‘\leuron,

Vol. 6, 595-606,

April,

1991, Copyright

0 1991 by Cell Press

‘Transfected F3/Fll Neuronal Cell Surface Mediates Intercellular Adhesion and Promotes Neurite Outgrowth Cianfranco Cennarini,*+ Pascale Durbec,* Annie Boned,* Genevieve Rougon,* and Christo Goridis* *Centre d’lmmunologie INSERM-CNRS de Marseille-Luminy Case 906 F-l 3288 Marseille Cedex 9 France tlstituto di Fisiologia Umana FacoltP di Medicina e Chirurgia Universita di Bari I-70124 Bari Italy ‘Biologic de la Differentiation Cellulaire URA CNRS 179 Case 901 F-13288 Marseille Cedex 9 France

Summary The mouse neuronal F3 glycoprotein and its chicken homolog Fll belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F34ransfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/Fll and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance. Introduction Cell adhesion plays a central role in many stages of neurogenesis including the migration of neuroblasts, the guidance and fasciculation of axons, and myelination (for reviews see Edelman, 1984; Rutishauser and Jessell, 1988; Jessell, 1988; Doherty and Walsh, 1989). Various cell surface proteins have been identified in nervous tissue that mediate adhesive interactions between neural cells or their processes when tested in appropriate in vitro systems. On the basis of their predicted primary structure, adhesive proteins have been grouped into larger families, the members of which include both neuronally and nonneuronally expressed molecules. Most cell-cell adhesion molecules known to date that are expressed in nervous

Protein

tissue belong to the immunoglobulin superfamily (Williams and Barclay, 1988). These include NCAM (neural cell adhesion molecule) (Barthels et al., 1987; Cunningham et al., 1987), Ll (Moos et al., 1988), MAG (myelin-associated glycoprotein) (Salzer et al., 1987; Lai et al., 1987; Johnson et al., 1989), and PO (Lemke and Axel, 1985; Filbin et al., 1990) in vertebrates, and several cell adhesion molecules from insects (Harrelson and Goodman, 1988). The recent cloning and sequencing of the neuronal surface proteins, Fll in chicken (Brummendorf et al., 1989), F3 in mouse(Gennarini et al., 1989b), and TAG-l in the rat (Furley et al., 1990), have led to the definition of a new subgroup of proteins among the members of the immunoglobulin superfamily. They share the same overall structure with six immunoglobulin domains of the C2 type (Williams and Barclay, 1988) in their NH*-terminal half, a premembrane region containing segmentswith sequence similaritytofibronectin type III repeats and a glycophosphatidylinositol (GPI) membrane anchor, and exhibit extensive nucleotide and protein sequence homologies. The sequence of the chicken neuronal protein contactin (Ranscht, 1988) matches the Fll sequence over the extracellular domains, but has been reported to possessatrans-membraneandacytoplasmicdomain.The high sequence similarity between F3 and Fll (77% at the protein level) makes it likely that the two proteins are species homologs, and we propose to use the term F31Fll for both of them. The expression of these proteins is higher in developing than in mature nervous tissue. F3/Fll mRNA expression in the mouseforebrain,for instance, peaks during the first two postnatal weeks followed by an 8-fold drop into adulthood (Gennarini et al., 1989b). All four proteins are more prominently expressed on neuronal processes than on the corresponding cell bodies. This has been particularly well documented for TAG-I, which is detected on commissural axons of the embryonic spinal cord but is absent from their cell bodies (Dodd et al., 1988). Moreover, TAG-1 expression seems to be confined to the initial part of these axons before they cross the midline. Although such a pronounced specificity of localization has not been demonstrated for F3/Fll, these proteins were also found to be markedly enriched in neuropil and fiber tracts of the two central nervous system regions studied, retina and cerebellum (Rathjen et al., 1987; Gennarini et al., 1989a). Furthermore, F3/Fll seemed to be confined to neurites and neurite bundles in mouse fetal brain cultures grown in synthetic medium (Gennarini et al., 1989b). The FYFlliTAG-1 group of proteins is significantly more closely related to the neuronal adhesion molecule Ll than to other members of the immunoglobulin superfamily (Gennarini et al., 1989b; Briimmendorf et al., 1989; Furley et al., 1990).

NeUrOll 596

Ll also ization Bartsch

shareswith them (Schachner et et al., 1989).

a predominantly al., 1985; Dodd

axonal et al.,

local1988;

Few studies have addressed the function of the F3/ FlliTAG-1 set of proteins, although their structural similarity to known adhesion molecules suggests that they are likely to mediate adhesive interactions. Antibody perturbation experiments in the chick indicate that F3/Fll may be involved in the elongation of neurites from sympathetic neurons on other neurites (Chang et al., 1987) and in the fasciculation of retinal axons (Rathjen et al., 1987). Purified TAG-l, on the other hand, has been shown to be a potent substrate for neurite outgrowth (Furley et al., 1990). In the present study, we probed the function of F3/Fll using a DNA transfection approach. Mouse F3/Fll cDNA was transfected into a line of Chinese hamster ovary (CHO) cells selected for a relatively low spontaneous tendency to form aggregates (Benchimol et al., 1989). We found that CHO cells expressing the protein at thecell surface, but not F3negativecelIs, formed large aggregates in suspension and aggregated with faster kinetics than did the parental cells or an F3-negative clone obtained in the same transfection. When used as a substrate for the growth of sensory neurons, the F3-expressing CHO cells stimulated neurite outgrowth as compared with control CHO cells. In addition, biochemical analysis of the transfected cells establishes that F3 cDNA codes for a GPI-anchored protein with an apparent M, of 130 kd for its N-deglycosylated form.

Figure

1. Antibody

Staining

of Transfected

LR-73 Cells

Live cultures of clone A10 were stained with anti-F3 fusion protein antibodies and photographed through phase (A) and fluorescence (B) optics. Bar = 20 pm.

