Neuron,

Vol. 4, 633-642,

April,

1990, Copyright

0 1990 by Cell Press

Degradation of Underlying Extracellular Matrix by Sensory Neurons during Neurite Outgrowth Paul C. McGuire* and Nicholas Department of Biochemistry, Genetics University of Colorado Health Denver, Colorado 80262

W. Seeds Biophysics,

and

Sciences

Center

Summary The ability of differentiating sensory neurons to remodel a fibronectin substratum was examined. During the early stages of neurite outgrowth, fibronectin was cleared from areas beneath the neuronal soma and processes. The removal of fibronectin occurred in the presence and absence of plasminogen and was associated with the release of fibronectin fragments into the culture medium. The degradation of fibronectin was dependent upon neuronal contact with the substratum. Extraction of cells with the nonionic detergent Triton X-114 identified plasminogen activator and plasmin associated with the cell surface. These findings suggest that the plasminogen activatorlplasmin system may play an important role in the interaction of differentiating sensory neurons with the extracellular matrix during axonal outgrowth.

that both CNS and PNS neurons, as well as certain neural-derived tumors, produce the serine protease plasminogen activator (Krystosek and Seeds, 1981a, 1981b, 1984; Moonen et al., 1982; Pittman, 1985; Baronvan Evercooren et al., 1987) and metalloprotease (Pittman and Williams, 1988; Machida et al., 1989) activities. These and other proteases may influence the interactions of neuronal processes with the matrix and cell surface in a number of ways, including the degradation of the surrounding matrix to “clear a path” through the extracellular space, the selective remodeling of the extracellular matrix and cell surface components to alter their composition and thereby change the regulatory information that they contain (i.e., guidance, migratory, and adhesive cues), and the facilitation of a series of attachments and detachments between a growth cone and the substratum by a limited and selective cleavage of matrix or cell adhesion molecules or the turnover of specific cell surface receptors. The studies described here were designed to examinewhether remodeling of the extracellular matrix occurs during the early stages of neuronal differentiation and neurite outgrowth and which proteases might be involved.

Introduction Results Two important cellular events involved in the differentiation of sensory neurons are the extension of processes or neurite outgrowth and the recognition of targets and synapse formation. During the projection of neurites, complex interactions are likely to occur between the neuronal cell surface and the variety of cellular and extracellular macromolecules encountered in the various migratory pathways. In this respect, a number of recent studies have shown that neurons and their processes specifically interact with a variety of cell surface adhesion molecules (N-CAM, N-cadherin, Ll, and TAG-I) and components of the extracellular matrix (laminin, fibronectin, thrombospondin, tenascin, and heparin sulfate proteoglycan; Rogers et al., 1985; Hatta et al., 1985; Lander, 1987; HantazAmbroise et al., 1987; Rogers et al., 1988; Werz and Schachner, 1988; Chamak and Prochiantz, 1989). The contacts formed between a neuronal growth cone and the matrix or ceil surface must be of a transient nature to facilitate sustained movement of the growth cone. These transient contacts of the neurite with its surroundings would thus involve a series of alternating phases of adhesion and release. One mechanism that may be involved in neuronal migration and neurite outgrowth is the production of extracellular proteases and their localization near sites of cell-substratum interaction. Previous studies have demonstrated *Present Mexico

address: Department School of Medicine,

of Anatomy, Albuquerque,

University New Mexico

of New 83171.

Substrate Remodeling by Differentiating Sensory Neurons When sensory neurons from neonatal mice were plated onto coverslips coated with fibronectin, the cells rapidly responded by extending neurites within 2-4 hr (Figure IA). By 16 hr in culture, a rich network of neurites had formed over the surface of the coverslip (Figure IB). It was during this early stage of differentiation in vitro that we investigated the extent to which neurons were able to reorganize or remodel the substratum. Cell-free substrata were prepared and stained with antibodies to fibronectin and a fluorescently labeled second antibody. Using this technique, it was possible to visualize the areas of the substratum that had been altered by the neuron and its processes. Clearing of fibronectin from the coverslip surface was seen in areas occupied by the neuronal soma as well as by many individual neurites (Figure 2). When cells were grown on FITC-labeled fibronectin and examined with the cells still attached to the coverslip, a similar pattern of clearing beneath the cells was observed (data not shown). Cells grown in serum-free medium (Figure 2A) produced areas of clearing around neurites that were generally thin, except in areas where the neurite branched. These areas of slightly more extensive clearing may represent a change in the morphology of the growth cone or its interaction with the substrate at various stages of outgrowth. indeed,

