WWI [IHIWHU W. 126 HMO WM 9.4:: :9 LIEBRARY Michigan State University This is to certify that the thesis entitled "PC12 Neurite Outgrowth in the Absence of Microtubules on Extracellular Matrix" presented by Vivian Lynn Steel has been accepted towards fulfillment of the requirements for M. S .1 Physiology degree in Major professor Date January 20, 1989 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution _‘,..__.__.4--.-———-— v-—— - IV1£SI_J RETURNING MATERIALS: Place in book drop to usamuas remove this checkout from n your record. FINES will be charged if book is returned after the date stamped below. PC12 NEURITE OUTGROWTH IN THE ABSENCE OF MICROTUBULES ON EXTRACELLULAR MATRIX BY Vivian Lynn Steel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1989 ABSTRACT PC12 NEURITE OUTGROWTH IN THE ABSENCE OF MICROTUBULES ON EXTRACELLULAR MATRIX BY Vivian Lynn Steel The cytoskeleton has an important structural role in neurite outgrowth of PC12 cells. Actin and microtubules are in a complementary force interaction; actin is under tension which is supported partly by compression of microtubules and partly by attachment to the substratum. We wished to determine the contributions of each component of compressive support during PC12 neurite outgrowth on extracellular matrix (ECM). NSF-"primed" and "unprimed" PC12 cells exhibited extensive neurite outgrowth during days 1 to 5 on ECM compared to controls. 78% of unprimed and 77% of primed ECM-grown neurites exhibited normal microtubule arrays. Previous work demonstrated Li+-inhibition of NGF-induced PC12 neurite outgrowth, presumably due to alterations in microtubule-associated proteins required for microtubule assembly. However, we induced neurite outgrowth of both primed and unprimed PC12 cells on ECM_in the presence of LiCl: 31% of both primed and unprimed neurites examined lacked microtubules entirely. This is first evidence to date indicating neurite outgrowth in the absence of microtubules. ACKNOWLEDGEMENTS I am extremely grateful to my thesis advisor, Steve Heidemann, for his guidance, encouragement and patience during the past two and a half years. I would also like to thank my other thesis committee members, Dr. Birgit Zipser, Dr. Melvin Schindler and Dr. Seth Hootman for their help and critique of my thesis. The time I've spent as a graduate student at Michigan State would never have been the same without the fabulous people that I’ve come to know. Very special thanks (alphabetically) to Dr. Thomas Adams, Mary Lynn Bajt, John Behm, John Chimoskey, Joe Clark, Tim Dennerll, Nancy Eicker, Jeanne Foley, Harish Joshi, Phil Lamoureux, Jerry Lepar, Dave Levier, Gary Sinclair, Bob Stephenson, Leigh White, Jing Zheng, and the Physioslugs. Thank you Mom and Dad for all of your support and sage advice through my first twenty eight years...this one's for you! Finally, special thanks to my friend Steven P.H. Alberts for his patience, support and encouragement. ii TABLE OF CONTENTS Page Acknowledgements ................................... ii List of tables... ..... ... .............. .... ........ iv List of figures ...................... . ............. v Introduction: Basics of neuronal growth..... ................... 1 PC12 cells and NGF effects....................... 3 NGF induction of cytoskeletal elements........... 5 Adhesion, ECM and neurite outgrowth.............. 7 Cytoskeletal functions; complementary force model for neurite outgrowth............................ 10 PC12 neurite outgrowth in the absence of microtubules on extracellular matrix: Introduction..................................... 13 Materials and methods............. ....... . ....... 16 Results.......... ...... ........... ............... 18 Discussion........................ ...... . ........ 33 List of References..... .......................... 36 iii LIST OF TABLES page Table 1. Percentage of the number of neurites per number of cell bodies of "unprimed" PC12 cells grown on polylysine in the presence of nerve growth factor or on extracellular matrix in the absence of nerve growth factor for five days ..... 19 Table 2. Percentage of day 3 or 4 neurites containing microtubules from "primed" and "unprimed" PC12 cells grown on extracellular matrix in the presence or absence of LiCl. ....... 21 Table 3. Percentage of the number of neurites per number of cell bodies of "primed" PC12 cells grown on polylysine or on extracellular matrix in the absence of NGF for five days.............. 24 Table 4. Percentage of the number of neurites per number of cell bodies of "primed" and "unprimed" PC12 cells plated on extracellular matrix in the presence of LiCl for five days................... 28 - iv LIST OF FIGURES Electron micrograph of a "normal" microtubule array in an "unprimed" PC12 neurite grown on extracellular matrix............................ Electron micrograph of microtubule "loops" in the nerve terminal of an unprimed PC12 neurite grown on extracellular matrix................... Day 1 neurite outgrowth by "primed" PC12 cells plated onto extracellular matrix in the absence of nerve growth factor.......................... Electron micrograph of microtubule "loops" in the nerve terminal of a "primed" PC12 neurite grown on extracellular matrix................... Day 4 neurite outgrowth by "unprimed" PC12 cells grown on extracellular matrix in the presence of LiCl......................... ....... Electron micrograph of an "unprimed" PC12 neurite grown on extracellular matrix in the presence Of LiCIOOOOOOOOOOOOOOO0.0.0.00.0... Electron micrograph of a "primed" PC12 neurite grown on extracellular matrix in the presence of LiCl......................................... Page 22 23 25 27 29 3O 32 INTRODUCTION BASICS QE NEURONAL GROWTH The nervous system develops by extending numerous, long cytoplasmic outgrowths, referred to as axons and dendrites, from neuronal cell bodies. The highly asymmetric neuronal cell shape is supported by an internal "cytoskeleton" consisting of actin, neurofilaments, microtubules and a subcellular matrix (Bray and Gilbert, 1981; Ellisman and Porter, 1980: Brady et a1., 1984; Hirokawa, 1982; Schnapp and Reese, 1982; Yams .971). The role of the cytoskeleton during neuronal elong H has been a subject of interest for many years. Due to numerous complicating factors involved in the study of nerve growth in vivo, cultured nerve cells have frequently been used as a model for neuronal elongation (Alberts et a1., 1983). Many cultured cells extend processes that are difficult to distinguish as axons or dendrites and are thus referred to as "neurites". At the leading edge of the developing nerve cell process (or neurite) is an enlarged structure with an irregular spiky shape called the "growth cone", which is continuously active. It consists of a flattened region from which numerous, long microspikes 2 alternately extend, adhere to the substratum and then retract back into the growth cone (Landis, 1983). As the growth cone crawls forward it leaves the cell body in place, causing the neurite to grow in length (Alberts et a1., 1983). A process necessary for neurite elongation is the synthesis and assembly of cytoskeletal elements. Current evidence suggests that microtubules are assembled by addition of newly synthesized tubulin subunits at the distal end of the neurite (Bamburg et a1., 1986; Hirokawa et a1., 1988). New membrane is also probably added at the neurite tip. The importance of specific cytoskeletal components has been determined using drugs which inhibit their polymerization. Cytochalasin B, for example, prevents polymerization of actin into filaments. Addition of cytochalasin B to culture medium inhibits growth cone activity but allows the neurite to remain extended (Heidemann et a1., 1985; Joshi et a1., 1985; Solomon and Magendantz, 1981; Yamada et a1., 1971; Landis, 1983; Bray and Gilbert, 1981). Colchicine (which disrupts microtubules) causes the neurite to retract into the cell body, but does not inhibit growth cone activity or effect new growth cone formation (Daniels, 1972, 1973, 1975: Bray et a1., 1978: Heidemann et a1., 1985; Jacobs et al., 1986a, Joshi et a1., 1985; Solomon and Magendantz, 1981). Thus microtubules appear to stabilize the developing neurite. Substrate adhesion is another factor important during neurite elongation (Letourneau, 1975, 1979). The growth cone shows a preference for highly adhesive substrata. For example, the negatively charged plasma membrane is strongly attracted to positively charged polymers such as polyornithine and polylysine. Neurons are also strongly adherent to extracellular matrix, a naturally produced complex molecular array (Hay, 1982). It is thought to guide growth cones and control their branching, in vivg (Sanes, 1983; Hay, 1982). Besides being guided by contact interactions, the growth cone is susceptible to the effects of molecules dissolved in the extracellular fluid. An excellent example is that of nerve growth factor (NGF). Peripheral sensory neurons and many sympathetic neurons require NGF for their survival, as well as to extend neurites (Theonen et a1., 1980). NGF has been shown to act directly on the growth cone: a concentration gradient of NGF guides growth cones towards it (Theonen et a1., 1980). PC12 CELLS ND NGF EFEEQIS PC12 is a clonal cell line derived from a rat pheochromocytoma (Greene and Tischler, 1976). When cultured in the presence of NGF for several days, PC12 cells differentiate into cholinergic sympathetic neurons (Theonen et a1., 1980). They have thus proven useful as a model system for the study of NGF action on the developing neuron. 