Results Expression of F3 Protein by CHO Cells A construct encompassing the entire F3 cDNA coding sequence was cloned into the pRC/CMV vector downstream of the strong cytomegalovirus (CMV) promoter. CHO cells from the LR-73 line were chosen for transfecting the construct since they readily express GPI-anchored moleculesat their cell surface and have been selected for their low tendency to form aggregates in suspension, thus providing a suitable model for

functional studies (Benchimol et al., 1989). Of 16 independent transfectants obtained by growth in G41Scontaining medium, 9 expressed detectable levels of the F3 glycoprotein at the cell surface. Clones A10 and E12, which consistently showed high cell surface expression, were used throughout. An example of immunostaining of A10 cells is shown in Figure 1. The parental LR-73 cell line was negative with both first and second generation F3 antibodies (results not shown). As previously reported (Gennarini et al., 1989a, 1989b), in mouse brain extracts anti-F3 sera detect a major 135 kd glycoprotein species that exists both in soluble and membrane-associated forms. To study the molecular forms expressed by the CHO transfectants, detergent extracts of the cells and their culture super-

natants

were

analyzed

by immunoblotting

with

F3 an-

tibodies. A doublet composed of 142 and 135 kd bands was observed in the cell extracts (Figure 2A, lanes b and d), while culture supernatants contained only the larger component (lanes c and e). The lower band of the doublet comigrated with the endogenous protein expressed in mouse brain (lane a). Since identical amounts of protein were loaded on all slots of the gel shown in Figure 2A, similar levels of F3 are expressed by brain and transfected CHO cells, and the functional results shown below cannot be attributed to overexpression of the protein. No immunoreactive material was detected in the parental cells and their culture supernatants whether first generation (lanes f and g) or anti-fusion protein (data not shown) antibodies were used. To test the possibility that the two bands revealed in the transfected cells are glycosylation variants, the material isolated by immunoprecipitation from detergent extracts of the El2 cell line was digested with glycopeptidase F (PNGase F). After enzymatic deglycosylation, the 142 and 135 kd components migrated as a single species with an M, of 130 kd (Figure 2A, lane i), suggesting that the two forms differed in the extent

F3/Fll 597

Functions

in Cell Adhesion

and Neurite

Outgrowth

Figure Protein tants

2. Biochemical Analysis of the F3 Expressed by CHO Cell Transfec-

(A) lmmunoblot analysis and effect of deglycosylation. Monolayers of the two 142 kD transfected cell lines Al0 (lanes b and c) 135 kD and El2 (lanes d and e) and the parental LR-73 line (lanes f and g) were harvested with EDTA and NP-40 extracts of the ceils (lanes b, d, and f) or their culture supernatants (lanes c, e, and g) analyzed by immub c d e f Dab c d noblotting with first generation F3 antiserum. Lane a shows the pattern given by an extract of adult mouse brain. In parallel, F3-immunoreactive material isolated from El2 extracts by immunoprecipitation with 142 kD first generation F3 antibodies was incu135 kD bated in the presence or absence of PNCase F and then analyzed by immunoblotting with the same antibodies (lanes h and 0 30 min 90 min Trypsin 0 0.1 1 10 1 PIPLC _ + i). In the latter experiments, a 170 kd band C SN C SN P SN P S’N was also detected. The status of this band ,s not clear at present. It was only observed in immunoblots after prior enrichment by immunoprecipitation and may represent material !hat binds nonspecifically to immunoglobulin since it was also detected when preimmune serum was used instead of anti-F3 serum idata not shown). (B) Biosynthetic labeling of the F3 glycoprotein expressed by the CHO transfectants. Monolayers of clone El2 were pulse-labeled for IO min with [3SS]methionine. The cells were then either immediately recovered or chased in complete medium containing 2 mM rnethionine for 30 and 90 min periods. NP-40 extracts of the cells (lanes a, b,and d) or culture supernatants (lanes c and e) were then analyzed byimmunoprecipitation with the anti-fusion protein serum. C = cells,SN = culture supernatants. (C) Trypsin sensitivity of the 142 kd chain on intact cells. Cells (3 x 105) were incubated (1 hr at room temperature) in the absence (lane h) or in the presence of trypsin at 0.1, 1, or 10 mg/ml as indicated. After stopping the digestion with soybean trypsin inhibitor, detergent c>xtracts of the cells wereanalyzed by immunoblotting with anti-F3 fusion protein antibodies. For control, the digestion (1 mg/ml trypsin) was done in the presence of 1% NP-40 (lane f). Lane a represents the pattern obtained with anti-fusion protein antibodies on mouse brain detergent extracts. (D) The 142, but not the 135 kd chain, is released by PI-PLC. The cells (3 x 105 in 100 pl of PI-PLC buffer) were incubated in the absence (lanes a and b) or presence (c and d) of 0.1 U of PI-PLC. NP-40 extracts of the cells (P, pellet; lanes a and c) and their incubation medium (iN, supernatants; lanes b and d) were then analyzed by immunoblotting with the anti-fusion protein serum.