merely reorganization of the substratum. After 16 hr in culture, the conditioned media and substratum were collected, and the percentage of the total radioactivity released into the medium was determined. As seen in Figure 3, 1 x IO5 neurons grown in serumfree medium without added plasminogen released approximately 12% of the total 12sl-labeled fibronectin into the medium. When plasminogen (4 ngiml) was added to the media, a significant increase in the amount of substrate material released (38.6% of the total radioactivity) was seen. In both media f-t/plasminogen) the extent of neurite outgrowth appeared similar. The conditioned media were subsequently analyzed by SDS-PAGE (Laemmli, 1970) and autoradiography. The conditioned media from cells grown in the presence of plasminogen contained a number of fibronectin cleavage products as a result of the action of plasmin that had been generated by the cellderived plasminogen activators (Figure 4, lane C). Only a small amount of the intact molecule could be detected. The media from cell cultures grown in the absenceof plasminogen or in plasminogen plus apro-

Figure 1. Phase Photomicrograph sory Neurons at Various Stages Fibronectin

of Dissociated of

Neurite

Mouse Outgrowth

Senon

Substratum

By 2 hr in culture, many of the neurons have begun to extend processes (A). After 16 hr, a rich network of neurites is seen over the surface of the substratum (B). Bar, IO pm.

others have shown that the morphology of the growth cone may change from a simple to a more complex filopodial form when the directionality of the advancing neurite changes (i.e., branching and turning; Bovolenta and Mason, 1987). When plasminogen was added to the culture medium, the removal of fibronectin appeared overall more extensive in the areas of clearing (Figure 28). With this technique, it also appeared that the pattern of fibronectin clearing was confined to the area immediately adjacent to the cell surfaces, as the remainder of the substratum around the cells and their processes appeared to be intact. Fibronectin Is Degraded by Differentiating Sensory Neurons Neurons were grown on coverslips coated with radiolabeled substrates to determine whether the observed clearing of fibronectin represented degradation or

a

tinin were also analyzed and found to contain material in the range of 440 kd (indicative of intact or neariy intact fibronectin), as well as a relative increase in the monomer forms of the molecule (220 and 210 kd). In addition, a band of lower molecular mass, at 200 kd, was also seen (Figure 4, lane B). As a control, an aliquot of the 1251-labeled fibronectin was incubated at 37OC for 16 hr and was seen to consist mainly of the 440 kd form with a very small amount of the 210 and 220 kd monomers (Figure 4, lane A); however, no 200 kd protein was seen. As seen in Figures 2A and 2B, the degradation and clearing of fibronectin by the neurons appeared to occur close to the cell surface. To investigate this possibility further, we compared the release of radiolabeled fibronectin from coverslips in direct contact with cells with that from coverslips exposed only to the conditioned rnedia of neurons grown on unlabeled substrates. Alone, the conditioned media from neurons grown in the absence of plasminogen released a small percentage of the total radioactivity (4.4%) compared with that released when the cells were grown directly on the radiolabeled macromolecule (Figure 5). Exposure to media from cells grown in the presence of plasminogen resulted in an increase in the percent release (12.2%); however, it was again considerably less than that observed when the cells were in contact with the substrate. These results suggest that the proteases involved with fibronectin degradation may be localized to the neuronal cell surface and/or its associated substratum and that the action of proteases secreted into the media may account for only a small degree of matrix degradation. Characterization To begin to differentiating

of Neuronal Proteases characterize the proteases produced sensory neurons, we attempted

to

by in-

Matrix 635

Degradation

Figure

2. Fluorescent

by Sensory

Micrographs

The absence of fibronectin minogen. (B) + plasminogen. 5 urn.

