ous movements of filopodia and lamellipodia and the formation of contacts with other cells and surfaces by the nerve tip (Harrison, 1910; Letourneau, 1979; ...
DEVELOPMENTAL
BIOLOGY
85, 113-122 (1981)
lmmunocytochemical Evidence for Colocalization in Neurite Growth Cones of Actin and Myosin and Their Relationship to Cell-Substratum Adhesions PAULC. LETOURNEAU Department
of Anatomy,
h-135 Jackson Hall,
321 Church Street SE, University
of Minnesota,
Minneapolis,
Minnesota
55455
Received August 25, 1980; accepted December 15, 1980
Sensory neurons from 8- to II-day chick embryos were cultured on polyornithine-treated coverslips, fixed with glutaraldehyde, and stained for immunofluorescent localization of actin. Actin was distributed in a fibrous form in the growth cones, extending into filopodia and lamellipodial expansions of the growth cone margin. Often, these actin fibers were located at sites of linear adhesions to the glass substratum, as viewed by interference reflection optics. Our antisera to myosin did not recognize myosin in glutaraldehyde-fixed cells, and paraformaldehyde, which preserves the antigenicity of myosin, did not fix embryonic neurons well. Thus, myosin was localized in NGF-stimulated PC12 cells, whose morphology is better preserved by paraformaldehyde. Within the growth cones of PC12 neurites, actin and myosin are distributed into fibrous arrays which resemble the actin fibers seen in the growth cones of sensory neurons. Thus, actomyosin-like contractile forces may be exerted in neurite growth cones. These forces may act in concert with cell-substratum adhesive bonds to move the growth cone across the substratum or move organelles within the growth cone. INTRODUCTION
movements of the growth cone and nerve fiber elongation. Recently, glutaraldehyde fixation, followed by reduction of unreacted aldehyde groups, and stepwise alcohol dehydration have been used to improve preservation of cell morphology for immunofluorescence and immunoelectron microscopy (Eckert and Snyder, 1978; Weber et al., 1978). We used this technique for immunofluorescence cytochemistry and found that actin is organized into fine linear arrays in the locomotor-y portions of growth cones extended by embryonic sensory neurons. Linear adhesive contacts of the motile growth cone periphery to the coverslip coincide with the positions of some linear arrays of actin (Letourneau, 1979). Unfortunately, our antiserum to myosin did not stain glutaraldehyde-fixed cells, though we did observe linear staining with our myosin antisera in the growth cones of paraformaldehyde-fixed NGF-treated cells of the clonal pheochromocytoma line, PC12. Actin was also present in these linear arrays within the growth cones of PC12 neurites. Thus, actomyosin interactions can potentially generate mechanical forces within growth cones, and adhesive contacts may provide stable points from which tension is exerted upon neurite structures.
The tip of an elongating nerve fiber has been long regarded as extremely important in nerve fiber growth. Initially called the growth cone by Cajal, it was believed responsible for leading the growing nerve fiber to synaptic targets (Ramon y Cajal, 1917). The most striking features to an observer of nerve fiber growth are the vigorous movements of filopodia and lamellipodia and the formation of contacts with other cells and surfaces by the nerve tip (Harrison, 1910; Letourneau, 1979; Nakai and Kawasaki, 1959; Speidel, 1933; Yamada et al., 1971). However, a critical question remains unanswered: How is this motility related to the mechanism of elongation of a nerve fiber? One approach to this problem is to examine the growth cone for molecules capable of exerting mechanical force within the nerve tip. Previous cytochemical studies have demonstrated