of N-glycosy1ation.A possible precursor-product relationship between them was explored by pulse-chase experiments. El2 transfectants were pulse-labeled with [“5S]m$hionine and harvested after different chase periods. F3 antigen was then isolated by immunoprecipitation from cell extracts or culture supernatants. As shown in Figure 2B, only the 135 kd component could be detected immediately after the pulse; the 142 kd component had appeared in cell extracts after 30 min of chase, while no band was yet detected in the culture supernatant. The 142 kd component became the predominant molecular species after a 90 minchaseperiodwhen itwasalsofoundintheculture supernatant. We then asked whether one or both proteins were expressed at the cell surface. To this end, CHO transfectants were treated in suspension with a range 0.i: trypsin concentrations, and the F3 chains present were analyzed by immunoblotting. The 135 kd component was hardly affected by up to IO mg/ml trypsin unless digestion was performed in the presence of detergents, whereas the 142 kd species had already become faint at 0.1 mg/ml and had disappeared at 1 mg/ml (Figure 2C). Hence, only the larger of the two species seems to be exposed at the cell surface. This conclusion was confirmed by phospholipase C

digestion experiments of intact cells. F3/Fll has been shown to beanchored in the membrane by phosphatidylinositol and to be released in soluble form upon digestion by phosphatidylinositol-specific phospholipase C (PI-PLC) (Gennarini et al., 1989b; Wolff et al., 1989). Incubation of the El2 transfectants in the presence of PI-PLC dramatically increased the release of the 142 kd form into the medium (compare lanes b and d of Figure 2D). In the same experiment, a soluble form of the 135 kd component was not detected. The 142 kd species still recovered with the cells after PI-PLC treatment resolved into two closely spaced bands. It may be that a minor, PI-PLC-resistant variant form exists, the presence of which is masked in untreated cells by the large excess of the PI-PLC-sensitive molecules. Similar PI-PLC-resistant forms of GPIanchored molecules have been detected in other systems (Futerman et al., 1985). Together, the above results show that transfected CHO lines produce two major glycosylation variants from the transfected cDNA, of which the 142 kd species represents the mature cell surface-expressed form. F3 Expression Correlates with increased Adhesiveness Mouse F3 and its chicken homolog Fll have been

Neuron 598

Figure 3. Aggregation Patterns CHO Cells and F3 Transfectants

of Parental

Monolayers of parental (A) or transfected (B) cells (clone E1’2) were dissociated in EDTA and incubated (2 x IO6 cells/ml in completemedium) in bacteriological grade petri dishes for 4 hr in a CO, incubator before being observed through phase optics.

suspected to mediate cell-cell adhesion because of their structural similarity with known cell adhesion molecules (Gennarini et al., 1989b; Briimmendorf et al., 1989) and becauseof the inhibition of axon fasciculation by anti-F11 antibodies (Rathjen et al., 1987). We therefore investigated whether expression of F3 cDNA conferred new adhesive properties to the parental LR-73 cells. In a first series of experiments, single-cell suspensions were prepared by trituration in the presence of EDTA, a treatment that leaves cell surface F3 molecules intact (see Figure 2A), resuspended in complete medium, and incubated for various times in bacteriological grade petri dishes that do not allow cell attachment. In these conditions, F3-expressing El2 cells showed a marked propensity for aggregation (Figure 3B), while the parental LR-73 cells (Figure 3A) or C418-resistant F3-negative transfectants (data not shown) formed much fewer and smaller aggregates. After a4 hr incubation period, most control cells had settled to the bottom of the dish as single cells forming only occasional aggregates, whereas most of the transfectants had assembled into large aggregates. When the mean size of the aggregates that had formed was determined, values of 11.7 + 0.7 and 22.8 t 1.4 cells per aggregate were obtained for control and El2 cells, respectively (mean f standard error of the mean [SEMI for 22 aggregates). Similar differences in the aggregation behavior could already be observed after a 1 hr incubation. To provide quantitative data on the increased adhesiveness of F3-expressing CHO cells, a widely used assay for adhesion (see, for example, Brackenbury et al., 1981) was applied that measures the rate of disappearance of single cells over time. Cells dissociated as described above were left to aggregate in polystyrene tubes with or without application of rotational forces. When the tubes were either rotated at different

speeds or the cell suspension agitated by means of a stirring bar, only marginal differences in aggregate formation between F3-positive and -negative cells were observed (data not shown). These assay conditions, while increasing the probability of intercellular collisions, also generate sheer forces that may disrupt relativelyweak interactions between cell surface molecules. A similar situation has been described for NCAM-mediated adhesion of 3T3 cells transfected with NCAM cDNA (Pizzey et al., 1989). However, when the assay was conducted in still medium, a consistent difference in adhesiveness between F3-positive transfectants and parental LR-73 cells or control transfectants was reproducibly observed. The kinetics of disappearance of single cells are shown in Figure 4A for untransfected cells and clone E12. A statistically significant difference in the aggregation kinetics between both types of cells was already observed at the 15 min time point. As shown in Figure 4B for the 30 min time point, the cells of another F3-expressing transfectant (clone AIO) aggregated with very similar kinetics as did El2 cells, whereas there was no difference between the aggregation of a sibling clone (clone 41) recovered from the same transfection that had become F3 negative over time and that of the parental cells. Also, prior incubation of El2 cells in the presence of PI-PLC abolished the difference between them and the control cells (Figure4B), as expected if the increased adhesiveness of the transfectants is mediated by the 142 kd cell surface-expressed form of F3. Finally, we tested whether Fab’ fragments prepared from anti-fusion protein antiserum would impair the enhanced aggregation of the El2 cells. In the presence of the antibodies, inhibition of aggregate formation by the transfectants was indeed observed (Figure4C). At theconcentration used (0.5 mglml), the inhibition of aggregation was

.3/Fll ;99

Functions

in Cell Adhesion

and Neurite

Outgrowth

A 100

*

LR-73

U

El2

E121LR73 lh30

(I) Ii Y

60

s z

40

8 8

20

Y

800 LIT!!!? 0

15

30

45

60 MIN

O-20

20-40

40-60

60-80

80-100

I3

% OF LABELED

100 l/l j

60

Figure 5. Analysis of the Composition Mixtures of Parental and Transfected

60

El2 transfectants either unlabeled batedforeither formed reveals

Y y W ,$ b 8

40

CELLS of Aggregates CHO Cells

Formed

by

were labeled with dil, mixed at a I:1 ratio with parental or F3-transfected CHO cells, and incu1.5or4 hr.Thepercentdistributionofaggregates a unimodal distribution in all conditions.

