©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cytoplasmic Domain of the Drosophila Cell Adhesion Molecule Neuroglian Is Not Essential for Its Homophilic Adhesive Properties in S2 Cells (*)

(Received for publication, January 25, 1995; and in revised form, May 23, 1995)

Michael Hortsch (1)(§),   Yu-mei Eureka Wang (2) Yasmin Marikar (1) Allan J. Bieber (2)

From the (1)Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor, Michigan 48109 and the (2)Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Drosophila neuroglian is a transmembrane glycoprotein that has strong structural and sequence homology to the vertebrate L1 gene family of cell adhesion molecules (Bieber, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, J. R., Traquina, Z. R., Schilling, J., and Goodman, C. S.(1989) Cell 59, 447-460). Two different neuroglian protein forms that are generated by a differential splicing process are expressed in a tissue-specific fashion by embryonic and larval cells (Hortsch, M., Bieber, A. J., Patel, N. H., and Goodman, C. S. (1990) Neuron 4, 697-709). The two neuroglian polypeptides differ only in their cytoplasmic domains. Both of these neuroglian species, when transfected into and expressed in Drosophila S2 cells, induce the calcium-independent, homophilic aggregation of transformed cells. A third artificial neuroglian protein form was constructed by substituting the neuroglian transmembrane segment and cytoplasmic domains with the glycosyl phosphatidylinositol attachment signal of the Drosophila fasciclin I protein. This cDNA construct generates a glycosyl phosphatidylinositol-anchored form of neuroglian, which retains the ability to induce homophilic cell aggregation when expressed in S2 cells, and was able to interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that neuroglian mediates a calcium-independent, homophilic cell adhesion activity and that neither cytoplasmic neuroglian domains nor a direct interaction with cytoskeletal elements is essential for this property.


INTRODUCTION

A growing number of putative neural cell adhesion molecules (CAMs) (^1)with immunoglobulin and fibronectin type III protein domains are currently being identified and characterized from a wide range of different species. Among the best studied examples are representatives of the NCAM and the L1 CAM families. These molecules have been implicated in many different developmental and cellular processes including neurite outgrowth, axonal fasciculation, neuronal cell migration, muscle formation and innervation, neuron-Schwann cell interactions(1, 2, 3, 4, 5) .

Drosophila neuroglian is a member of the L1 CAM gene family (6) . It encodes a transmembrane polypeptide with six immunoglobulin and five fibronectin type III protein domains and has strong sequence homology to the mouse L1 glycoprotein. Differential splicing of the primary neuroglian transcript in Drosophila embryos results in the production of two polypeptide species differing only in their cytoplasmic domains(7) . The larger of the neuroglian protein forms (designated neuroglian, in accordance with its apparent molecular mass in kDa) is restricted to a subset of neuronal cells in the peripheral and central nervous system, whereas the smaller neuroglian polypeptide (neuroglian) is also expressed by many non-neuronal cell types and tissues. Multiple interchangeable cytoplasmic protein domains have also been reported for a number of other immunoglobulin-type CAMs including NCAM(8, 9) , L1(10, 11) , and myelin-associated glycoprotein (MAG)(12, 13) . This diversity of cytoplasmic domains, and therefore protein forms, could result in differential interactions with cytoskeletal elements, different involvement in cellular second messenger systems, or different degrees of intracellular regulation of the extracellular adhesive properties of these molecules.

The importance of the cytoplasmic domain for interactions with the cellular cytoskeleton and ligand binding and specificity has been studied in detail for some members of the integrin and cadherin CAM families. Deletions of the cytoplasmic domain of the lymphocyte function associated antigen-1 (LFA-1) integrin beta(1)-subunit do not interfere with cell surface expression or heterodimer assembly, but abolish binding to its intercellular adhesion molecule-1 ligand and disrupt the association of the LFA-1 integrin with the actin cytoskeleton(14) . L-CAM and E-cadherin both depend on the integrity of their cytoplasmic protein domains for their calcium-dependent homophilic adhesive function(15, 16) .