4 The responses of PC12 cells to NGF include both short- term (occurring within seconds or minutes following exposure) and long-term (occurring hours to days after exposure) effects (Greene and Tischler, 1982). The short term effects include the appearance of ruffles on the dorsal cell surface (Connolly et a1., 1979), increase in the number of coated pits per unit area of membrane (Connolly et a1., 1979, 1980), and increased cell-cell and cell-substrate adhesion (Schubert and Whitlock, 1977; Schubert et a1., 1978). In addition to these rapid onset surface alterations, PC12 cells exhibit a significant increase in the rate of transport of amino acids (McGuire and Greene, 1979) and increased phosphorylation of several proteins (Yu et a1., 1980; Halegoua and Patrick, 1980). By 24 hours following exposure to NGF, longer term effects are manifested: the cells "flatten" and exhibit short cytoplasmic spikes. Beyond twenty-four hours, the cells extend long, branching neurites (Greene and Tischler, 1976; Tischler and Greene, 1978) and somatic volume and total protein content per PC12 cell are increased (Greene and Tischler, 1976: Greene and McGuire, 1978). Electron microscopic examination of PC12 neurites exposed to NGF demonstrates the presence of parallel microtubule arrays and clusters of synaptic vesicles (Greene and Tischler, 1976; Tischler and Greene, 1978: Luckenbill-Edds et a1., 1979). Following 24-hour exposure, PC12 cells also cease cell division, become electrically excitable, and exhibit 5 increased synthesis of several proteins (Greene and Tischler, 1976, 1982; McGuire and Greene, 1980). N F INDUCTION Q_ CYTOS A ELEMENTS One of the intracellular events that appears to be essential for neurite outgrowth is the formation and extension of microtubule bundles (Daniels, 1973; Luckinbill- Edds et a1., 1979; Yamada et a1., 1970). PC12 cells exposed to NGF exhibit long, branched neurites containing extensive parallel microtubule bundles, which appear to provide the principal architectural framework for PC12 neurites (Black and Greene, 1982; Greene et a1., 1982; Luckinbill-Edds et a1., 1979; Tischler and Greene, 1978). In the absence of NGF, PC12 cells are rounded in shape, lack neuritic processes and parallel microtubule arrays (Luckinbill-Edds et a1., 1979; Tischler and Greene, 1978). The close temporal relationship between the appearance of compact microtubule bundles and outgrowth of long neurites suggests a causal relationship between these events. Various pharmacological experiments have shown that agents which prevent the formation of microtubule bundles also prevent neurite outgrowth in PC12 cells and other cultured neurons (Greene et a1., 1982; Daniels, 1972, 1973; Yamada et a1., 1970, 1971). Tubulin and microtubule associated proteins (MAPs) are the major reactants in microtubule formation. Microtubule associated proteins (MAPs) have been shown to enhance 6 microtubule assembly and stability. Several studies have shown that the abundance of tubulin and MAPS in PC12 cells increases following treatment with NGF. Greene et a1. (1983) first demonstrated that MAP-1 is synthesized and phosphorylated during neurite outgrowth. Drubin et al. (1984, 1985) found that during the first 10 days after exposure to NGF, MAP1 and tau increase 20-fold, while the tubulin pool changes less than 2.5% over this time. Black et a1. (1986) demonstrated that long-term NGF treatment increased the level of MAP-2 and influenced post- translational modification of three microtubule associated proteins referred to as the "chartins" in PC12 cells (Mr = 64K, 72K and 80K). Further investigations using purified microtubule associated proteins (MAPS) from brain or cultured cells indicate that they enhance microtubule stability (Cleveland et a1., 1977; Job et a1., 1985; Sloboda and Rosenbaum,. 1979). MAPS also apparently influence microtubule spacing (Brown and Berlin, 1985; Kim et a1., 1979) and are capable of cross-linking microtubules with other cytoskeletal components (Griffith and Pollard, 1982; Hirokawa et a1., 1985; LeTerrier et a1., 1982; Sattilaro et a1., 1981). Thus, the effects on MAPS brought about by NGF appear to contribute to the complex process of microtubule bundle formation and stability. Black and Greene (1982), observed that neurites grown for several days contained microtubules which were more 7 resistant to microtubule-depolymerizing drugs than younger neurites. Greene (1984) suggested the enhanced stability could be due to NGF-induction of MAPS and subSequent MAP- microtubule interactions known to occur after at least 3 days of growth. Indeed, NGF-induction of MAP-1 Species, MAP- 2, chartins and tau MAPS occur in parallel with NGF-induced neurite outgrowth and microtubule stability (Black