20

30 MIN

U -)*

0

t igure 4. Short-Term trol CHO Cells

20

40

Aggregation

El2 + Fab control E12+FabF3 LR-73 + Fab control -R-73 + Fab F3

60 MIN of F3 Transfectants

and Con-

(A) Kinetics of aggregation. Cells (clone E12, squares; parental l-R-73 cells, triangles) were dissociated in EDTA and incubated (2 < IO6 cells/ml in complete medium) in FCS-coated polystyrene tubes for 60 min at 37°C. Aliquots were withdrawn at 15 min intervals and single cells counted in a hemocytometer. The results are expressed as the number of single cells that remained ;It each time point as the percentage of single cells at time zero. Values shown are the mean * SEM for six independent experiments. Cell viability as judged bytrypan blueexclusion remained (lose to 100% over the whole time period. (!3) Comparison between different clones and effect of PI-PLC. 7 he fraction of cells remaining as single cells after a 30 min incubation was determined for untransfected LR-73 cells, the FZnegatIvetransfectantclone41,andclonesAlOand E12. ElZand LR-73 tells were treated in suspension with PI-PLC (0.5 U/ml for 1 hr zlt 37“C) before being repelleted and distributed into polystyrene tubes. Values shown are the mean of triplicate determinations. (!C) Effect of anti-F3 antibodies. Cells were pretreated for 15 min with 0.5 mg/ml Fab’ fragments prepared from either anti-F3 fusion protein serum (Fab F3, closed symbols) or preimmune serum (Fab control, open symbols), and the test was performed in tne presence of the antibodies. The fraction remaining as single tells was counted at various times for El2 transfectants or LR-73 control cells as indicated. The values shown are the mean of t~.iplicate determinations.

not complete, but the same change in aggregation kinetics was reproducibly observed in three independent experiments. It must be kept in mind that the antiserum has been raised against the immunoglobulin domains of the molecule expressed as a fusion protein in Escherichia coli, and this may explain why only partial inhibition was observed. For example, although theantibodies and their Fab’fragments recognized the native cell surface-expressed molecules, those directed against conformation-dependent epitopes mediating cell adhesion may constitute only a minor fraction of the anti-F3 antibodies present or bind with low affinity. An alternative possibility is that sequences not included in the fusion protein, for example the fibronectin type III repeats, contribute to the strength of F3-mediated adhesion, thus making it difficult to inhibit aggregation completely. The results presented up to this point show that F3 expression correlates with increased adhesiveness but do not address the question of whether F3 molecules bind to each other via a homophilic adhesion mechanism or to a different molecule that might be constitutively expressed by LR-73 cells. In an attempt to distinguish between these two possibilities, we examined the ability of nontransfected LR-73 cells to form mixed aggregates with transfected cells. To do this, we mixed El2 cells labeled with the carbocyanine dye dil (Honig and Hume, 1989) at a I:1 ratio with parental cells, allowed for aggregate formation in petri dishes, and scored the composition of randomly selected aggregates. In this test, homophilic binding would be indicated by the formation of aggregates composed mainly of labeled cells. We found instead that the vast majority of the aggregates were composed of both labeled and unlabeled cells. As shown in Figure 5 the distribution of labeled and unlabeled cells within the aggregates was unimodal (x* test of homogeneity), irrespectiveofwhethertheywerecomposed of both transfected and nontransfected cells or

Neuron 600

Figure

6. DRG

Neurons

Extend

Longer

Neurites

and a Denser

Neuritic

Network

on A10 Transfectants

Than

on Parental

Cells

Cultures of postnatal day 1 mouse DRC cells were grown on A10 ([A], [C], and [D]) and LR-73 ([B], [El, and [F]) cells. After 72 hr the cultures were fixed and stained either with methylene blue ([A] and [B]; bar = 40 vm) or reacted with P31 monoclonal antibodies followed by rhodamineconjugated goat anti-rat immunoglobulin antibodies ([Cl to [F]) and photographed through fluorescence ([Cl and [EJ) or

FVFII 61H

Functions

in Cell Adhesion

and Neurite

Outgrowth

of transfected cells only and whether incubation was for 4or 1.5 hr. The results obtained with nontransfected cells were the same as those with the labeled cell population (data not shown). One interpretation of this data would be that CHO cells express a ligand with which F3 is able to interact. Alternatively, F3-mediated adhesion may still be homophilic, but the srrength of interaction may be too weak to result in a bimodal distribution of transfected and parental cells in the aggregates.

E

2500.

