Growth Cones Contain Myosin II Bipolar Filament Arrays

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1 Cell Motility and the Cytoskeleton 52:91 96 (2002) Growth Cones Contain Myosin II Bipolar Filament Arrays Paul C. Bridgman* Dept. of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B) that have overlapping but distinct distributions are found in growth cones. It appears that both myosin IIA and IIB may be necessary for normal nerve outgrowth and motility, but the molecular interactions responsible for their activity remain unclear. For instance, it is unknown if these myosin II isoforms produce bipolar minifilaments in growth cones similar to those observed in other nonmuscle cells. To determine if minifilaments are present in growth cones, we modified the electron microscopy preparative procedures used to detect minifilaments in other cell types. We found structures that appeared very similar to bipolar minifilaments found in noneuronal cells. They also labeled with antibodies to either myosin IIA or IIB. Thus, the activity of myosin II in growth cones is likely to be similar to that in other nonmuscle cells. Bipolar filaments interacting with oppositely oriented actin filaments will produce localized contractions or exert tension on actin networks. This activity will be responsible for the myosin II dependent motility in growth cones. Cell Motil. Cytoskeleton 52:91 96, Wiley-Liss, Inc. Key words: motility; cytoskeleton; actin; traction force; nerve outgrowth INTRODUCTION Myosin II is abundant in growth cones and the two isoforms (IIA and IIB) have overlapping but different distributions [Rochlin et al., 1995]. Myosin IIA and IIB have distinct enzymatic activities and F-actin sliding speeds [Kelley et al., 1996]. Recently a model called the dynamic contractile mechanism has been described to explain how myosin II organized as bipolar filaments could produce retrograde flow [Svitkina et al., 1997], which has been linked to traction force generation [Lin et al., 1996; Suter et al., 1998; Suter and Forscher, 2000]. In this model, myosin II pulls peripheral actin networks rearward and consolidates them into stabilized centrally located bundles. Myosin II interaction with peripheral actin arrays linked to the substrate by adhesion proteins provides a model for generating traction force. Although this model focused on the broad lamellipodia of fish keratocytes, variations of the model for the more complicated morphologies of growth cones are possible [Heidemann and Buxbaum, 1998]. Consistent with the Svitkina model, we have found that growth cone filopodia 2002 Wiley-Liss, Inc. from myosin IIB knockout mice generate significantly reduced amounts of traction force [Bridgman et al., 2001; Tullio et al., 1996]. Partially as a consequence of the reduced traction force, axonal outgrowth rates of knockout neurons are slowed [Tullio et al., 2001; Bridgman et al., 2001]. During the force production, filopodia of wild type growth cones frequently shortened in length. We think that is because the flexible substrate allows the shortening. We have not observed similar shortenings when growth cones are moving on a rigid glass substrate. It seems likely that both myosin IIA and IIB contribute to Contract grant sponsor: NIH; Contract grant number: NS *Correspondence to: Paul C. Bridgman, Washington University School of Medicine, Dept. of Anatomy and Neurobiology, 660 S. Euclid Ave, St. Louis, MO bridgmap@pcg.wustl.edu Received 3 January 2002; Accepted 8 February 2002 Published online in Wiley InterScience ( com). DOI: /cm.10038

2 92 Bridgman capable of generating force through conventional contractile schemes using bipolar filament arrays. Thus it is possible that myosin II produces the traction force required for growth cone crawling activity through a contractile mechanism. However, it is unknown if myosin II forms bipolar filaments in growth cones allowing for interaction with oppositely oriented actin filaments and contraction. To determine if bipolar filaments are present in growth cones, we modified the techniques of Svitkina et al. [1989, 1995] that allow direct visualization of bipolar filaments in rotary shadowed preparations. We found bipolar-like filament arrays that resemble those seen in fibroblasts. Furthermore, these filament arrays label for antibodies to myosin IIA and IIB. Thus, it appears both isoforms of myosin II known to be present in growth cones form bipolar filaments and thus can cause localized contractions that could contribute to traction force. MATERIALS AND METHODS Cell Culture Superior cervical ganglion neurons were cultured on laminin-coated glass coverslips as explants from embryonic rats or mice as previously described [Rochlin et al., 1995]. Fig. 1. A: A low magnification rotary shadowed image of a growth cone showing the preservation of structure including the actin cytoskeleton. B: A video-enhanced DIC sequence showing the affects of permeabilization and gelsolin treatment. 