et a1., 1986). Burstein et a1. (1985), demonstrated that Li+ inhibits NGF-induced neurite outgrowth from PC12 cells as well as regeneration of neurites by sympathetic neurons and NGF-primed PC12 cells. Li+ also Specifically inhibits short- term phosphorylation of three proteins (Mr = 64K, 72K and 80K) which have been identified as the chartin MAPS, but not phosphorylation of tubulin or MAP 1.2. This suggests a possible role of phosphorylated chartins in neurite outgrowth. ADHESION, Egg AND NEURITE OUTGROWTH Cell growth and differentiation in vivo is the result of a complex balance between cell-cell and cell-substrate interactions. These interactions are mediated by a unique arrangement of macromolecules which constitute the ECM. Extracellular matrix is the material that occupies and maintains Spaces between cells. In recent years, several of its components have been characterized (Sanes, 1983) and its role in neuronal development has been investigated (Hawrot, 1980; Loring et a1., 1982; Akers et a1., 1981; Baron Van- 8 Evercooren et al., 1982a,b; Carbonetto et al., 1983; Rogers et al., 1983; Edgar et a1., 1984; Lander et al., 1982; Letourneau, 1975; Carbonetto, 1984). These and other studies have established that an important function of ECM is that of cell adhesion: this is a step thought to be necessary for neuronal differentiation, outgrowth and guidance to occur. Effects of the culture substratum on neurite outgrowth are well documented. Letourneau (1975) correlated increased neuron-substratum adhesion with rapid and significant neurite outgrowth. On more adhesive substrates, the growth cone flattens out and spreads further than on poorly adhesive substrates (Letourneau, 1979). Davis et al. (1985), found that dissociated E8 chick ciliary ganglion neurons exhibited enhanced neurite initiation time and total neuritic output per neuron on laminin compared to other surfaces. Additionally, Bray et al. (1987) found that sensory neurons dissociated from lumbar dorsal root ganglia of chick embryos exhibited enhanced growth on surfaces prepared with laminin or conditioned medium compared to glass or collagen surfaces. Laminin and fibronectin, predominant glycoproteins in ECM have been shown to enhance neurite elongation of both central and peripheral neurons (Akers et al., 1981; Baron Van-Evercooren et al., 1982a; Faivre-Bauman et al., 1984; Manthorpe et a1., 1983; Rogers et al., 1983). Regional distributions of these molecules suggest their role in determination of preferred peripheral nervous system 9 neuronal pathways (Rogers et al., 1986). Although several experiments have demonstrated that laminin is the preferred substrate for neurite elongation (Smalheiser et al., 1984; Hammarback et a1., 1985; Gundersen, 1987), direct adhesion measurements Show that laminin decreases overall adhesion, arguing against the importance of adhesion during neurite elongation (Gundersen, 1987). The absence of contaminating nonneuronal cells also renders the PC12 cell line useful for investigations of neuronal adhesion mechanisms (Luduena, 1973). NGF has been Shown to enhance both cell-cell and cell-substratum adhesion in PC12 cells (Schubert and Whitlock, 1977; Chandler and Herschman, 1980; Fujii et al., 1982). Schubert (1979) suggested that neurite outgrowth could be caused Specifically by increased cell adhesiveness. However, when plated in the presence of epidermal growth factor (EGF), although PC12 cells exhibit increased cell-substratum attachment, they lack neurite outgrowth (Chandler and Herschmann, 1980), demonstrating that adhesion alone is not sufficient to explain the neurite outgrowth-promoting aspects of NGF. Turner et al. (1987) found that PC12 cells extended neurites on laminin, native collagens I/III, II and IV and on denatured collagen IV, but not on denatured collagens I/III or II, nerve growth factor or wheat germ agglutinin. 10 QIIQfiKELEIAL Eflflgilgflfit QQMELEMENIABX £9393 MQDEL £93 NEUELIE OUIQRQWTH The cytoskeleton is thought to provide the mechanical basis for cell Shape, (Alberts et al., 1983), including the highly asymmetric structure of neuronal cells (Landis, 1983). The two most important proteins present in the cytoskeleton are actin and tubulin. Specific intracellular mechanisms exist which control the assembly of these and accessory proteins from soluble protein pools in the cytoplasm into an actin network and microtubules (Alberts et al., 1983). Actin and microtubules are crucial to various cell functions, such as cell division, motility and cytoplasmic extension (Alberts et a1., 1983). Numerous studies on cultured neurons have indicated a clear cut "division of labor" of microtubules and actin filaments during growth cone motility and axonal elongation (Landis, 1983; Yamada et al., 1970). Drugs