-

5 E

2000

-

z CL E

1500

-

1000

-

P cu 0 .E 2

0

q 0

500

q

A10 LR-73 AlO+FabF3 LR-73+FabF3

-

z 0-

FIbExpressing CHO Transfectants Stimulate hieurite Extension The preferential localization of F31Fll on neuronal processes suggests that it may play a role in axonal growth and guidance. To test this possibilitywe investigated whether monolayers of F3-transfected CHO cells would stimulate the extension of neurites when used as a substrate for dissociated dorsal root ganglion (DRG) neurons. DRG cells were plated on transfected A10 or parental LR-73 cells, and the cultures were observed at various times thereafter either through phase optics after staining with methylene blue or through fluorescence optics after staining with anti-P31 antibodies. There were no morphological differences between monolayers of transfected and nontransfected cells. Neuronal attachmentwasestimated to bearound 50% with no significant difference between parental LR-73 and transfected cells. Both types of cells were more effective in supporting neuronal attachment than poly+-lysine-coated plastic (data not shown). Neurite outgrowth had begun on both cellular substrates at 2-1 hr and reached a maximum at around 72 hr. The sizes of neuronal cell bodies were homogeneous and not significantly different in the two conditions (measured diameter of neuronal cell bodies = 37.5 + 0.86 and 33 + 2.8 pm on LR-73 and A10 cells, respectively; mean + SEM for 300 neurons). It is thus likely that the same population of neurons was surviving on both types of cells, at least as far as large versus small DRG neurons are concerned (Sommer et al., 1985). There were marked differences in the ability of the two monolayers (i.e., LR-73 and A10 cells) to promote neurite outgrowth. When examined after 72 hr of coculture, DRG neurons extended longer neurites and a denser neuritic network on transfected than on control cells. Typical morphological aspects of such cultures are illustrated in Figure 6, showing the very long branching neurites on A10 cells in contrast to shorter, mainly unbranched processes that grow on control monolayers. On F3-transfected cells over 80% of the neurons had neurites in excess of 200 Fm, and this percentage was only 42% on the parental cells. The most striking differences were observed at the level

p’lase optics ([D] and [F]); bar = 60 pm. Arrows nc:urons grown on LR-73 as opposed to neurons

indicate grown

Figure 7. Analysis of Neurite Outgrowth Grown on A10 Transfectants or Parental ence and Absence of Anti-F3 Antibodies

from DRC Neurons LR-73 Cells in the Pres-

Neurite outgrowth was quantified by image analysis of 72 hr cultures fluorescently labeled with P31 and rhodamine-conjugated anti-rat immunoglobulin antibodies and expressed as the total neuritic output per neuron, which was calculated as the ratio of the sum of the lengths of the neurites over the number of cell bodies. Fab’fragments of anti-F3fusion protein antibodies (250 pg/mI) were added at the beginning of the culture period and after 24and 48 hr. Fab’fragments prepared from preimmune serum were without effect on neurite growth, and the values from these experiments were pooled with those of experiments done in the absence of antibodies. The values recorded for neurons grown in the absence of specific antibodies were 2370 + 120 pm on A10 monolayers (AIO, mean f SEM for 300 neurons analyzed in eight independent experiments) and 722 k 86 pm on LR-73 monolayers (LR-73,80 neurons analyzed in eight independent experiments). In the presence of anti-F3 Fab’ these values became 730 f 112 pm (A10 + Fab F3, 90 neurons analyzed in three independent experiments) and 679 3: 81 pm (LR-73 + Fab F3,45 neurons analyzed in three independent experiments) on A10 and LR-73 monolayers, respectively. The asterisk indicates a significant difference (p < 0.01) with the LR-73 value (bar with horizontal stripes) according to Duncan’s multiple range test (Duncan, 1955).

of the density of the neuritic network. When neurite outgrowth was quantified by image analysis of 72 hr cultures, the total neuritic output per neuron on F3transfected cells was found to be over three times higher than on LR-73 cells (Figure 7). To ascertain that the observed effect on neurite extension was indeed mediated by F3 expressed at the surface of the transfected cells, the experiment was done in the continuous presence of Fab’fragments of anti-F3 antibodies raised against the immunoglobulin domains of the molecule. Addition of the antibodies reduced the neuritic output to a level very similar to the one observed on control monolayers (Figure 7). In these experiments, complete inhibition of the enhanced neurite outgrowth was observed in contrast to the previous results showing only partial antibody blockade of aggregation in an adhesion test. Several factors could account for the greater sensitivity of process outgrowth as compared with aggregate forma-

tips of the neurites. on A10 cells.

Note

the difference

in lengths

of the

neurites

between

NWVNI 602

tion: a better accessibility of the molecule on monolayers,thecontinuouspresenceoftheantibodiesover a 3 day period, or the involvement of a different epitope. Whatever the correct explanation, the results strongly suggest that F3 stimulates neurite outgrowth of developing sensory neurons and that this effect is mediated by an epitope located within the immunoglobulin domains of the molecule. However, our observations do not indicate whether the cell surface or released forms of F3 are involved in this process. Discussion We previously described the characterization and cloning of mouse F3/Fll, a neuronal cell surfaceglycoprotein expressed in the central and peripheral nervoussystem.Thecommon structuralfeaturesthisprotein shares with known cell-cell adhesion molecules together with its conspicuous localization in neuropil and fiber tracts led us to propose that it may also be involved in mediating intercellular adhesion (Gennarini et al., 1989b). The results presented here provide direct evidence that F3/Fll expression confers increased adhesiveness to transfected CHO cells. Sensory neurons responded to FYFII-expressing CHO cells by extending longer neurites, suggesting that promotion of neurite outgrowth may be one of the consequences of FYFII-mediated adhesion. Our experimental approach has been modeled after studies in which cell surface proteins have been expressed from cloned cDNAs to test their potential adhesive properties or neurite outgrowth-promoting activities (for examples see Seed, 1987; Nagafuchi et al., 1987; Edelman et al., 1987; Snow et al., 1989; Johnson et al., 1989; Doherty et al., 1989). In all of the above examples, cell surface expression of functional molecules was obtained. We have expressed a complete F31Fll cDNA under control of the CMV promoter in CHO cells. As judged by immunofluorescence analysis, the clones obtained expressed the molecule at the cell surface in amounts comparable with that found on fetal brain or DRG neurons (data not shown). However, the protein produced by CHO cells was more extensively glycosylated than F3/Fll from mouse brain. Higher levels of carbohydrate on CHO cellexpressed molecules than on brain-derived molecules have also been reported for MAC (Johnson et al., 1989). In contrast to brain F3/Fll, the molecule produced by CHO cells separated into two components that differed by the extent of glycosylation and of which only the higher M, form appeared at the cell surface. Possibly, such a glycosylation precursor exists also in brain but does not separate from the mature form in SDS gels. In any event, our results establish that thecDNA we have isolated does encode a GPI-linked 130 kd polypeptide. CHO cells transfected with a single cDNA produce both GPI-linked, membrane-associated F31Fll and molecules released into the medium. It is thus not necessary to assume the existence of a distinct mRNA, perhaps generated