the presence of actin and myosin in growth cones and neurites, but due to the small size of many growth cones and their poor preservation by standard procedures, a more precise intracellular distribution of these proteins resisted elucidation (Chang and Goldman, 1973; Kuczmarski and Rosenbaum, 1979a,b; Marchisio et al., 1978). Are actin and myosin situated in growth cones such that they can interact and generate force to advance the nerve tip or to move precursors of the growing MATERIALS AND METHODS neurite? Several laboratories report that myosin is not seen in ruffling membranes of fibroblasts (Heggeness et Cell Culture al., 1977; Zigmond et al., 1979), a structure with the Dorsal root ganglia were dissected from 8- to 11-day same motile properties as the growth cone. If so, acto- chick embryos and dissociated as described previously myosin-like contractile activities may not account for the (Letourneau, 1975). The cells were plated onto polyor0012-1606/81/090113-10$02.09/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
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nithine-treated coverslips (Letourneau, 1975), placed in 35-mm plastic petri dishes (Falcon Plastics). The culture medium was 50% fresh Ham’s nutrient mixture F12 supplemented with 10% fetal calf serum, 100 pm/ml streptomycin, 100 U/ml penicillin, and 0.25 pg/ml fungizone (all media, sera, antibiotics; from Gibco) and 50% heart conditioned medium (Helfand et al., 1976). Nerve growth factor (p-NGF), a gift from Dr. Eric Shooter, was also added at 10 rig/ml. Cells were cultured in a humidified 5% CO, incubator at 37°C. PC12 cells, a clonal pheochromocytoma line (Greene and Tischler, 1976), were obtained from Dr. Eric Shooter. Cells were cultured in Dulbecco’s modified minimum essential medium supplemented with antibiotics as above and with 10% fetal calf serum and 5% horse serum. Cells were induced to form neurites by adding 10 rig/ml P-NGF and 1 n-&f dibutyryl cyclic adenosine monophosphate (Sigma Chemical Co.) to the culture medium. For immunofluorescence studies PC12 cells were plated onto polyornithine-treated coverslips at l-2 X 104 cells/cm2. Cultures of smooth muscle cells from bovine umbilical veins were generously provided by Ms. Pat Anaya. Interference Reflection Microscopy
Fixation of embryonic sensory neurons and PC12 cells was done in two ways. Sensory neurons were fixed with 4% paraformaldehyde and 1.0% glutaraldehyde in Sorensen’s buffer, pH 7.4, containing 0.12 M sucrose. PC12 cells were fixed with 4% paraformaldehyde in Sorensen’s buffer, pH 7.4, containing 0.12 M sucrose. In all cases, fixation was at 37°C for 20 min. After fixation the coverslips were washed twice with phosphate-buffered saline (PBS), pH 7.4, at 22”C, and the cells were made permeable by dehydration through 50, 70, and 95% ethanol in H,O at 4°C. Sodium borohydride (0.5 mg/ml) was added to the 95% ethanol for three rinses of 4 min each. Rehydration was through 50% ethanol at 4°C then 20 and 10% ethanol at 22°C (Weber et al., 1978). Primary antisera were placed on the coverslips at l/50 or l/100 dilutions in PBS for 45 min at 37°C. The coverslips were rinsed by three immersions in PBS, followed by draining on filter paper. This rinse was repeated three times. The fluorescent secondary antibodies (fluoresceinconjugated goat anti-rabbit serum, Miles Laboratories) were placed on the coverslips at l/100 dilution in PBS for 45 min at 37°C. The coverslips were then rinsed in PBS as above, rinsed in HzO, and mounted on slides in Elvanol (DuPont de Nemours and Co., Inc., Wilmington, Del.). Observations of immunofluorescence were done on a Zeiss IM microscope, using an HBO 100-W mercury illuminator. Photographs were taken on 35-mm Tri-X film (Kodak) with exposures of 15-30 sec.