that disrupt microtubules (colchicine and colcemid) cause neurite retraction, but have no effect on growth cone motility (Daniels, 1972, 1973 and 1975; Bray et al., 1978; Heidemann et al., 1985; Jacobs et al., 1986b; Joshi et a1., 1985; Solomon and Magendantz, 1981). Cytochalasins act by inhibiting polymerization of actin filaments (Flanagin and Lin, 1980; Brown and Spudich, 1979). They have been Shown to stabilize the neurite to retraction, but also to inhibit filopodial contractility (Bray et al., 1978; Heidemann et al., 1985; Joshi et al., 1985; Solomon and Magendantz, 1981; ll Yamada et al., 1971; Bray and Gilbert, 1981; Landis, 1983). This contractility is presumably responsible for tension development in cultured neurites, which influences growth cone motility (Bray, 1979). Additionally, drugs that augment microtubule assembly stabilize neurites to retraction and Sometimes cause extension, while drugs that augment actin assembly cause retraction (Corvaja et al., 1982; Joshi et a1., 1985; Letourneau et a1., 1987; Solomon and Magendantz, 1981). Several lines of evidence have demonstrated the importance of mechanical force during axonal elongation. Bray (1979, 1984) originally demonstrated that neurites are under tension and induced neurite outgrowth using experimentally applied tension. Heidemann et al.(1985) observed neurite retraction in response to microtubule depolymerization, consistent with microtubules being under compression. Joshi et al. (1985), used retraction as an assay for neurite tension and drugs to manipulate various- cytoskeletal elements. They demonstrated that microtubules in PC12 neurites are under compression, supporting tension in the actin network: disruption of the actin network inhibited neurite retraction, while actin polymerization facilitated retraction. Depolymerization of microtubules resulted in neurite retraction, while microtubule stabilization did the opposite. Recently, direct tension measurements made in PC12 neurites using force calibrated needles supported these observations (Dennerll et al., 12 1988). This evidence supports the hypothesis that actin and microtubules are in a complementary force interaction: the actin network is under tension, which is supported in part by compression of microtubules. This complementary force interaction is similar to the force balance among structural elements that underlies the "tensegrity" (tensioned integrity) architectural principle, first developed and named by Buckminster Fuller (1961). We have used the PC12 cell line as a model system to examine the morphological and cytoskeletal changes that occur in cells grown on various substrata (extracellular matrix, polylysine) and in the presence of various molecules (nerve growth factor, lithium) dissolved in the extracellular fluid. The importance of the cytoskeleton to neuronal structure and axonal elongation is well documented (Yamada et a1., 1970; Bray and Gilbert, 1981). Most evidence suggests that axonal elongation of cultured neurons occurs by the motility and mass addition at the growth cone (Landis, 1983; Goldburg- and Burmeister, 1986; Bamburg et al., 1987). The motility of the growth cone and its ability to advance is widely regarded to depend upon actin function (Yamada et al., 1971; Landis, 1983; Forscher and Smith, 1988). Many experiments had shown that addition of cytochalasin D to elongating neurons inhibited growth cone motility and subsequent elongation (Yamada et a1., 1971; Forscher and Smith, 1988; Bray 1984, 1985; Joshi et al., 1985; Letourneau et al., 1987; Solomon and Magenstanz, 1981). However, Marsh and Letourneau (1984) demonstrated neurite outgrowth by cultured chick dorsal root ganglion cells in the presence of cytochalasin on highly adhesive substrata. The neurites observed were abnormal, being markedly curved in contrast to the straight growth typical of neurites cultured withoutl cytochalasin. We have recently studied the structural roles of the cytoskeleton in neurite outgrowth of PC12 cells. We find that actin and microtubules of PC12 neurites are in a complementary force interaction; actin iS under tension which is supported in part by compression of microtubules 13 14 (Joshi et al., 1985; Dennerll et al., 1988). In principle, neurite elongation could occur through an actin-based tensile mechanism (pulling) or through a microtubule-based compressive mechanism (Buxbaum and Heidemann, 1988). Indeed, whether the growth cone is pulling or pushing has been a source of recent controversy (Letourneau et al., 1987; Bray and White, 1988; Goldburg and Burmeister, 1986). Our data (Dennerll et al., 1988) suggests that neurite elongtion in the presence of cytochalasin (Marsh and Letourneau, 1984) or cytochalasin and taxol (Spero and Roisen, 1985; Letourneau