by alternative splicing as shown in the NCAM system (Cower et al., 1988), to account for the soluble form present in the brain (Gennarini et al., 1989b). As determined by a quantitative adhesion assay, stable F3/Fll expressors aggregated with faster kinetics than did either the parental or transfected F3/Fllnegative cells. Significant differences were already measured at the 15 min time point, implying that the protein is involved in initiating rather than in stabilizing intercellular contacts. As the test had to bedone in still medium to reveal consistent differences between F3/Fll-expressing and control cells, the protein appears to mediate low affinity interactions, at least in the assay employed. This again suggests that F3/Fll may be involved in initial, weak adhesions between cells. In fact, the most striking effects of its expression on aggregation were revealed when the cells were left undisturbed in nonadherent culture dishes and their aggregation behavior was scored by microscopic observation. However, there is the possibility that secondary factors may augment the intrinsic adhesiveness of the molecule on neural cells. lmmunoglobulin superfamily members may mediate adhesion through homophilic or heterophilic interactions. The former mechanism has been demonstrated for NCAM (Rutishauser et al., 1982; Edelman et al., 1987), Ll (Kadmon et al., 1990), and PO (Filbin et al., 1990); examples for the latter include MAC (Poltorak et al., 1987) and the CD2-LFA3 ligand-receptor couple (Seed, 1987). In principle, this issue can be addressed by performing mixing experiments between transfected and parental cells. In our hands, transfected FS-expressing cells formed mixed aggregates with parental LR-73 cells. One explanation for this finding would be that CHO cells fortuitously express a ligand for F3/Fll. The other possibility is that the interactions mediated by F3/Fll are too weak as compared with the intrinsic adhesion mechanism of LR-73 cells to result in detectable segregation in spite of a homophilic binding mechanism. A further possibility that must be kept in mind is that the protein is spontaneously released into the medium and that soluble F3/Fll retained at the surface of the parental cells may mediate heterotypic interactions in this form. Distinguishing among these possibilities obviously requires further analysis. Mouse F3 shares the same overall structure and substantial sequence conservation with chicken Fll (Briimmendorf et al., 1989) and rat TAG-l (Furley et al., 1990). It is, however, more similar to Fll (77% sequence identity overall) than to TAG-l (49.5% overall sequence identity) (data not shown). Because of their preferential association with axons and fiber-rich areas, these molecules have been proposed to be implicated in processes such as axon or neurite outgrowth, axon fasciculation,orgrowthconeguidance. Previous studies have shown that anti-F11 antibodies have defasciculating properties (Rathjen et al., 1987) and perturb the elongation of neurites on other neurites (Chang et al., 1987). Purified TAG-l, on the other hand,

F::/Fll 6( :3

Functions

in Cell Adhesion

and Neurite

Outgrowth

was an effective substrate for neurite extension (Furley et al., 1990). Consistent with a role of this set of neuronal surface proteins in promoting axonal growth and guidance, we found that sensory neurons respond to FYFII-expressing transfectants used as a substrate with increased neurite outgrowth, an effect that could be blocked by monovalent anti-F3 antibodies raised against the immunoglobulin domain region of the protein. Although we did not test this, the long, branched processes that develop on the transfectants most likely represent axons. Most DRG neurons express F31Fll at the cell surface (C. R. and G. G., unpublished data). As anti-F3 antibodies inhibited neurite extension on transfected but not on parental cells, a homophilic binding mechanism seems most likely in this system. It remains to be seen, however, whether F3 and, by inference, its close relatives Fll and TAG-l act mainly mechanically by increasing adhesive interactions or whether binding to their respective ligands results in trans-membrane signaling. The transfection-based assays presented here will undoubtedly be useful systems for addressing such questions and for mapping the domains responsible for adhesion and for stimulation of neuriteoutgrowth. Our results add yet another molecule to the list of cell-cell adhesion molecules that are expressed on neurons. At least in the cerebellum, the same neuron, the granule cell, expresses at least three of them: NCAM (Langley et al., 1983), Ll (Faissner et al., 1984), and F31Fll (Rathjen et al., 1987; Gennarini et al., 1989a). Is remains a challenge for the future to learn how a given neuron may be able to integrate and respond to t’ie cues presented by different adhesion molecules. Experimental

Procedures

Construction of F3 cDNA in the Expression Vector pRC/CMV A full-length F3 cDNA was constructed by ligating restriction fragments derived from the @tlO cDNA clones 7.1.1 and 19.1 (Gennarini et al., 1989b). In brief, a5.1 kb Hindlll-Smal fragment oi the first clone, including 2.5 kb of coding region, 500 bp of 3’ untranslated region, and 2 kb of 1 vector were cloned into pCEM7Zf(+) (Promega Biotech). A 800 bp BamHI-Ncol fragment from pGEM 19.1 (Cennarini et al., 1989b) containing 200 bp of 5 untranslated and 600 bp of coding region was then joined via a unique Ncol site. From this construct, the full-length F3 cDNA was excised by cutting at the unique Hindlll site located 50 bp upstream of the translation initiation site and by partial digestion a! the Xmnl site located 100 bp downstream of the termination codon. The pRC/CMV vector (Invitrogen Corporation) was chosen for expressing the F3 cDNA. This vector contains the strong CMV enhancer/promoter, the polyadenylation signal of the bovine growth hormone gene, and the bacterial neomycin resistance gene under control of the SV40 early promoter for selection in G418-containing media. To create compatible ends for cloning, the vector was cut in the polylinker at the unique Apal site, made blunt with T4 DNA polymerase, and then cut again at the Hindlll site. Cell Culture and Transfection The CHO cell line LR-73 (Pollard and Stanners, 1979) (kindly provided by Dr. C. Stanners, McGill University, Montreal, Canada) was cultured in aMEM medium containing 10% fetal calf serum (KS), glutamine, and antibiotics (complete medium). Stable transfectants, obtained by the calcium phosphate precipitation method (Gorman, 1985), were selected in G418 (800 pglml) and