Cell-substratum contacts were visualized by interference reflection microscopy on a Zeiss IM microscope (Letourneau, 1979). An HBO 100-W illuminator and a 100x epiplan POL objective were used. Photographs were taken on 35-mm Panatomic X film (Kodak). Chambers were constructed for interference reflection and immunofluorescence observations of the same RESULTS growth cones. Coverslips were glued over 2-cm holes drilled in 60-mm plastic petri dishes (Falcon Plastics; Actin Localization in Cultured Neurons Dow-Corning silicone cement). Cell culture, fixation, all Figures 1 and 2 are a phase-contrast image and an imfollowing procedures and photography were carried out munofluorescence localization of actin in a glutaraldein these dishes. hyde-fixed sensory neuron. The rounded cell soma is intensely fluorescent, being much thicker than the neurites, growth cones, or flattened cell margins. Actin Immunojluorescence Staining is present throughout the cytoplasm of the cell, but atAntisera to chicken gizzard smooth muscle actin and tention is drawn to sites of linear distribution of actin in myosin, preimmune serum, and specific antigen-ab- the growth cones (Figs. 2, 6, 3). These linear accumulasorbed sera were prepared and characterized as de- tions of actin measure approximately 0.2-0.3 pm wide scribed in Schollmeyer (1980). In brief, 75 pg of homoge- and 10 pm long and may correspond to small bundles of neous purified antigen was emulsified in Freund’s microfilaments observed by transmission electron miadjuvant and injected into the popliteal lymph nodes of croscopy of whole-mounted growth cones (Letourneau, separate rabbits. Sera were harvested at weekly inter- 1979; Nuttall and Wessells, 1979). These structures are vals from 21 days after the initial injection. IgG fractions narrower and much shorter than the stress fibers or actin cables seen in fibroblastic cells (Brown et al., 1976; Lawere obtained by DEAE-cellulose chromatography (Schollmeyer, submitted) and, finally, monospecific anti- zarides and Weber, 1974), and the actin staining of bodies were prepared by affinity chromatography (Cua- growth cones is not as striking as that of fibroblastic trecasas et al., 1968). Only monospecific antibodies were actin cables. However, the staining in growth cones is used in these studies. These sera were generously pro- linear and will be tentatively called actin fibers, pending vided b-v Dr. Judith Schollmever. ultrastructural study. "
PAULC.LETOURNEAU Actin and Myosin in Growth Cones
The specificity of this stain for actin was indicated by demonstrations that cells treated with identical dilutions of preimmune rabbit serum or with immune serum absorbed with smooth muscle actin showed only weak fluorescence and no fibrous staining in the growth cones (not shown). This means that fibrous staining was not due to nonspecific trapping of antisera within cells or merely to local differences in thickness of the growth cone. That antibody recognition of actin occurs in glutaraldehydefixed cells was indicated by the obvious immunofluorescent staining of myofibrils in umbilical vein smooth muscle cells fixed with 0.5 or 1.0% glutaraldehyde or 4.0% paraformaldehyde and treated with antisera to actin (not shown). The possibility that these actin fibers in growth cones are composed of microfilaments is supported by the effects of cytochalasin B (CB) on the staining of growth cones. Cultures of ganglionic cells were treated with 2.5 pg/ml CB for 10 min at 37”C, then fixed and stained for actin. Growth cones appeared shrunken and lacked distinct linear staining (Fig. 3). Instead, patches or aggregates of fluorescence, as reported previously (Weber et al., 1976), were seen. These patches may correspond to the areas of dense material seen in thin sections of CBtreated cells, which are interpreted as condensed or disorganized microfilaments (Weber et al., 1976; Yamada et al., 1971). Unlike the actin fibers of growth cones, the stress fibers of ganglionic nonneuronal cells stained and appeared intact after our short CB treatment (Fig. 4), although fluorescent patches were also seen. This is consistent with electron microscopic findings that the sheath microfilaments and long microfilament bundles of ganglionic nonneuronal cells are not broken down by CB treatment (Spooner et al., 1971), and supports proposals that the organization of actin is more stable in stress fibers than in the microfilament lattice