Neurons

of Fibronectin

staining is seen (C) Low-power

Clearing

by Differentiating

Sensory

Neurons

in areas occupied by both the neuronal processes (arrow) and the cell soma (S). (A) - plasview of fibronectin clearing by two sensory neurons in the absence of plasminogen. Bar,

NfXKln 636

.:

:::. ‘. lY!YzTL

:.: :., ,.::‘/:;

“’

” .’ :

.;:

!

z i

OLASMINOGEN Figure 3. Histogram Showing nectin from the Substratum sory Neurons

+PLASMINOCEN

the Release of lLsI-Labeled Fibrointo the Medium by 1 x IO5 Sen-

The total amount of ‘251-labeled fibronectin on each coverslip prior to the addition of cells was approximately 50,000 cpm (3.5 ug). Background, nonspecific release of ‘Z51-labeled fibronectin was 3%-5% of the total counts per minute. Values are the percentage of the total radioactivity associated with the coverslip with background release subtracted.

A

B

hibit the release of 125i-labeled fibronectin and its fragments using a variety of proteinase inhibitors at concentrations that were nontoxic when added to the culture medium (Table 1). inhibitors of thrombin (hirudin) and metalloproteases (FIBACITIMP) had no effect on the amount of 1251-labeled fibronectin release in the assay described. In this respect we have also been unable to detect metalloprotease activity in the culture medium of these cells by gelatin zymography (data not shown). When aprotinin (a strong inhibitor of plasmin) was added to cultures, it was found to inhibit the “plasminogen-dependent” release of 125l-labeled fibronectin, but had no significant inhibitory effect on the “plasminogen-independent” release, even at very high concentrations. Leupeptin and aminocaproic acid had a similar effect, Of all the inhibitors tested, only protease nexin (an inhibitor of thrombin, plasmin, and urokinase) significantly reduced the degree of 12sl-labeled fibronectin release in the absence of plasminogen. An antibody to the mouse tissue-type plasminogen activator was also tested and found to have no effect on the release of radioactivity

C

In 205 -

116-

men!

9?-

66-

45-

Figure

4. Biochemical

Autoradiograph and sensory neurons in alone incubated at kd band. Molecular

Analysis

of Released

‘251-Labeled

Fibronectin

associated densitometric analysis of the fibronectin fragments released into the conditioned medium the absence (lane B, 1800 cpm) or presence (lane C, 1500 cpm) of plasminogen. Lane A is rL’I-iabeled 37OC for 16 hr (1800 cpm). The arrow in tracing B of the densitometric scan indicates the appearance mass in kilodaltons is indicated.

by 1 x IO5 fibronectin of the 200

Matrix 637

Degradation

by Sensory

Neurons

50 -

40 -

m [3

-PLAStlINOGEN +PLASMINOFEN

I

30 -

go20 -

66-

IO -

380 CELL CONTACT

tlEDlA

Figure 5. Histogram Showing the Dependence on the Release of ‘Z51-Labeled Fibronectin from into the Medium by 1 x IO5 Sensory Neurons

of Cell Contact the Substratum

The total amount of ‘Wabeled fibronectin on each coverslip prior to the addition of cells was approximately 50,000 cpm (3.5 ug). Background, nonspecific release of ‘Wabeled fibronectin was 3%-5% of the total counts per minute. The values are the percentage of the total radioactivity associated with the coverslip with background release subtracted.

in the absence of plasminogen. The urokinase inhibitor amiloride was also tested; however, it was toxic to live ceils at all concentrations used (0.5,1, and 10 mM). We have also begun to characterize the neuronal protease activity by zymography in SDS gels contain-

Table

1. Characterization

of Neuronal % of Total

Protease cmp

Activity Released

Treatment

- Plasminogen

+ Plasminogen

None Aprotinin (300 U/ml) (3000 U/ml) Leupeptin (I mM) Aminocaproic acid (3 mM) Hirudin (IO U/ml) TIMP (IO Bglml) PAI(0.5 pg/ml) (1.0 ug/ml) Protease nexin Human (I vgiml) (IO &ml) Recombinant (IO ugiml) C6 glioma cell-conditioned media