cloned twice by limiting dilution. The isolated clones were probed for surfaceexpressionofthe F3glycoprotein byimmunofluorescence labeling using either the first generation F3 antibodies (Gennarini et al., 1989a) or the anti-F3 fusion protein serum described below. Antibodies An antiserum was raised in rabbits against the N-terminal 785 amino acids of F3 expressed in E. coli using the PET system (Rosenberg et al., 1987). A 2.4 kb fragment of the 5’ region of the F3 cDNA was excised and, after adding BamHl linkers, inserted in the correct orientation and reading frame in the unique BamHl siteof the pET3avector (a kind gift of Dr. W. Studier, Brookhaven National Laboratory, Upton Long Island, NY). In this vector, the protein is expressed under control of a T7 RNA polymerase promoter as a fusion protein starting with 12 amino acids coded by vector sequences. The protein was expressed in E. coli BL21 (DU, pLys.S) (Studier et al., 1990) after induction with 0.4 mM IPTG. To purify the fusion protein, induced cells (I liter culture) were harvested and lysed in 100 ml of lysis buffer (50 mM Tris-HCI [pH 81, containing 100 mM NaCI, 2 mM EDTA, and protease inhibitors). Lysis was achieved by endogenous T7 lysozyme by a single freeze/thaw cycle followed by a 30 min agitation at room temperature after adding 1% Triton X-100 and 10 pg/ml DNAase. The insoluble material was recovered by centrifugation (9000 rpm for 30 min) and was further extracted for 30 min in lysis buffer containing 2 M urea. The material that remained insoluble was dissolved by sonication (five times for 1 min in a Branson sonicatar) in lysis buffer containing 8 M urea, 0.5 M NaCI, and 100 mM mercaptoethanol. After a 2 hr centrifugation at 200,000 x g, the supernatant was dialyzed overnight against two changes of phosphate-buffered saline. Following dialysis, a heavy precipitate formed in which the expressed protein of the expected size (90 kd), which could be revealed by immunoblotting with first generation F3 antibodies, represented 80% of the protein present(datanotshown).Aliquotscontainingabout0.5 mgof protein were emulsified with complete Freund’s adjuvant and used to immunize rabbits at 2 week intervals over a 3 month period. When tested by immunoblotting on mouse brain extracts, the immune serum recognized a single 135 kd species (see Figure 2C). It did not react with F3-negative tissues and cells such as the nontransfected CHO LR-73 line (data not shown). Monovalent Fab’ fragments were prepared from IgC fractions of the serum as described (Brackenbury et al., 1977). The preparation and specificity of the first generation anti-F3 rabbit serum have been described (Cennarini et al., 1989a, 1989133. lmmunofluorescence Staining Cells, grown overnight on poly-t-lysine-treated coverslips were incubated with a 1/250dilution of primary antiserum in complete medium for 60 min at 4OC. After three washes in medium, the coverslips were incubated with fluoresceinor rhodamine-conjugated goat anti-rabbit IgG F(ab)* fragments (Immunotech Marseille, France) at a l/50 dilution in medium. After three more washes, the cells were fixed with 5% acetic acid/95% ethanol and examined under a Zeiss fluorescence microscope. Cell Aggregation Assays Subconfluent monolayers of transfected or parental CHO cells were detached from the culture dish in IO ml of EDTA (GIBCO) (15 min at room temperature) and dissociated into single cells by gentle pipetting with a fire-polished Pasteur pipette. After a further wash in EDTA, the cells were resuspended in complete medium at 2 x 106 cells per ml. For quantitation of aggregate formation, they were transferred to 15 ml polystyrene tubes (Falcon 2051) that had been coated with FCS for 1 hr at 37OC. The testwasperformed byincubating2mlaliquotsofthecellsuspension at 37OC in a 7.5% CO? atmosphere without agitation. Aliquots were withdrawn at 15 min intervals after mixing by gentle inversion, and the single cells remaining were counted in a hemocytometer. For microscopical observation of aggregate formation, 2 ml aliquots were incubated in bacteriological petri