1,2: Images of the live growth cone prior to saponin treatment. 3: Immediately following saponin treatment. 4 6: A sequence showing the affects of the gelsolin treatment (3-min intervals). The growth cone appears to slowly dissolve. C: A low magnification rotary shadowed image of a growth cone following gelsolin treatment. The actin has been removed, but neurofilaments and microtubules remain along with smaller filament networks. Scale bars (A) 3.2 m, (B) 16 m, (C) 1.4 m. traction force because growth cone filopodia from the myosin IIB knockout mice still produce traction force (at reduced levels) and also shorten on flexible substrates. Shortening suggests a contractile mechanism. Myosin II is the only myosin known to be present in growth cones Preparation for Electron Microscopy We used a procedure modified from Svitkina et al. [1995]. Cells were permeabilized with a warm solution containing 0.01% saponin and 6.6 M rhodamine phalloidin in PHEM buffer (60 mm Pipes, 25 mm Hepes, 10 mm EGTA, 2 mm MgCl 2, ph 6.9) for 2 3 min. The phalloidin was necessary to prevent retraction of growth cones. It also allowed us to determine the minimum time required to remove the actin using gelsolin. Gelsolin (20 U/ml) dissolved in MES buffer [50 mm 2-(n-morpholino) ethane sulfonic acid, 0.2 mm CaCl 2, 0.5 mm DTT, 2mMMgCl 2, ph 6.5] was then added to the permeabilized cultures after mixing with an equal volume of PHEM buffer containing 0.01% saponin. Incubation was for min. The cultures were rinsed carefully with PHEM. For antibody-labeled preparations, primary antibodies were incubated for 45 min in PHEM followed by colloidal gold conjugated secondary antibodies in PHEM 0.01% Triton X 100 for 40 min. Controls were incubated with secondary antibody only. The cells were then further extracted and fixed with a solution containing 2.5% glutaraldehyde and 0.1% Triton X 100 in PHEM. The solution was changed to KM (70 mm KCl, 5mMMgCL 2 ) with multiple rinses to prevent precipitation of tannic acid. The fixed cultures were treated with 0.1% tannic acid in KM for 30 min, rinsed 4, and then

3 Bipolar Filaments in Growth Cones 93 Fig. 2. Rotary shadowed images from a gelsolin-treated endothelial cell. Regular arrays of bipolar-like filaments can be seen. Inset (top): A higher magnification image from a thick area of the cell showing the regular arrays of filaments that resembled the arrays of myosin II seen in muscle. In contrast to muscle, the arrays are compacted without a gap that would normally be associated with actin filaments. This indicates a structural head-to-head type of interaction. Inset (bottom): Irregular networks of filaments from a thin peripheral area of the same cell. Again the filaments appear to interact through head to head contacts. Scale bar 1 m; (insets) 200 nm. treated with 0.1% uranyl acetate in KM for 30 min. The samples were rinsed with water, dehydrated in ethanol, and then switched to acetone prior to critical point drying. Critical point drying was performed as previously described [Bridgman and Dailey, 1989]. Observations were made on four separate sets of cultures. Myosin II was purified from bovine brain using the method of Burridge and Bray [1975]. Rotary shadowing of purified brain myosin was performed using the glycerol spray method. Samples were sprayed onto cleaved mica, allowed to dry, and then shadowed. All samples were rotary shadowed with platinum and carbon using a Balzers freeze-etch device. They were photographed at 100 KV in a JEOL 1200EX electron microscope. Negatives were digitized using an Agfa Duoscan scanner. The visualization of gold particles in digitized images was enhanced using a segmentation protocol on selected regions of interest. This was done because digitization resulted in a reduction of the dynamic range in the images. The accuracy of the procedure was determined by checking the gold particle position on 10 