12.92 (0)

38.61

(0)

12.16 (6.4) 11.35 (12.2) 14.30 (0) 12.63 (2.3) 13.30 (0) 12.50 (3.3)

13.10 12.36 11.02 14.34 36.60 ND

(65.8) (68.0) (71.5) (62.9) (5.3)

13.51 11.53

(0) (11)

ND ND

10.35 3.20 4.40 6.18

(19.9) (75.3) (66.0) (52.2)

13.76 (64.4) 8.37 (78.4) 10.88 (71.9) ND

Values represent the mean of three experiments. Those in parentheses are the percent inhibition for the given inhibitor compared with no treatment. The total amount of 1251-labeled fibronectin on each coverslip prior to the addition of cells was approximately 50,000 cpm (3.5 ug). Background, nonspecific release of 1251-labeled fibronectin was 3%-5% of the total counts per minute. The effectiveness of the above inhibitors was shown to be more than capable of inhibiting the appropriate purified enzyme in the presence of matrix substrates at the indicated concentrations. Inhibitors were incubated with an appropriate enzyme (urokinase and plasminogen, thrombin, or collagenase) in wells coated with either ‘251-labeled fibronectin or a collagen gel and incubated for 16 hr at 37OC. The release of radioactivity into the medium and the digestion of the collagen were monitored.

Figure 6. Detection Cultures

of Proteases

Associated

with

Neuronal

Cell

Dissociated sensory neurons were grown on fibronectin-coated coverslips for 16 hr in serum-free medium with added plasminogen. The conditioned medium and cell layer were collected and examined by zymography in casein-containing gels with and without incorporated plasminogen. Lane 1, conditioned medium; lane 2, Triton X-114 pellet; lane 3, Triton X-114 supernatant. (A) + plasminogen in the gel. (8) - plasminogen in the gel. Molecular mass in kilodaltons is indicated.

ing either casein or fibronectin as substrates. Proteases associated with the conditioned media as well as the membrane and soluble fractions of the neuronal cells were examined. When cells are grown in serum-free medium to which plasminogen was added, three major bands of proteolytic activity (molecular masses of 90,66, and 38 kd) were seen associated with the conditioned media (Figure 6A, lane 1). Cells grown in serum-free medium without added plasminogen showed only the 66 and 38 kd activities (data not shown). In both cases the appearance of the 66 and 38 kd activities was dependent on the presence of

plasminogen in the gel (compare Figures 6A and 6B, lane 1). In addition, the 66 kd activity was inhibited by antibodies to tissue-type plasminogen activator and the 38 kd activity was inhibited by the urokinase-type plasminogen activator inhibitor amiloride (data not shown). The 90 kd activitywas present only in the conditioned media of cells grown in the presence of plasminogen. In zymography gels, the 90 kd activity was present with or without added plasminogen (compare Figures 6A and 6B, lane 1) and was completely inhibited with the plasmin inhibitor aprotinin. Extraction of cells with the nonionic detergent Triton X-114 followed by heat-induced separation of the solution resulted in the production of two separate phases. Integral membrane proteins and molecules associated with these proteins partition wiith the nonionic detergent phase; free hydrophilic proteins remain in the aqueous phase (Bordier, 1981; Pryde, 1986). Using this technique, the 3866, and 90 kd proteolytic activities were seen in the detergent phase of the cell extract (Figure 6A, lane 2), suggesting that these three proteases may be bound to molecules on the cell surface. Again, when plasminogen was deleted from the zymography gels, only the 90 kd activity appeared (Figure 6B, lane 2). The aqueous phase (representing both the cytoplasmic compartment and the extracellular substratum) contained only the 38 and 66 kd activities (Figure 6A, lane 3). The membrane-associated proteolytic activity (Triton X-114 detergent phase) was also examined in zymography gels containing r251-labeled fibronectin as a substrate. In gels containing plasminogen, the 38, 66, and 90 kd activities became apparent (Figure 7, lane A), whereas only the 90 kd activity corresponding to plasmin was evident in gels lacking added plasminogen (Figure 7, lane B). Bythis technique, no other areas of proteolysis that might function in the plasminogen-independent cleavage of fibronectin were observed. Discussion One of the earliest events in the differentiation of neuronal cells is the production of neuritic processes. In the case of developing sensory neurons, these processes with their leading growth cones navigate through a complex and changing environment containing a number of different extracellular matrix macromolecules and cell surface components. One possible mechanism involved with the transient interaction of the growth cone with various molecules may be the production of a variety of extracellular proteases and protease inhibitors (for review see Monard, 1988). In the nervous system, plasminogen activators have been shown to be involved in the migration of granule cells in the developing cerebellum (Krystosek and Seeds, 1981b; Moonen et al., 1982). In addition, neurons of the PNS grown in vitro produce plasminogen activators that localize to the growth cone (Krystosek