NellrOn

604

dishes, and photographs were taken at different times afterward. To test the effect of monovalent anti-F3 antibodies, ceils were preincubated for 15 min at 37OC in the presence of 0.5 mglml Fab’ fragments in complete medium. The aggregates that had already formed were dissociated by gentle pipetting and the cells, left in the same medium, were incubated and counted as described above. The adhesive specificity of F3 transfectants versus the parental CHO cells was probed by examining the composition of the aggregates formed by incubating both cell types over a 1 to 4 hr period. LR-73 or El2 cells were labeled by overnight incubation of monolayers with the lipophilic carbocyanine dye dil (Honig and Hume, 1989). The ceils were then dissociated in EDTA as above and mixed in a I:1 ratio with similarly prepared suspensions of unlabeled ceils at 2 x IO” cells per ml in complete medium and allowed to aggregate in petri dishes in a COz incubator. Theculturedishes werethen either observed and photographed directly using an inverted fluorescence microscope or aliquots of 0.5 ml were gently removed from the dishes, fixed in 3.5% formaldehyde/0.5% glutaraldehyde, spotted on microscope slides, and observed through fluorescence and phase optics. The distribution of labeled and unlabeled cells within randomly selected aggregates was determined and plotted against the total number of aggregates that had been evaluated. Only clumpsof fourcellsor morewere included intheanalysis.Values for the percentage of aggregation were found to fit a Gaussian distribution and were compared using Student’s t test. For statistical evaluation of the formation of mixed aggregates, we tested whether the observed distribution of labeled and unlabeled cells within aggregates differed significantly from the theoretical distribution of a homogeneous population using a x2 test of homogeneity according to Bass (1974). Biochemical Techniques lmmunoblots, immunoprecipitations, and PNGase F (Boehringer Mannheim; glycopeptide: N-glycosidase F) digestion were performed in the same conditions as previously described (Gennarini et al., 1989a, 1989b). For PI-PLC digestion the cells were collected in EDTA and resuspended in PLC buffer (50 mM Tris-HCI [pH 7.51, containing 150 mM NaCl and protease inhibitors) at 3 x 10’ cells/ml. Aliquots of 100 ~1 were incubated for 40 min at 37OC in the presence or absence of 1 U/ml PI-PLC from Bacillus thuringiensis (Immunotech). After digestion, cells were pelleted and extracted with 100 ~1 of Nonidet P-40 (NP-40) buffer as described (Gennarini et al., 1989a). Cell extracts and PI-PLC digestion supernatants were then analyzed by immunoblots with anti-F3 fusion protein antibodies. For trypsin digestion, 3 x IO5 cells in 100 ~1 of aMEM medium were incubated (1 hr at room temperature) with 0.1 to 10 mg/ml trypsin (Type Ill, Sigma). The digestion was stopped by adding a 2-fold molar excess of soybean trypsin inhibitor and, after pelleting, the cells were extracted in NP-40 buffer and prepared for electrophoresis as above. Metabolic labeling experiments were done as follows. Subconfluent monolayers were preincubated for 1 hr in medium without methionine (GIBCO) containing 10% dialyzed FCS. The ceils were then pulse-labeled for 10 min in the presence of 1 mCi of [??]methionine and recovered either immediately or after chase periods of 30 and 90 min, respectively. Detergent extracts of the labeled cells and culture supernatants were then analyzed by immunoprecipitation using first generation F3 antibodies. Neurite Outgrowth Assay Parental or F3-transfected CHO cells (clones A10 and E12) were seeded in 9Gwell tissue culture-treated plastic plates (Falcon) and grown for approximately 24 hr in Dulbecco’s modified Eagle’s medium containing 10% FCS and antibiotics until near confluency. Some experiments were also conducted in aMEM medium with identical results. Postnatal day 1 mouse DRGs were dissected out and dissociated as described (Rougon et al., 1983), except that the cell suspension was triturated through a 21G gauge needle in order to

maximize the proportion of single cells. The cells (0.5-I x IO’ cells per well) were then seeded onto the CHO cell monolayers in the presence or absence of 250 pglml Fab’fragments of anti-F3 fusion protein IgG or of IgG isolated from preimmune serum. The same amount of antibodies was readded every 24 hr. After various time intervals (3, 24,48, and 72 hr) wells with control or transfected CHO cells were gently rinsed with medium without FCS, fixed with 4% paraformaldehyde, and stained with 1% methylene blue to visualize neuronal cell bodies. Alternatively, double immunofluorescence staining was performed on fixed cells using anti-P31 rat monoclonal antibody and rabbit anti-F3 fusion protein serum. The anti-P31 antibody recognizes a surfacedeterminant expressed by virtually all DRC neurons at this stage of development (Pierres et al., 1987; C. R., unpublished data) but absent from CHO cells and was used as a marker to visualize neurites (Figure 6). Anti-F3 serum labels a subpopulation of large DRG neurons (G. R. and C. G., unpublished data) and virtually all F3-expressing transfectants. Bound antibodies were revealed by goat anti-rat and goat anti-rabbit F(abJz fragments conjugated to rhodamine and fluorescein, respectively (Jackson Laboratories). Microscopical observations were made on an inverted ICM 405 Zeiss microscope. Photographs were taken using a 20x fluorolphase lens on 400 Asa TriX-films. The 35 mm negatives were projected at a fixed magnification onto the screen of an image analyzer (Visiosoft, Pontoise, France), and the lengths of neurites were measured using a digitized BiolabTM program (written by P. Rage, LNB Marseille, France, with Macintosh Programming Workshop for Macll). Neurite lengths were determined as the distance between the edge of the cell body and the tip of the neurite. The neurite had to meet the following requirements: it must emerge from a distinguishable cell body (not a clump of neurons), it must be longer than the diameter of the cell body, and it must be identifiable over its entire length. Approximately 15 to 20 neurites were measured within an area of 1 mm2. The data were pooled and an estimation of the total neuritic output per cell was calculated as the ratio of the sum of the lengths of neurites over the number of cell bodies. The criteria chosen for measurement of neurite lengths underestimate the overall length, as about 50% of neurons had neurites that fasciculated with neurites of adjacent neurons that, by the above criteria, were excluded from the analysis. Values for the longest neurites were calculated from measurements made on nonfasciculated neurites emerging from cells in isolation. Statistical significance was determined using one-way analysis of variance followed by Duncan’s multiple range test (Duncan, 1955). Acknowledgments We thank C. Di Benedetta for support and encouragement, P. Golstein and P. Naquet for critical reading of the manuscript, and C. Beziers La Fosse for artwork. This work was supported by institutional grants from CNRS to the Centre d’lmmunologie and URA 179, from INSERM to the Centred’lmmunologie,fromMinisteroPubblicaInstruzioneand Consiglio Nazionale delle Richerche to the lstituto di Fisiologia Umana, and by specific grants from CNR to C. C., from Association Francaise de Lutte contre les Myopathies et Association pour la Recherche contre le Cancer to C. G. and G. R. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenr’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

11, 1990; revised

January

16, 1991

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