magnifications of each original negative. RESULTS We adapted the method of Svitkina et al. [1995] that allows visualization of the intact or modified cytoskeleton. The procedure was successful in preserving the overall morphology and cytoskeleton of growth cones (Fig. 1A). Gelsolin treatment of permeabilized growth cones caused little detectable distortion or loss of overall growth cone morphology, but successfully removed actin filaments (Fig. 1B,C). Gelsolin-treated endothelial cells prepared by our procedure had regular arrays of filaments in thick areas normally associated with stress fibers, but loosely interconnected networks of filaments in thin peripheral areas (Fig. 2). Thus, the filaments in thin areas resembled myosin II bipolar filaments that were interacting through

4 94 Bridgman Fig. 3. Comparison of purified brain myosin II filaments to filament networks observed in gelsolin-treated growth cones. A: Rotary shadowed purified brain myosin II. Individual myosin dimers (arrowheads indicate head regions) form a loosely associated bipolar filament that has spread onto the mica surface. B: A single bipolar-like filament from a rotary shadowed growth cone. The filament is labeled at its center (arrow) with an antibody to the myosin IIB c-terminus detected by the gold particle. C: From a region of a growth cone showing a network of bipolar-like filaments. Filaments are labeled with an affinity-purified antiserum to myosin IIB. The gold particles (arrows) label the filaments approximately midway between the head-like expansions. Scale bars (A,B) 120 nm, (C) 160 nm. their head regions in fibroblast lamellae as previously described [Verkhovsky et al., 1995]. Growth cones did not have regular arrays of filaments like those observed in thick areas of endothelial cells, but most did have filament networks that resembled those seen in thin areas. Two types of filaments were observed. In the first, the individual elements of the filament networks were short thin filaments connected to expanded globular regions at both ends. These have similar dimensions to bipolar filaments that form in purified brain myosin II (Fig. 3A,B). Often the globular ends contacted other filaments forming an interconnected

5 Bipolar Filaments in Growth Cones 95 Fig. 4. A: High magnification images of filament networks labeled with an affinity-purified antiserum to myosin IIA. Similar to the labeling of myosin IIB, the gold particles tend to label the filaments between the head-like expansions. B: An image from the central region of a cone showing neurofilaments and a more elongated filament type labeled with the antiserum to myosin IIA. Scale bar 230 nm. network. These networks were always found immediately peripheral to the looping ends of microtubules suggesting that they were in the transition zone between the thin peripheral and thicker central zones of the cone [Rochlin et al., 1995]. Individual isolated bipolar-like filaments were found more peripherally (Fig. 3B). Because the gelsolin treatment removes all actin filaments, it was not possible to determine the exact location of these filaments relative to specific structures such as filopodia. When gelsolin-treated growth cones were labeled with an affinity-purified antiserum specific for myosin IIB carboxy-terminal end [Rochlin et al., 1995], many of the filament networks of this type were labeled (Fig. 3C). Four growth cones from each preparation (and each condition, myosin IIB labeled, myosin IIA labeled, and control) that had detectable filament networks were photographed at high magnification. Myosin IIB gold label was usually found near the middle of filaments halfway between the two globular regions. This is the expected location for carboxy-terminal labeling, which is located at the end of the myosin tail. The interconnected network of filaments also labeled to a lesser extent with an affinity-purified antibody specific for myosin IIA carboxy-terminal end (Fig. 4A). Controls showed a very low gold particle density ( 1 particle/10 m 2 ) and no label of filament networks. In one experiment, we prepared SCG cultures from a myosin IIB knockout mouse [Bridgman et al., 2001; Tullio et al., 1996] and labeled the preparation with the antibody specific for myosin IIB. No label was seen associated with filament networks. The second type of filament observed was longer and had period enlargements along its length consistent with a bipolar filament