et al., 1987) occurs under net compression. Curved growth is expected because pushing within the flexible neurite is like pushing on a rope. Under more normal growth conditions for cultured neurons, this complementary force hypothesis suggests that microtubules would need to be rather stable in order to support the tension of the actin network because compression destabilizes microtubules, tending to cause disassembly (Hill and Kirschner, 1982; Buxbaum and Heidemann, 1988). Alterations in microtubule assembly accompanying NGF-induced neurite outgrowth of PC12 cells is consistent with the need for increased microtubule stability during neurite elongation; PC12 neurite outgrowth in response to NGF is accompanied by an increase in microtubule stability to anti-microtubule drugs (Heidemann et al., 1985; Black et al., 1986), an increase in tubulin synthesis (Drubin et al., 1985; Black et al., 1986) and a marked increase in synthesis of assembly-promoting microtubule 15 associated proteins (Drubin et al., 1985; Black et al., 1986; Greene et al., 1983; Peng et al., 1985; Magendantz and Solomon, 1985). We found the reports of Vlodavsky et al. (1982) and Fujii et a1. (1982) particularly interesting in respect to our complementary force model and the role of microtubules in neurite elongation. These workers showed that PC12 cells grown on extracellular matrix (ECM) in the absence of NGF induced rapid but transient neurite outgrowth. They concluded that ECM mimics some of the activity of NGF. We were interested in the role of microtubules in this rapid neurite outgrowth. However, neurite outgrowth on ECM appears to be too rapid to require the transcriptionally-dependent changes in microtubule stability that accompany NGF-induced neurite outgrowth. Does the increased adhesion of PC12 cells to ECM relative to other substrates (Sanes, 1983) permit all of the putative compressive support to be provided by attachment to the dish? If so, are microtubules now expendable for neuronal growth? We have extended the work of Vlodavsky et al. (1982) and Fujii et al. (1982) with particular emphasis on the occurrence and need for microtubules in ECM-induced outgrowth of PC12 neurites. MAIEBLALé AND MEIEQQfi PC12 cells were cultured as previously described (Heidemann et al., 1985). "Primed" cells (Burstein and Greene, 1978) were grown for 6 days in RPMI-l640 medium (Gibco Laboratories, Grand Island, NY) containing 10% horse serum, 5% fetal bovine serum and 50 ng/ml 7s NGF. Cultures were maintained at 37° C in a humidified atmosphere containing 10% C02. "Unprimed" cells were grown under the same conditions excluding NGF. At the beginning of an experiment, cells (primed or unprimed) were triturated from the surface of the dish and replated at a density of 5 x 104 onto 60 mm tissue culture dishes with varying substratum treatments. Preliminary experiments Showed that at this density neurite outgrowth was maximized on extracellular matrix. One experimental group was grown on tissue culture dishes (Corning Medical and Scientific Corporation, Medfield, MA) treated with 1 mg/ml polylysine for 30 minutes and then rinsed twice with sterile distilled water. Another group was grown on commercially obtained dishes with extracellular matrix coated culture surfaces (Extracell dishes, coated with extracellular matrix produced from corneal endothelial cells; Accurate Chemical and Scientific Corporation, Westbury, NY). All cells were maintained in RPMI-1640 containing horse and fetal bovine sera as above in the presence or absence of additional experimental 16 17 variables, 50 ng/ml NGF and 10 mM LiCl, as described in Results. Neurite outgrowth was assessed quantitatively by an assay similar to that previously described (Heidemann et a1., 1985). On day 1 of the experiment, cultures were examined using an inverted phase microscope and four 1 mm circles were marked on the bottom of culture dishes. On days 1-5, the total number of neurites and cell bodies for a given experimental treatment were counted from photographs that surveyed the circled regions. Cells were fixed, processed and observed by transmission electron microscopy as previously described (Baas and Heidemann, 1986). RESULTS We confirmed the results of Vlodavsky et al. (1982) and Fujii et al. (1982) that ECM stimulates a rapid, but temporary, outgrowth of PC12 neurites in the absence of nerve growth factor (NGF). Table 1 summarizes experiments comparing neurite outgrowth of PC12 cells treated with nerve growth factor plated on polylysine with the neurite outgrowth of PC12 on extracellular matrix (ECM) without nerve growth factor. An outgrowth extending from the cell body was specified as a neurite if it was greater than two cell bodies in length; these were more selective criteria than