present in the cell cortex and within growth cones (Lazarides, 1976; Spooner et al., 1971). Actin fibers in sensory neurons are largely restricted to the neurite growth cones, especially at the peripheral margin where protrusive activity occurs. Fibrous staining is not visible in the central axis of neurites or in the flattened margins of cell somata (Fig. 2). The central soma region is too intensely fluorescent to rule out the possible presence of fibrous actin staining at the lower cell surface. Apparently, these actin fibers are only in the locomotory portions of neurons and may represent a functional state of actin in terms of cell motility. Relationship of Actin Distribution Cone-Substratum Adhesion
to Growth
The distribution of actin fibers resembles that of linear adhesive contacts of growth cones to polyornithine-
115
treated coverslips (Letourneau, 1979). In order to assess the relationship of these similar images, interference reflection microscopy and immunofluorescence localization of actin were carried out on the same neurons. Fixation with glutaraldehyde does not alter the interference reflection images of growth cone-substratum adhesions (unpublished data), but alcohol dehydration does reduce the apparent adhesive contacts, possibly because plasmalemmal lipids are removed. Thus, the cells were fixed, then interference reflection images of selected growth cones were photographed, and the positions of these cells were marked, so they could be found after the immunofluorescence procedures. A striking example of the correlation of adhesive contacts and actin fibers is presented in Figs. 5 and 6. The black lines which radiate outward beneath the growth cone and continue beneath filopodia are the closest contacts with the substratum (Letourneau, 1979; Izzard and Lockner, 1976). Linear adhesive contacts are also seen at the edges of lamellar expansions of the growth cone margin. When compared to the immunofluorescence image, one sees that each linear adhesive contact coincides with an actin fiber which extends outward within the growth cone and into a filopodium or along the edge of a lamellipodium (Figs. 5,6). Many of the actin fibers at the center of lamellar expansions of the growth cone margin are not located at close contacts, but in every instance we have seen, linear adhesive contacts coincide with intracellular actin fibers. Often, filopodia do not have a continuous adhesive contact, but rather are adherent only at their tips (Figs. 7, 8). These filopodia, too, contain actin fibers which continue centripetally within the growth cone. Many regions of sensory neurons make extensive, but nonlinear, adhesive contacts with the coverslip. These regions do not contain actin fibers. The central or proximal portions of many growth cones make large contacts at an approximately 300-&separation from the substratum (Figs. 5, 7), and these areas contain only diffuse actin (Figs. 6,8). The growth cone pictured in Fig. 9 has an unusual shape, being round and flattened, and has a very large adhesive contact interrupted by small (white) areas of further separation. This growth cone contains mostly diffusely distributed actin, with some small punctate concentrations, except at the edge where a few filopodia are extended (Fig. 10). It would be interesting to compare the motility of this morphological type of growth cone to those pictured in Figs. 6 and 8, which contain many actin fibers in their margins. The interference reflection image in Fig. 11 is typical of a neuronal cell soma, showing an extensive adhesive contact interrupted by small nonadhesive areas. The cell soma of this neuron was rounded and uniformly fluorescent, so actin fibers, if present, may have been obscured. However, actin fibers are not present where a better view is possi-
116
FIG. 1. Phase-contrast image of a sensory neuron prepared for immunofluorescence localization of actin. One neurite (N) and growth cone (GC) are extended and two possibly newly initiated neurites (N) with growth cones are also seen. ~6’75. FIG. 2. Immunofluorescence localization of actin in this neuron. Note the fibrous concentrations of actin in the growth cones (arrowheads). Actin fibers are not seen in flattened margin of the cell soma (M). x675. FIG. 3. Localization of actin in a growth cone treated for 10 min with 2.5 pg/ml cytochalasin B (CB) before fixation. Note large fluorescent masses of actin (arrows), but no actin fibers. x 10’75.