A

B

go66-

38-

Figure 7. DetectIon of Neuronal “il-Labeied Fibronectin

Proteases

in Gels

Containing

Lane A, conditjoned medium; lane 6, Tutor- X-114 pellet. Molecular mass in kilodaltons 15 ;ndlcated. This figure represents a reversed image of the autoradiograph.

and Seeds, 1981a). Plasminogen activators function through the conversion of the inactive zymogen plasminogen to the broad spectrum protease plasmin. However, work by Quigley et al. (1987) suggests that other molecules (specifically fibronectin) may be alternative substrates for the plasminogen activator urokinase. The degradation of extracellular matrix at sites of contact between a growth cone and the substratum may thus help to facilitate the sustained movement of the neurite toward its target cells. Using a modification of the technique described by Chen et al. (1984; Chen and Chen, 1987), we have demonstrated the ability of differentiating sensory neurons to remodel a fibronectin substratum. We observed that sensory neurons produced areas of substrate clearing in the region of the neurite and growth cone during the initial phases of neurite outgrowth. A similar result was obtained when cells were grown in the presence of inhibitors. As this is a nonquantitative assay, it was not possible to determine whether these inhibitors had an effect on the clearing of fibronectin. In addition, using a radiolabeled substrate, we were able to confirm that this clearing involved the production of fibronectin fragments and thus degradation. Although fibronectin release was enhanced when a source of plasminogen was made available to the cells, fibronectin clearing and release also occurred in the absence of plasminogen and in the presence of plasmin inhibitors. In addition to plasminogen activators, other proteases have been implicated in neurite growth; these include thrombin (Monard et al., 1983; Curwitz and Cunningham, 1988) and metalloproteases (Pittman and Williams, 1988; Ma.chida et al., 1989). To begin to characterize the matrix-degrading activity in differen-

Matrix 639

Degradation

by Sensory

Neurons

tiating neurons shown here, we examined the release of radiolabeled fibronectin by sensory neurons grown in the presence of a variety of protease inhibitors (Table 1). Thrombin and metalloprotease inhibitors had no effect on either the plasminogen-dependent or -independent release of fibronectin. Various serine protease inhibitors that inhibit plasmin in addition to other proteases reduced the levels of plasminogendependent degradation. However, only protease nexin, a specific inhibitor of urokinase, thrombin, and plasmin (Scott and Baker, 1983; Eaton and Baker, 1983; Scott et al., 1985), was able to reduce the level of plasminogen-independent fibronectin degradation significantly. An activity (similar to protease nexin) in the conditioned media from the C6 glioma cell line also inhibited the fibronectin turnover in plasminogen-free media. This inhibitor, referred to as glialderived nexin (Guenther et al., 1985; Zurn et al., 1988), has been shown to form a complex with and inhibit the action of urokinase, while being a poor inhibitor of tissue-type plasminogen activator (Stone et al., 1987). The urokinase inhibitor amiloride was not useful in these studies as it was toxic to cells. Other studies using amiloride to inhibit cell-derived urokinase were done with fixed cells, in which toxicity was