Some adhesion molecules belonging to the immunoglobulin gene superfamily have been functionally tested for their requirement for an intact cytoplasmic domain. One example of functional differences between alternate cytoplasmic domains is seen for the major protein forms of NCAM. In comparison to the smaller transmembrane NCAM form (NCAM 140) the largest NCAM protein form (NCAM 180) is less able to support neurite outgrowth from rat cerebellar neurons when expressed in 3T3 fibroblast cells(17) . The NCAM 180 form also interacts with the cellular spectrin-actin cytoskeleton, in contrast to the other two major NCAM species, which do not(18) . Besides the two major transmembrane forms of NCAM, a naturally occurring, GPI-anchored form has also been described(9, 19) . Lesley et al.(20) reported that the CD44 receptor loses its ability to bind hyaluronic acid after deletion of its cytoplasmic domain. Smaller deletions in the CD44 cytoplasmic protein domain that abolish CD44 binding to the cytoskeleton adapter molecule ankyrin have similar effects on its adhesive ability(21) . Similarly, PECAM-1 molecules with a complete deletion of the cytoplasmic domain do not support cell-cell adhesion of transformed mouse L-cells(22) . Interestingly, DeLisser and co-workers (22) found that partial deletions of the PECAM-1 cytoplasmic domain result in a basic change of adhesion mechanism from a calcium-dependent, heterophilic mode to a calcium-independent, homophilic mode. Carboxyl-terminal deletions of the myelin P(0) cytoplasmic protein domain abolish P(0)-mediated homophilic adhesion of transformed cells(23) .

These and other studies strongly support the notion that the adhesive function of many different types of CAMs often depend on, or is regulated by, the cytoplasmic portions of these membrane molecules. By expressing different protein forms of Drosophila neuroglian in Drosophila S2 cells, we have analyzed the adhesive properties of this invertebrate member of the L1 CAM gene family. S2 cells have been used as host cells for the functional characterization of a number of Drosophila and vertebrate CAMs(24, 25, 26) . The experiments reported here show that both of the naturally occurring forms of neuroglian act as homophilic CAMs. Furthermore, the substitution of the neuroglian transmembrane segment and the cytoplasmic domains, with the GPI anchor signal of the Drosophila CAM fasciclin I, produces a novel neuroglian polypeptide (neuroglian) that is anchored in the plasma membrane by a GPI moiety and is still able to induce homophilic cell aggregation of transformed S2 cells. These findings indicate that neither of the two neuroglian cytoplasmic domains is required for neuroglian-dependent homophilic cell adhesion.


EXPERIMENTAL PROCEDURES

Materials and Antibodies

The BP-104 mAb specifically recognizes the neuron-specific protein form of Drosophila neuroglian (neuroglian)(7) , and the 3F4 mAb recognizes an epitope on the extracellular domain that is shared by both naturally occurring neuroglian protein forms. (^2)A rat polyclonal antiserum specific for the neuroglian polypeptide was raised against a decapeptide (NH(2)-C-norL-G-Q-Y-G-R-K-G-L-COOH) that contains the 8 carboxyl-terminal amino acid residues of neuroglian(6) , after coupling to bovine thyroglobulin as a carrier protein. Goat anti-mouse and rat IgG peroxidase-conjugated antibodies were purchased from Jackson ImmunoResearch, West Grove, PA. 1,1`-Dioctadecyl-3,3,3`-tetramethylindocarbocyanine perchlorate (DiI) was from Molecular Probes, Eugene, OR. Schneider's medium, penicillin/streptomycin stock solution, and heat-inactivated fetal calf serum were from Life Technologies, Inc.

Construction of cDNA Expression Vectors

cDNAs encoding the two naturally occurring forms of neuroglian were identified and characterized as described(6, 7) . Full-length cDNAs were excised from isolated gt11 DNA by partial EcoR I digestion and cloned into the KpnI site of the expression vector pRmHa3, using KpnI/EcoRI linkers. Expression of cDNAs inserted into the polylinker of pRmHa3 is under the control of the Drosophila metallothionein promoter(27) .

A GPI-anchored form of neuroglian was constructed as outlined in Fig.1. A 3.4-kb KpnI/AvaII cDNA fragment, encoding the amino-terminal 1121 amino acid residues of the 1138-amino acid neuroglian extracellular domain, was isolated from a neuroglian cDNA. The Drosophila fasciclin I GPI anchor signal (28) was derived by PCR from a fasciclin I cDNA in Bluescript SK. In the PCR the universal sequencing primer was used along with a 23-mer oligonucleotide (5`-GATCAACGTATACGGACCCGATG-3`) that was identical to a small part of the coding strand of the fasciclin I cDNA in all but 2 nucleotide residues (introducing an AvaII site underlined in the sequence). PCR products were digested with AvaII and SalI, and an 1.1-kb DNA fragment was isolated from an agarose gel. This fasciclin I cDNA-derived PCR product encodes the carboxyl-terminal 53 amino acids of the Drosophila fasciclin I open reading frame and most of the 3`-untranslated region. Both the 3.4-kb neuroglian cDNA fragment and the 1.1-kb fasciclin I PCR product were subcloned into KpnI/SalI-cut pRmHa3 vector DNA.