PAUL C. LETOURNEAU
Actin and Myosin in Growth Cones
ble; in the flattened margin of the cell soma shown in Fig. 2. Localization of Myosin in Growth Cones Fibrous staining is not seen in glutaraldehyde-fixed growth cones stained for localization of myosin. We wondered whether myosin in glutaraldehyde-fixed cells is recognized by our antisera to myosin. This concern was tested by staining umbilical vein smooth muscle cells for myosin, just as previously done for actin, after fixation by several ways; with 4% paraformaldehyde, with 0.5% glutaraldehyde, or with 1.0% glutaraldehyde. The myofibrils of paraformaldehyde-fixed cells stained intensely for myosin, but the glutaraldehyde-fixed cells showed no fibrous staining, though myofibrils were clearly visible with phase contrast optics (not shown). Thus, we may not be able to localize myosin in glutaraldehyde-fixed growth cones with this antisera. Unfortunately, paraformaldehyde does not preserve the growth cones of chick embryo sensory neurons. Typical growth cones pictured in Figs. 12, 13, 14, and 15 appear shrunken or retracted and their filopodia seem disrupted after paraformaldehyde fixation. These growth cones stained positively for actin and myosin (Figs. 13 and 15, respectively), but no fibers are seen. Since we know that actin fibers are present in the betterpreserved growth cones fixed by glutaraldehyde, these results say little about the organization of myosin in growth cones of sensory neurons. Another attempt to localize myosin in neurite growth cones utilized the clonal line of rat pheochromocytoma cells, PC12, which respond to NGF and/or cyclic AMP by extending neurites (Greene and Tischler, 19’76;Schubert et al., 1978). The morphology of PC12 cells is preserved much better by paraformaldehyde than are chick sensory neurons. When NGF and dibutyryl cyclic AMP-treated PC12 cells were fixed and stained for actin, actin fibers were seen in growth cones and in the actively protrusive areas of the cell margin (Figs. 16-18). The growth cones of PC12 cells are much smaller than those of chick embryo neurons (compare Figs. 6 and 8 with Figs. 17 and 21), however, the general distribution of actin fibers was the same. Small ruffles, appearing black in phase con-
117
trast, stained intensely for actin and can be distinguished from actin fibers in flattened areas of the cell margin by careful focusing. Myosin, too, had a fibrous distribution, similar to that of actin, in paraformaldehyde-fixed PC12 cells (Figs. 19-21). PC12 cells stained with preimmune sera or with immune sera absorbed with actin or myosin, respectively, contained no fibrous staining. As suggested above for sensory neurons, this shows the specificity of these immunofluorescence pictures. We did not have the capacity to stain PC12 cells simultaneously for actin and myosin to show that the two proteins are colocalized. However, the actin- and myosincontaining fibers occupy the same positions in growth cones, when the patterns are compared (Figs. 16-21). In both cases, fibers extend into filopodia and are positioned at the sides of lamellipodia. This seems good evidence for the colocalization of actin and myosin. DISCUSSION
Using immunohistochemical procedures we have evidence suggesting that actin and myosin are organized into fibrous complexes, probably the same complexes, within neurite growth cones. In addition, these fibers are frequently located where growth cones make linear adhesive contacts with the coverslip. We will discuss the morphology of these fibrous structures, how they may act to generate force in the growth cone and what potential roles they have in nerve fiber growth. It should be stressed that we have not biochemically characterized the actin and myosin of PC12 cells and sensory neurons. However, our use of monospecific antibodies suggests that we have localized molecules in PC12 cells and sensory neurons which contain antigenic determinants of smooth muscle actin and myosin, respectively. Structural Organization of Actin and Myosin The likely ultrastructural correlates of these actin- and myosin-containing fibers are small bundles of microfilaments seen in whole growth cones by transmission electron microscopy (Letourneau, 1979; Nuttall and Wessells, 1979). Growth cones and similar motile regions of other cells contain a network or lattice of microfilaments
FIG. 4. Localization of actin in a ganglionic nonneuronal cell treated for 10 min with 2.5 pg/ml CB before fixation. Aggregates of actin are present within the cell (arrows), but actin-containing stress fibers (s) have remained. x675. FIG. 5. Interference reflection image of growth cone-substratum adhesion. Darkest black lines (arrows) are close contacts at approximately 100-200 A separation from the substratum. Dark grey area (G) is adhesive contact of 300-A-separation. White areas (W) and areas with the same tone as the reflection outside of the growth cone are not adherent to the substratum. x1075. FIG. 6. Localization of actin in growth cone. Many of the actin fibers within filopodia and lamellipodia are located at sites of linear close adhesive contacts (arrowheads) shown in Fig. 5. x 1075. FIG. 7. Interference reflection image of growth cone-substratum adhesion. Several filopodia and lamellipodia are largely nonadherent, but have adhesive contacts (arrows) at their distal ends. x 1075. FIG. 8. Localization of actin in the growth cone of Fig. 7. Many actin fibers extend centrally from filopodia or lamellipodia into the flattened cell margin. X 1075.