used in past studies (Heidemann et al., 1985). Data collection in some experiments was conducted only between days 3 to 5 due to culture contamination. The quantitative decreased number of cells observed on Day 4 were due to photographic difficulties during one experiment. On ECM, the apparent peak of neurite outgrowth occurred on day 3 when there were nearly as many neurites as cell bodies, compared to about 20% frequency of neurites: cell body on nerve growth factor stimulated cells. We were surprised by the high, but transient, neurite frequency for NGF-treated cells on day 2; this was a combined value of four experiments which Showed some numerical variability on this day. We examined by transmission electron microscopy five neurites grown on ECM for three or four days. Consistent with the results of Fujii et al. (1982), 78% had a 18 Table 1: Percentage of the number of neurites per number of cell bodies of "unprimed" PC12 cells grown on polylysine in the presence of nerve growth factor (NGF) or on extracellular matrix in the absence of NGF for five days. Extracellular matrix w/o NGF Polylysine Day w/ NGF 1 34/474 7% 2 201/445 45% 3 170/1091 16% 4 74/608 12% 5 226/1276 18% 232/637 333/660 751/769 345/568 232/316 36% 50% 98% 61% 73% 19 20 microtubule array (Table 2, Fig. 1) that we judged as being within the normal density range based on our previous ultrastructural observations of NGF-induced neurites (Joshi et al., 1985). We were able to observe the terminal of seven neurites and found that two of these contained "loops" of microtubules (Fig. 2), similar to those previously observed by Tsui et al. (1984) and Letourneau and Ressler (1984). PC12 cells that have previously been exposed to NGF for several days are able to regrow significant numbers of neurites within a day (Burstein and Greene, 1978). This regrowth of "primed" PC12 cells on collagen or polylysine treated surfaces requires the presence of NGF (Burstein et a1., 1978). We wished to determine whether ECM could substitute for NGF in the rapid regrowth of primed PC12 cells. Replating primed PC12 cells onto ECM in the absence of NGF produced neurite outgrowth, in marked contrast to primed cells plated on polylysine without NGF (Table 3). In many experiments, the neurites stimulated by ECM were the longest and most rapidly growing we have seen for PC12, even after only one day of outgrowth (Fig. 3). These neurites contained microtubule arrays of apparently normal density: 37 of the 48 neurites examined (77%) contained normal microtubule arrays, similar to those of unprimed PC12 cells grown on ECM (Table 2). Of the 39 primed nerve terminals examined ultrastructurally, 44% of the terminals on ECM contained "loops" of microtubules, similar to the loops observed in unprimed cells (Tsui et al., 1984; Letourneau Table 2: Percentage of day 3 or 4 neurites containing microtubules from "primed" and "unprimed" PC12 cells grown on extracellular matrix in the presence or absence of LiCl. Treatment + - o "unprimed" on ECM (n=5) 78% 22% 0% "primed" on ECM (n=48) 77% 19% 4% "unprimed" on ECM 45% 24% - 31% + LiCl (n=29) "primed" on ECM 36% 33% 31% + LiCl (n=31) + = presence of "average" number of microtubules - = negligible number of microtubules o = absence of microtubules 21 array in an "unprimed" PC12 neurite grown on Figure 1. Electron micrograph of a "normal" microtubule extracellular matrix. Figure 2. Electron micrograph of microtubule "loops" in the nerve terminal of an unprimed PC12 neurite grown on extracellular matrix. 23 Table 3: Percentage of the number of neurites per number of cell bodies of "primed" PC12 cells grown on polylysine or on extracellular matrix in the absence of NGF for five days. Extracellular Day Polylysine matrix 1 0/73 0% 53/109 49% 2 0/76 0% 47/152 31% 3 0/123 0% 91/215 42% 4 0/129 0% 54/203 26% 5 0/105 0% 47/204 23% 24 Figure 3. Day 1 neurite outgrowth of_"primed" PC12 cells plated onto extracellular matrix in the absence of nerve growth factor. 25 26 and Ressler, 1984; Fig. 4). Several nerve terminals also contained a large number of ribosomes (Fig. 4), Similar to those described by Fujii et al. (1982). LiCl has been shown to block neurite outgrowth, apparently by its ability to selectively inhibit phosphorylation of three microtubule associated proteins (MAPs) identified as the "chartins", which have been Shown to have a role in neurite outgrowth and appear to stimulate MT assembly when phosphorylated (Burstein et al., 1985). Addition of 10 uM LiCl to medium containing NGF suppressed neurite outgrowth on polylysine treated surfaces; less than 10% of cell bodies produced neurites during five days of observation (data not Shown), as previously reported (Burstein et al., 1985). However, both primed and unprimed PC12 cells plated in