not a problem (Pepper et al., 1987). Protease nexin, as well as other inhibitors, has been reported to influence neurite outgrowth by regulating the degree of proteolytic activity, thereby preventing the excess degradation of substrate macromolecules and promoting neurite adhesion (Hawkins and Seeds, 1986,1989; Knauer et al., 1987; Monard, 1988; Zurn et al., 1988). This protective function of protease nexin has been suggested by Reinhard et al. (1988); they showed that glial-derived nexin is present in the olfactory system associated with extracellular matrix in areas through which the processes of olfactory neurons migrate. The plasminogen activator-specific inhibitor PAI(Van Mourik et al., 1984; Kruithof et al., 1986) was unable to inhibit 1251-labeled fibronectin release and was toxic to cells at higher concentrations (10 @ml). However, this may be due to a difference in the accessibility of the cell-associated protease to this inhibitor. The results of these experiments suggest that the plasminogen-independent release of fibronectin by sensory neurons may be due to the direct action of urokinase on the substrate, similar to that previously shown in a “test-tube” assay (Quigley et al., 1987). Indeed, when the released radioactivity was examined by SDS-PAGE and autoradiography, intact and large fragments of fibronectin were present. The production of fragments with molecular masses of 220-200 kd is similar to that reported by Quigley and coworkers, who used purified preparations of urokinase and fibronectin, and is consistent with limited proteolysis of the molecule. A more recent report by Gold et al. (1988, J. Cell Biol., abstract) suggests that two sites which may be cleaved directly by urokinase exist in the fibronectin molecule. One is in the C-terminal

region immediately before the interchain disulfide bonds, and the other is at position 260-261 in the N-terminal of the molecule. Such limited cleavage would be consistent with the reduced molecular mass of the fibronectin chains seen in Figure 4, lane B. Proteolysis before the interchain disulfide bonds would permit the conversion of the dimeric fibronectin molecule into two monomeric chains, thereby breaking the cell-substratum attachments. The role of these proteases in modifying the extracellular matrix and specifically cell-matrix interactions is supported by the observation that the clearing of fibronectin appeared to be confined to the regions near the cell membrane. In addition, the release of radiolabeled fibronectin appeared to occur most dramatically when the cells were in direct contact with the substrate. By solubilizing the neurons with Triton X-114 and subjecting the material to zymography, we were able to show that urokinase, tissue-type plasminogen activator, and plasmin were all associated with the neuronal cell membrane, whereas only urokinase and tissue-type plasminogen activator were associated with the cytoplasmic compartment and/or the substratum. This suggests that sensory neurons may, like other cell types (Vassalli et al., 1985; Plow et al., 1986; Miles et al., 1988; Stephens et al., 1989; Estreicher et al., 1989), have specific binding sites or receptors on their surface for plasminogen activator and plasminogen. Such receptors may aid in the localization of the enzymes to a specific region of the cell surface so that only those substrates in the immediate area of the cell are degraded. In this respect, recent work by Verall and Seeds (1988,198V) has revealed the presence of specific tissue-type plasminogen activator binding sites on the surface of cerebellar granule cells. The present study has demonstrated that plasminogen activator in primary cultures of mouse sensory neurons can bring about the degradation of fibronectin in the vicinity of the cells by the production of plasmin. In the absence of plasminogen these cells appear also to degrade fibronectin, although to a more limited extent; this may be due to the direct action of urokinase on the fibronectin substrate. Additional studies with more specific inhibitors of urokinase (i.e., antibodies) may provide more direct evidence for this possibility. These results suggest that once the fibronectin substrate has fulfilled its function of promoting cell attachment and neurite outgrowth, it is subsequently removed by cell-associated proteases from areas of focal contact beneath the neurite. In addition, the cleavage of fibronectin by neuronal proteases may be an important step in releasing the growth cone from its attachment to the substratum, thus allowing the growth cone to proceed through the tissue in search of its target. Experimental