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FIG. 9. Interference reflection image of growth cone-substratum adhesion. Large gray adhesive contact with small (white) nonadherent spots is seen, but no linear adhesions are present, except under a filopodium (arrow). x 1075. FIG. 10. Localization of actin in growth cone shown in Fig. 9. Actin fibers are not present within the flattened growth cone. x 1075. FIG. 11. Interference reflection image of cell-substratum adhesion of a neuronal cell soma (S). Much of the soma is adherent to the substratum, but linear adhesions are not present. x 1075. FIG. 12. Phase-contrast image of a neurite and growth cone of a sensory neuron fixed with formaldehyde and dehydrated for actin localization. Cell morphology is indistinct and distorted. X1075. FIG. 13. Localization of actin in growth cone shown in Fig. 12. Distinct actin fibers are not seen in the shriveled growth cone. x 1075. FIG. 14. Phase-contrast image of a formaldehyde-fixed growth cone, prepared for localization of myosin. The margin of the growth cone appears shrunken (S) or retracted. x 1075. FIG. 15. Localization of myosin in the growth cone shown in Fig. 14. Myosin is present, but a distinct fibrous distribution is not seen. X 1075.
whose organization is unclear, having been described as made of short, branched, or crisscrossing filaments, or filaments bent at sharp angles (Hueser and Kirschner, 1980; Spooner et al., 1971; Wolosewick and Porter, 1979; Yamada et al., 1971). An important feature for this discussion is that in the cell cortex, the microfilaments contact the plasma membrane at many points. In the
microfilament bundles of growth cones, the filaments are straighter or longer and more closely packed to form a denser assembly than elsewhere in the cell margin (Letourneau, 1979). Whether these bundles are reorganized from the surrounding microfilament network or are assembled de nova from monomers is unknown. It is not surprising that these bundles stain richly for
FIG. 16. Phase-contrast image of two formaldehyde-fixed PC12 cells, treated for 48 hr with 10 rig/ml NGF and 1 nnI4 dibutyryl cyclic AMP. One neurite (N) with a growth cone is seen, as well as several protruded areas of the cell margin which may become growth cones. x 1075. FIG. 17. Localization of actin in the cells shown in Fig. 16. Fibrous distribution (arrowheads) of actin in the growth cone resemble structures seen in the growth cones of sensory neurons. x 1075 FIG. 18. Localization of actin in PC12 cells. Actin fibers (arrowheads) are seen in protrusions from the cell margin. These actin fibers closely resemble those seen in growth cones. x 1075. FIG. 19. Localization of myosin in formaldehyde-fixed PC12 cells. Fibrous arrays of myosin (arrowheads) in a protrusions of the cell margin resemble the actin fibers also present in these protrusions. x 1075. FIG. 20. Phase-contrast image of formaldehyde-fixed PC12 cells treated for 24 hr with 10 rig/ml NGF and 1 m&f dibutyryl cyclic AMP. The black lines (arrows) visible in the growth cone (gc) are small ruffles in the flattened cell margin. x 1075. FIG. 21. Localization of myosin in the cell shown in Fig. 20. Fibrous distribution of myosin in the flattened cell margin is seen (arrowheads) as well as intense staining in the small ruffles (R). x 1075.
actin, as microfilaments have been shown ultrastructurally to be composed mainly of F-a&in (Chang and Goldman, 1973; Hueser and Kirschner, 1980; Kuczmarski and Rosenbaum, 1979b; Mooseker, 1976). The immunofluorescence localization of myosin cannot be similarly supported by ultrastructural identification of thick fila-
ments in motile areas of nonmuscle cells (Fugiwara and Pollard, 1976; Mooseker et al., 1978). It may be that nonmuscle forms of myosin assemble into filaments which, although short, have a similar diameter as actin-containing microfilaments (Lazarides and Revel, 1979). Possibly, myosin filaments are not seen because they lie on the
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cytoplasmic side of the plasma membrane and on other surfaces and do not project far into the cytoplasmic compartment (Shizuta et al., 1976; Willingham et al., 1974). In any case, myosin seems closely associated with the microfilaments, because myosin is enriched in the fibrous arrays to a similar extent as is actin. As discussed below, the location and associations of myosin are important to the action of actomyosin complexes in the growth cone. We also saw a correlation between actin fibers and linear adhesions of the growth cone to polyornithinetreated coverslips. This resembles observations that the larger actin cables of fibroblastic cells insert into the plasma membrane at the sites of adhesion plaques (Abercrombie et al., 1977; Heath and Dunn, 19’78). Not every actin fiber was located at an adhesive site, but every linear adhesion of the growth cone margin to the substratum was associated with an actin fiber. It is not known how this relationship arises. A filopodium or lamellipodium may adhere initially at a single point and then continue the adhesion of the lower surface forward or backward along the axis of an existing actin fiber. Adhesive contacts of the lower surface may precede and stimulate the organization of an actin fiber, but once formed, fibers may persist after the adhesion is detached. Whatever links these features, the correlation of linear adhesive contacts with actin fibers reflects the direct and dynamic associations of the plasmalemma with cytoskeletal components within the motile areas of cells (Ash et al., 1977; Bourguignon and Singer, 1977; Condeelis, 1979; Flanagan and Koch, 1978; Koch and Smith, 1978; Toh and Hard, 1977). Like the protrusions in which they are found, these fibrous concentrations of actin and myosin are transient, but important features of the growth cone margin. Contractile forces could be generated anywhere actin and myosin interact, but the close and parallel packing of microfilaments in these particular areas have potential to produce mechanical forces which are stronger or more directed than elsewhere in the growth cone margin. As such, these fibers may dominate the activity of the growth cone. Generation of Force by Actomyosin The sliding filament hypothesis and the organization of the sarcomere are models for describing how actomyosin generates force in nonmuscle cells (Huxley, 1973; Mooseker, 1976). As in the sarcomere, the actin and myosin filaments must be anchored to other structures to transfer the forces for cell movement. Although the intrinsic polarities of actin and myosin filaments determine the directions in which force can be exerted, the degree to which the actin and myosin filaments, respectively, slide in an actomyosin interaction depends on the masses of the cellular structures linked to the actin and myosin fila-
ments and on other forces to which these structures are subject. The best-documented linkage of contractile filaments in nonmuscle cells is the attachment of actin filaments to the plasma membrane with the same polarity as actin filaments are linked to the Z-line of a sarcomere (Begg et al., 1979; Mooseker, 1976; Small et al., 1978). An actomyosin contraction involving these filaments may pull on their attachments at the cell membrane to shorten, bend or retract filopodia and other cellular protrusions. If, however, the plasma membrane has strong adhesive bonds to another surface, the actin filament may not slide, but rather draw myosin and its associated structures forward. Some important attachments which myosin filaments may have in the growth cone are with- the plasma membrane, membrane-bound structures, microtubules, and neurofilaments, or with actin filaments of opposite polarities as in a sarcomere (Bray, 1973; Mooseker, 1976; Mooseker et al., 1978). Present observations do not suggest that actomyosin interactions are involved in the protrusion of filopodia, because microfilaments are not seen attached to the membrane in a polarity so that they could slide and push the tip of a filopodium outward (Begg et al., 1979; Mooseker et al., 1978). Actomyosin Action in Nerve Fiber Growth These actomyosin complexes could function in nerve