containing many more individual myosin molecules than the first type. This type of filament labeled only with the affinity-purified antiserum specific for myosin IIA (Fig. 4B). It was found in close association with neurofilaments and microtubules. DISCUSSION AND CONCLUSIONS The results indicate that both myosin IIA and IIB form bipolar filament networks in growth cones. Although the organization of these networks are less regular than the filament arrays seen in thick relatively stable regions of endothelial cells or fibroblasts, they resemble the more loosely organized networks that are seen in the more labile regions of these cells. This is consistent with the rapid changing and highly motile nature of growth cones. Similar to the arrangement of filament networks in fibroblasts [Verkhovsky et al., 1995], the individual filaments appear to make interconnecting structural networks through contacts by their head regions. The networks were found in the transition region between the peripheral and central zones of the cone. This is the location previously identified as having the highest concentration of myosin II A&B labeling [Rochlin et al.,

6 96 Bridgman 1995]. The existence and location of these bipolar filament networks in growth cones is consistent with their role in retrograde flow and traction force generation in lamellipodia similar to that proposed for fish keratocytes [Svitkina et al., 1997; Heidemann and Buxbaum 1998]. The more peripherally located individual bipolar filaments may be responsible for localized contractions including those that may underlie filopodial shortening observed in cultures grown on flexible substrates [Bridgman et al., 2001]. It is not clear why myosin IIA appears to organize into two different types of filaments. However, it may be related to the position and secondary interactions of myosin IIA. The elongated filament type was observed only in the central region of the cone adjacent to or intermixed with neurofilaments and microtubules. ACKNOWLEDGMENTS I thank Grady Phillips for expert technical assistance. Dr. Robert Wysolmerski provided the endothelial cell cultures used for Figure 2. REFERENCES Bridgman PC, Dailey ME The organization of myosin and actin in rapid frozen nerve growth cones. J Cell Biol 108: Bridgman PC, Dave S, Asnes CF, Tullio AN, Adelstein RS Myosin IIB is required for growth cone motility. J Neuroscience 21: Burridge K, Bray D Purification and structural analysis of myosins from brain and other non-muscle tissues. J Mol Biol 99:1 14. Heidemann SR, Buxbaum RE Cell crawling: first the motor and now the transmission. J Cell Biol 141:1 4. Kelley CA, Sellers JR, Gard DL, Dui D, Edelstein RS, Baines IC Xenopus non-muscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities. J Cell Biol 134: Lin CH, Espreafico EM, Mooseker MS, Forscher P Myosin drives retrograde F-actin flow in neuronal growth cones. Neuron 16: Rochlin MW, Itoh K, Adelstein RS, Bridgman PC Localization of myosin IIA and B isoforms in cultured neurons. J Cell Sci 108: Suter DM, Forscher P Substrate-cytoskeletal coupling as a mechanism for the regulation of growth cone motility and guidance. J Neurobiol 44: Suter DM, Errante LD, Belotserkovsky V, Forscher P The Ig superfamily cell adhesion molecule, apcam, mediates growth cone steering by substrate-cytoskeletal coupling. J Cell Biol 141: Svitkina TM, Surgucheva IG, Verkhovsky AB, Gelfand VI, Moeremans M, DeMey J Direct visualization of myosin bipolar filaments in stress fibers of cultured fibroblasts. Cell Motil Cytoskeleton 12: Svitkina TM, Verkhovsky AB, Borisy GG Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. J Struct Biol 115: Svitkina TM, Verkhovsky AB, McQuade KM, Borisy GG Analysis of the actin-myosin II system in fish epidermal keratocytes mechanism of cell body translocation. J Cell Biol 139: Tullio AN, Accili D, Ferrans VJ, Yu ZX, Takeda K, Grinberg A, Westphal H, Preston YA, Adelstein, RS Nonmuscle myosin II-B is required for normal development of the mouse heart. Proc Natl Acad Sci USA 94: Tullio AN, Bridgman PC, Tresser NJ, Chan C-C, Conti MA, Adelstein RS, Hara Y Structural abnormalities develop in brain after ablation of the gene encoding non-muscle myosin-ii-b heavy chain. J Comp Neurol 433: Verkhovsky AB, Svitkina TM, Borisy GG Myosin II filament assemblies in the active lamella of fibroblasts: their morphologies and role in the formation of actin filament bundles. J Cell Biol 131: