentrations of actin (Fig. 8). Sanger (51) has also localized actin in neuroblastoma cells using fluorescent HMM. In DRG nerve cells, the neu- rites and growth cones were labeled with F-S~ (Fig. 8), confirming the location of actin as deter- mined by ultrastructural methods (reference 62 and Fig. 2). Although neuroblastoma cells, DRG cells, and brain tissue are known to contain significant amounts of actin, it is only in neuroblastoma cells that typical 6-nm actin filaments can readily be FIGURE 12 Demonstration of specificity of anti-brain seen in thin section. Ultrastructural studies of myosin antibody using chicken heart fibroblasts. Phase DRG nerve cells revealed short 6-nm filaments micrograph on left, corresponding fluorescent micro- graph on right. (a, b) antibody staining was blocked by pre-incubating with immune serum. (c, d) staining was adsorbed with breast myosin, but in g and h it can be not affected by pre-incubating with pre-immune serum. seen that cells did not stain when the antiserum was (e, f) cells were stained by antiserum which had been adsorbed with brain myosin. • 1,250.

368 THE JOURNAL OF CELL BIOLOGY' VOLUME 80,1979 Fmulm 13 Staining of cardiac cell cultures with antibodies to brain or breast myosins. Phase micrograph on left, corresponding fluorescent micrograph on right. (A, B) cells stained with anti-brain myosin antibody. (C, D) myoblasts, but not fibroblasts, stained with antibody to skeletal (breast) muscle myosin. Note the characteristic staining pattern of the myofibrils, x 1,250. which formed a meshwork in the growth cone, interesting to note that cells which had been and only nondescript filamentous material adja- exposed to HMM contained many more filaments cent to the membrane of the neurite (62). In than cells which were incubated in buffer plus numerous electron microscope investigations of glycerol only (37). It is known that HMM stimu- nervous tissue, few, if any, 6-nm filaments have lates the polymerization of actin (17, 33, 63), and been visualized (5, 47), although LeBeaux (36) it is possible that glycerol, like sucrose (46), also claims to have seen a few short microfilaments in influences the state of actin polymerization. If, specially fixed nerve tissue, and an interesting indeed, neuronal actin is normally in a nonfila- array of filamentous meshworks is present in close mentous form, this would explain why actin fila- association with the membrane of the Purkinje ments are so rarely seen in this tissue. Evidence cell axon hillock (47). for membrane-associated nonfilamentous actin Recently, LeBeaux and Willemot (37, 38) have has been obtained in other systems (27, 54, 55). reported the presence of large numbers of actin filaments in neurons of the rat substantia nigra Myosin Localization and caudate nucleus. For these studies, small Myosin cannot readily by visualized by ultra- pieces of brain were incubated in a glycerol solu- structural means, although filaments thought to tion to render the cells permeable to the HMM be composed of myosin have been seen in some probe used to identify actin filaments. Decorated cells (3, 43, 53). In the present study, therefore, filaments were found in both axons and dendrites, antibody specific for myosin from chick brain was as well as in association with pre- and postsynaptic used to localize myosin in chicken DRG nerve membranes. In control preparations which were cells with fluorescence techniques. The antibody either incubated in glycerol-containing buffer or was shown to be specific for brain myosin by incubated with HMM in the presence of ATP, Ouchterlony double-diffusion and immunoelec- undecorated actin filaments were visible. It is trophoresis. The neurites and growth cones of

KUCZMARSKI AND ROSENB^tlM Localization of Nerve Actin and Myosin 369 DRG nerve cells stained specifically with the loot and cardiac tissue cultures, also stained car- fluorescent antibrain myosin antibody (Fig. 11 E diac myoblasts. Myoblasts exhibited both diffuse and F). This staining pattern was not seen when staining as well as staining of stress fibers. Myofi- the cells were reacted with fluorescently-labeled brils present in the cardiac myoblasts did not react nonimmune serum, or when the reaction was with the antibody to brain myosin, although they blocked by unlabeled immune serum. These re- could be stained by fluorescently labeled antibody suits indicated that myosin was present in the to chicken skeletal muscle myosin. Holtzer et al. nerve cell but that no discrete localization within (29) were also able to stain chick cardiac myo- any particular region or structure of the cell could blasts with antibody to chicken skeletal muscle be determined with the resolution provided by myosin, and others have demonstrated immuno- light microscopy. Because actin, like myosin, was logical cross-reactivity between chicken skeletal found throughout the neuron (Fig. 8), it is possi- and cardiac myosins (18, 21, 49). The fact that ble that they may interact to produce force. cardiac myoblasts were stained with the antibody Possible functions for the myosin include move- to brain myosin but that myosin in the myofibrils ment of the growth cone and axoplasmic trans- of these same cells did not stain suggests that the port. During nerve growth, the cell body and myoblasts contain both cytoplasmic and muscle neurite remain stationary while the growth cone myosins. Studies on prefusion skeletal muscle "crawls" forward over the substratum, often at myoblasts also suggest the presence of two types rates of up to 50/zm/h (28). The neurite does not of myosin in the same cell (30, 31). Actin provides grow from the base, but rather new material another example of both cytoplasmic and skeletal appears to be added at the growing tip (7). forms of a contractile protein being present in a Membrane, proteins, and other materials synthe- cell at the same time (60). sized in the cell body are transported along the The fact that both nerve and satellite cells neurite to the growth cone via axoplasmic trans- stained with the antibody to brain myosin sug- port, an active process which also may require gested either that (a) a large amount of the myosin (4, 35, 44). In radiotracer experiments, myosin isolated from the brain came from Lasek and Hoffman (35) have demonstrated that Schwann cells, glial cells, astrocytes, and other five proteins make up the majority of the slow non-neuronal cells or that (b) the cytoplasmic component. Two of these polypeptides appear to myosins present in nerve and fibroblast cells are be tubulin, while the remaining three (68,000, immunologically similar. It should be possible to 145,000, and 200,000 daltons) are thought to be distinguish between these two choices by using associated with neurofilaments. These researchers existing techniques. propose a model in which the 200,000-dalton component (possibly myosin) interacts with actin This work was submitted as partial fulfillment of the attached to the axonal membrane to move the requirements for the Ph.D. degree, Yale University. neurofilament-microtubule assembly down the These investigations were supported by USPHS-HD axon at 1 mm/d. Ochs (44) has invoked a similar 00032-13 to Yale University and American Cancer actomyosin mechanism to explain the fast trans- Society grant VC-129 and National Science Foundation port (400-500 mm/d) of proteins and particulate grant BMS 76-02605 to J. L. Rosenbaum. E. R. Ku- czmarksi was a recipient of a National Science Founda- matter. tion predoctoral fellowship and a Connecticut State The present study demonstrated that non-neu- Graduate Award. ronal cells were stained by the antibody to brain myosin; large stress fibers in the satellite cells and Received for publication 15 March 1977, and in revised fibroblasts present in DRG and cardiac cultures form 28 August 1978. were stained by the fluorescent brain myosin antibody (Figs. llA and B, and 12). Myosin REFERENCES staining along the length of a stress fiber was often 1. Aunt~'trr-B~, G. 1976. of spreading 3T3 cells: do periodic, giving rise to a striated appearance. they have a substrate..expinring function?J. 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Kucz.zoam~ Am) ROSF,~BAUM Localization of Nerve Actin and Myosin 371