Procedures

Cell Preparation and Culture Cultures of sensory neurons

were

prepared

as described

by Seil-

NWtOn 640

heimer and Schachner (1988). Briefly, dorsal root ganglia (80) were dissected from I- to 3.day-old mice and incubated in a solution of either 0.1% collagenase or 0.03% collagenase and 0.25% trypsin at 370C for 40 min. Similar results were obtained with erther enzyme treatment. The ganglia were rinsed with media containing 10% fetal calf serum to inactivate the enzymes and were mechanically dissociated into single cells. The cells were collected by centrifugation, washed twice in serum-containing medium, resuspended in a small volume of serum-free medium, layered onto a cushion of 35% Percoll (Pharmacia), and centrifuged at 200 x g for 15 min. The cell pellet was washed twice with serum-free medium, and approximately 1 x 1Oi ceils were plated onto a coverslip previously coated with an appropriate substrate (see below). These “neuron-enriched” cultures were grown in serum-free Eagle’s basal medium containing IO mM HEPES, 50 ngiml 2.5s NCF, and the ITS+ additives insulin, transferrin, selenious acid, bovine serum albumin, and linoleic acid (Collaborative Research, Waltham, MA). In some cases, human plasminogen (Sigma, St. Louis, MO) was added to the medium at a final concentration of 4 ngiml. The rat glioma cell line C6 (Guenther et al., 1985) was grown in DMEM containing 10% fetal calf serum until confluent. The ceils were washed extensively in DMEM and Incubated in medium containing the ITS+ additives for an additional 48 hr to collect conditioned medium. Detection of Matrix Clearing at Sites of Cell-Substrate Contact Substrate clearing by differentiating neurons was visualized by a modification of the technique of Chen et al. (1984). Coverslips were incubated with human plasma fibronectin (IO ugiml; Collaborative Research) in 50 mM carbonate buffer (pH 9.5) for l-2 hr at room temperature and rinsed with saline prior to the addition of cells. After 16 hr in culture, the cells and their processes were released from the coverslips by incubation in calciumand magnesium-free Hanks balanced salt solution (HBSS) at 4°C for l-2 hr. The resulting cell-free matrices were briefly rinsed with HBSS containing 0.01% Triton X-100 and subsequently incubated with anti-fibronectin antiserum (1:lOO) followed by a fiuorescently labeled second antibody. Areas of matrix clearing were visualized as regions of decreased fluorescence. A srmilar decrease in fluorescence was seen under cells that attached and grew directly on FITC-labeled fibronectin. Analysis of Matrix Degradation To quantify the extent of matrix degradation, cells were seeded onto radiolabeled substrata and the release of radioactivity into the media was monitored. Fibronectin was radiolabeled with rL51, using lodo-Beads (Pierce, Rockford, IL), to a specific activity of 3 x IO1 cpmlug. The rL51-iabeled fibronectin was diluted with unlabeled fibronectin in pH 9.5 buffer to yield a specific activity of approximately 14,000 cpmiug. After incubation for l-2 hr at room temperature, approximately 3.5 ttg of fibronectin was adsorbed to the coverslip surface. Neurons (1 x 105) were seeded onto the radiolabeled substrates and incubated at 37OC in the serum-free medium described above. After 16 hr, the conditioned media and substrates were collected, and the amount of radioactivity associated with each was determined. The amount of radioactivity released into the medium was expressed as a percentage of the total radioactivity on the coverslip. Duplicate coverslips were incubated with media only to determine the amount of spontaneous or background release of the radiolabeled proterns. The conditioned media were further analyzed by SDS-PAGE (laemmli, 1970) and autoradiography to determine whether the proteins released into the media by the cells were present in an intact form or as fragments. After 16 hr in culture, the media from 4-8 coverslips of cells in each growth medium (+Iplasminogen) were collected, pooled, dialyzed and concentrated. Aliquots containing approximately 1000-3000 cpm were mixed with SDS sample buffer, boiled, and electrophoresed under nonreducing conditions in a 7.5% polyacrylamide gel. The gel was stained with Coomassie (brilliant) blue, dried, and exposed to Kodak X-Omat film at -7OOC for I-3 days. The

resulting autoradrograph was analyzed by scanning densitometry. To determine whether cell contact was required for substrate release and degradation, coverslips were split; one side was coated with unlabeled fibronectin and the other side with ‘Lsllabeled fibronectin. The neurons were seeded onto the unlabeled substrate and allowed to attach. The two coverslips were placed side by side with media continuous between the two. Following incubation for 16 hr, the medium and substrates were collected, and the percentage of released radioactivity was determrned. In other experiments the conditioned media from 16 hr cultures of cells grown on native (unlabeled) substrates were collected and incubated on radiolabeled fibronectrn for an additional 16 hr, and the release of radioactrvity was monitored. Characterization of Proteolytic Activity When indicated, cells were grown on radrolabeled substrates in media containing the following protease inhibitors: aprotinin (300-3000 U/ml; Boehringer Mannheim, Indianapolis, IN), leupeptin (1 mM; Boehringer Mannheim), aminocaproic acid (3 mM; Sigma); hirudin (IO U/ml; Sigma), FIBAC, a tissue inhibitor of metalloprotease (IO Kg/ml; Synergen Corp., Boulder, CO), PAI-I (0.5-I pgiml; American Diagnostica, Greenwich, CT), protease nexin (purified human and recombinant alpha-PN; I-10 kg/ml; In Vitron Corp., Redwood City, CA), and an inhibitor produced by glioma cell line C6 (Guenther et al., 1985) maintained in the laboratory. Cellswere grown in the presence of the inhrbitors for 16 hr, and the amount of radioactivity released was determined. To test for toxicity, duplicate coverslips of cells on unlabeled substrates in the presence and absence of the inhibitors were incubated with 10,000 cpm of lH-labeled amino acids. After 16 hr, the medium was removed, and the cells were solubilized with 5% trichloroacetic acid. The precipitate was dissolved in 0.5 N NaOH, and the amount of radioactivity incorporated into trichloroacetic acid precipitable material was determined. The proteolytic activity associated with the differentiating sensory neurons was also examined in substrate-containing SDS gels as originally described by Heussen and Dowdle (1380). In these studies, 10% polyacrylamide minigels were prepared. Either casein (I me/ml), ‘2sI-labeled fibronectin (1.6 kg/ml), or gelatin (1 mg/ml) were copolymerired into the gels. Some gels also contained human plasminogen (0.04 U/ml). After electrophoresis, the gels were incubated in 2.5% Triton X-100, rinsed in water, and incubated in HBSS containing0.02% azideovernight at 37OC. Following incubation, gels containing casein were stained with 0.125% Coomassie C. The zones of degradation were evident as clear bands against a blue background. Gels containing ‘*sllabeled substrates weredried following incubation and exposed to X-ray film at -70°C for 3-5 hr. To localize the proteoiytic activity in these cultures further, samples of the conditioned media and the cell layer were individually analyzed by zymography. Proteases associated with the membranous, cytoplasmic, and extracellular (i.e., substratum) components of the cell layer were separated by using the nonionic detergent Triton X-114 (Calbiochem, LaJolla, CA). This detergent has been used to separate integral membrane proteins from the more hydrophilic proteins present in the cell cytoplasm and extracellular space (Bordier, 1981). Cells were solubilized at 4OC with 1% Triton X-114 in 10 mM Tris-HCI, 150 mM NaCI (pH 7.4). The solution was layered onto a cushion of 6% sucrose, 75 mM iris-HCI, 150 mM NaCI, 0.06% Triton X-114, incubated at 37OC for 3 min, and centrifuged at room temperature for 3 min at 300 x g. The resulting oily pellet (detergent phase) and top aqueous phasewere collected. The aqueous phase was adjusted to 0.5% Triton X-114, layered on top of the original cushion, incubated at 37’C, and centrifuged. The detergent phase was pooled with the first, and the aqueous phase was collected and concentrated. Acknowledgments We wish

to thank

In Vitron

Corp.

for the gift of protease

nexin,

Matrix 641

Degradation

by Sensory

Neurons

Synergen Corp. for the gift of FIBAC, Dr. Barbara Johnson-Wint for the gift of rabbit collagenase, and Dr. D. Monard for supplying the C6glioma cell line. Wethank Kathy Christensen for technical assistance. This work was supported by an NIH National Research Service Award NS 08436 to P. C. M., by NSF grant BNS 8607719, and by a grant from the Muscular Dystrophy Association. N. W. S. is a recipient of a Jacob Javits Investigator Award, NIH NS 09818. Received

September

7, 1989;

revised

February

6, 1990.

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