the presence of this Li+ concentration showed significant neurite outgrowth (Table 4, Fig. 5), although there was less neurite outgrowth than in Li+-free conditions (Tables 1 and 3; Fig. 3). Electron microscopic examination of Li+-treated neurites of unprimed PC12 cells grown on ECM revealed that only 45% of the neurites examined contained normal microtubule arrays compared to 78% in control ECM neurites. Additionally 9 of 29 Li+-treated, unprimed cells were substantially devoid of microtubules (Table 2; Fig. 6). Less than 5% of neurites grown in Li+-free conditions were similarly devoid of microtubules (Table 2). Similarly, primed PC12 cells exhibited a significant decrease in microtubules. Ten of 31 Figure 4. Electron micrograph of microtubule "loops" in the nerve terminal of a “primed" PC12 neurite grown on extracellular matrix. 27 Table 4. Percentage of the number of neurites per number of cell bodies of "primed" and "unprimed" PC12 cells plated on extracellular matrix in the presence of LiCl for five days. Day "unprimed" "primed" 1 127/567 22% 10/67 15% 2 239/560 43% 31/111 28% 3 200/598 33% 28/105 27% 4 162/300 54% ‘ 27/69 39% 5 39/238 17% 15/66 22% 28 Figure 5. Day 4 neurite outgrowth by "unprimed" PC12 cells grown on extracellular matrix in the presence of LiCl . 29 Figure 6. Electron micrograph of an "unprimed" PC12 neurite grown on extracellular matrix in the presence of LiCl. 30 31 neurites examined were substantially devoid of microtubules. AS in unprimed cells, only short, ambiguous tubular elements were observed (Fig. 7; Table 2).' Figure 7. Electron micrograph of a "primed" PC12 neurite grown on extracellular matrix in the presence of LiCl . 32 DI§§Q§§19N Our results confirm and extend those of Vlodavsky et al. (1982) and Fujii et al. (1982) that extracellular matrix (ECM) substitutes in some way for the action of nerve growth factor (NGF) in the initial stages of neurite outgrowth. PC12 cells on ECM that had not been previously exposed to NGF Showed a peak of neurite outgrowth on day 3 after plating; outgrowth declining rapidly thereafter. It is postulated that the delay in neurite outgrowth following exposure to NGF is due to an RNA transcription-dependent process called "priming" (Burstein et al., 1978). Plating NGF-primed cells onto ECM promotes rapid neurite outgrowth, again suggesting the substitution of ECM for the normally required continued presence of NGF. Indeed, outgrowth of neurites from primed cells on ECM was the most rapid and extensive that we have observed in PC12. Plating primed cells onto polylysine in the same NGF-free medium produced no neurite outgrowth as previously reported (Burstein et al., 1985). Because PC12 neurite outgrowth is closely correlated with microtubule assembly and stability (Black et al., 1986; Joshi et a1., 1985; Dennerll et a1., 1988), we postulated that the ECM effect might include an effect on promoting microtubule polymerization, organization and/or stability. Using electron microScopy, we found that 80% of the neurites of NGF or ECM grown cells, both primed and unprimed, 33 34 contained normally extensive arrays of microtubules. We take this to indicate that microtubule assembly on ECM is as least as active as with NGF. Additionally, a Significant fraction of ECM neurite terminals contained unusual microtubule "loops". Such loops were not observed in NGF- induced neurites in this nor in previous ultrastructural studies of PC12 (Joshi et al., 1985; Heidemann et al., 1985). Similar loops appear, however, in chick sensory neurons exposed to the MT-assembly-promoter, taxol (Letourneau and Ressler, 1974) and in embryonic chick retinal neurons (Tsui et al., 1984). Like these workers, we suggest microtubule loops occur due to increased microtubule polymerization. To our surprise, we have found that microtubule assembly is not required for neurite outgrowth: LiCl, which has been Shown to inhibit NGF-induced neurite outgrowth of PC12 cells, presumably by inhibiting phosphorylation of certain microtubule associated proteins, does not prevent neurite outgrowth on ECM. Approximately 30% of the neurites grown on ECM in the presence of LiCl appeared completely devoid of microtubules. To our knowledge, this is the first report of neurite outgrowth in the absence of microtubules. Mechanical measurements on PC12 neurites indicate that microtubules provide compressive support to the tensile neurites (Dennerll et al., 1988). Thermodynamics predicts that compression will destabilize microtubules and promote their disassembly (Hill and Kirschner, 1982; Buxbaum and 35 Heidemann, 1988). Experimentally, increased compression of PC12 neurites resulted in an increase in the soluble tubulin pool (Dennerll et al., 1988). 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