fiber growth in several ways. It is proposed that growth cones follow paths of high adhesivity to reach their synaptic targets (Letourneau, 1977). Proper pathways may be found through the exploratory activities of filopodia and lamellipodia as they extend, contact surfaces, and exert tension on these contact points (Albrecht-Buhler, 1976; Bray, 1979; Letourneau, 1979). If the adhesion is weak, the forces exerted by actin filaments on the filopodial membrane will break the adhesive bonds and pull the filopodium away. However, if the adhesion is strong, the actin filaments will not slide, but will draw the growth cone forward via associations with myosin filaments and other cell structures. Force may also be transmitted within the growth cone from stronger cell-substratum adhesions to break weaker adhesive sites in an intracellular tug-of-war, thus directing the growth cone onto the more adhesive surface (Bray, 1979). This is also suggested by observations that releasing the adhesions of filopodia at one side of a growth cone causes the growth cone to turn toward the unmanipulated side (Bray, 1979; Wessells and Nuttall, 1978). An important determinant of the pathways of axonal growth in viva is the growth of neurites along previously extended pioneer fibers (Constantine-Paton, 1979). Filopodial extensions of growth cones have been observed to adhere to neurites they meet and then pull on the contact point with sufficient force to distort the neurite (Nakai,
PAUL C. LETOURNEAU
Actin and Myosin
1960; Nakai and Kawasaki, 1959; Nakajima, 1965; Wessells et al., 1980). The strength of these initial contacts and their resistance to tension may determine whether a growth cone follows a particular pioneer fiber. Another action of actomyosin in the growth cone may be to move the various contents of the growth cone and adjoining distal neurite. Neurofilaments and microtubules may be diverted or pulled forward within the growth cone by their associations with actin and/or myosin filaments (Letourneau, 19’79). Such movement could influence the assembly site for precursors of these cytoskeletal elements or of the plasma membrane during neurite elongation. Again, the immobilization of actin filaments by linkage to areas of the plasma membrane which have strong adhesive contacts would affect the directions in which actomyosin forces move neurotubules and neurofilaments. This would constitute another way that adhesive contacts influence axonal growth. Collins (19’78) found that embryonic ciliary neurons sprout neurites within minutes of exposure to heart conditioned medium. Before neurites appear, however, the initial response is that filopodia extended from the cell margin become attached to the substratum. Perhaps actin filaments, inserted into the cell membrane at these adhesive sites, now resist sliding during actomyosin interactions. Thus, mechanical force is exerted in new directions, prompting the organization and extension of a neurite from cytoskeletal components of the cell soma. In summary, the colocalization of actin and myosin in growth cones suggests that actomyosin forces can be generated during nerve fiber growth. These contractile forces would be produced by the sliding of actin and myosin filaments, but the structures to which actin and myosin are linked may determine where sliding occurs. Thus, the associations of these actomyosin complexes with adherent portions of the cell membrane are potentially important influences on the use of mechanical energy to advance the nerve fiber and to transport intracellular structures. How these actomyosin fibers and linear adhesive contacts come to be formed in the motile areas of a nerve cell, and how the interaction of actin and myosin to generate force is regulated are important unanswered questions. The author is grateful to Dr. Judith Schollmeyer for antisera to actin and myosin and for her valuable advice. The photographic work of Gwen Watson and Tom Schuld and the secretarial skills of ‘Becca Vance were also appreciated. This work was supported by NSF Grant PCM7923907,by the Minnesota Medical Foundation, by the Graduate School of the University of Minnesota, and by the American Cancer Society Institutional Grant to the University of Minnesota. REFERENCES ABERCROMBIE,M., DUNN, G. A., and HEATH, J. P. (1977). The shape and movement of fibroblasts in culture. In “Cell and Tissue Interactions” (J. W. Lash and M. M. Burger, eds.), pp. 57-70. Raven Press, New York.
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ALBRECHT-BUHLER, G. (1976). Filopodia of spreading 3T3 cells. J. Cell Biol 69, 275-286.
ASH, J. F., LOUVARD, D., and SINGER, S. J. (1977). Antibody-induced linkages of plasma membrane proteins to intracellular actomyosincontaining filaments in cultured fibroblasts. Proc. Nat. Acad. Sci. USA 74,5584-5588.
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