Figure 1: Construction of a Drosophila neuroglian cDNA containing the GPI anchor signal of Drosophila fasciclin I. A 3.4-kb KpnI/AvaII Drosophila neuroglian cDNA fragment, encoding the first amino-terminal 1121 amino acid residues (comprising the signal sequence and almost the entire extracellular domain), and a 1.1-kb AvaII/BamHI PCR product, encoding the 53 carboxyl-terminal amino acid residues and the 3`-untranslated region of Drosophila fasciclin I, were ligated together into KpnI/BamHI-cut pRmHa3 vector DNA. An in-frame AvaII site was introduced into the fasciclin I cDNA by the 5` PCR primer. The resulting expression vector, pRmHa3-nrg, drives the expression of a hybrid protein consisting of almost the entire neuroglian extracellular domain bound to the membrane by a GPI anchor.



Cell Culture and Transfection

Schneider's line 2 (S2) cells (29) were maintained at 1 10^6 to 2 10^7 cells/ml, in Schneider's medium supplemented with 12.5% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, at 25 °C with air as the gas phase. Detailed methods for the transfection, selection, and cloning of S2 cells, as well as for cell aggregation assays, have previously been reported in detail (30) and are briefly described below.

S2 cells were transfected with DNA-calcium phosphate coprecipitates using the pPC4 plasmid to confer alpha-amanitin resistance as the selectable marker(31) . Individual subclones were expanded and assayed by Western blotting for high level expression of neuroglian protein. These cell lines are designated S2:pRmHa3-nrg, S2:pRmHa3-nrg, and S2:pRmHa3-nrg, respectively. Prior to assay, protein expression was induced by the addition of 0.7 mM CuSO(4) to the culture medium for 1-2 days.

Aggregation Assays

In assays for cell adhesion, cells were plated at 1 10^6 cells/ml in 60-mm Petri dishes and induced with 0.7 mM CuSO(4) for 24 h. After induction, the cells were gently dissociated by repeated pipetting and cell concentrations were readjusted with cell culture medium to 1 10^6 cells/ml. The cells were then shaken at 100 rpm on a rotating platform at room temperature. Aggregation of the cells was usually analyzed after 2 h.

In adhesion assays which required that two different cell lines be mixed to determine the extent of the reaction between their expressed surface molecules, one cell line was marked by labeling with the vital fluorescent membrane dye DiI . Cells were stained by adding 1 µl of a 2.5 mg/ml solution of DiI (in ethanol) for each ml of culture medium, and the cells were allowed to incorporate the dye for 2-12 h before washing the cells three times in culture medium to remove excess dye. Labeled cells were then mixed with unlabeled cells of the appropriate type, and the cells were induced together and assayed for aggregation. For all combinations of cell lines that were tested, several assays with different ratios of the two different cell types were examined, but the total cell concentration in all assays was 1 10^6 cells/ml.

Cell aggregates were visualized and photographed on a Zeiss Axiophot microscope with Nomarski interference optics or on a Nikon Diaphot with phase contrast optics. DiI epifluorescence was visualized on a Zeiss Axiophot microscope using a rhodamine optical filter set.

Quantitative Analysis of Cell Aggregation

At t = 0 h each cell line was diluted to 1 10^6 cells/ml, induced with 0.7 mM CuSO(4) and incubated on a rotary shaker at 100 rpm. Sufficient cells were prepared so that separate plates of cells were set up for each time point to be assayed. At the selected time points, aliquots of cells were analyzed for cell aggregation and the remaining cells were processed for Western blot analysis to determine neuroglian protein levels. Aggregation was quantified as the loss of single cells over time using the automatic object analysis programs of the Metamorph Imaging System. To enhance contrast for efficient detection by the imaging system, the cultures were labeled with Hoechst dye 33342 at 10 µl/ml for 15 min immediately prior to quantitation. Cell aliquots were mounted on a glass slide and viewed at 100 magnification with a DAPI (4`,6-diamidino-2-phenylindole) optical filter set. For each cell line and time point, images of six randomly chosen fields were digitally acquired, contrast enhanced with a thresholding application, and single cells counted using the object classifiers program. An average of about 1600 cells was counted for each data point. The standard errors for the data points in Fig.6usually represent <10-15% of the values plotted.


Figure 6: Quantitative analysis of neuroglian-induced cell aggregation. Cell aggregation was quantified as the loss of single cells over time. At t = 0 h, each cell line was diluted to 1 10^6 cells/ml and induced with 0.7 mM CuSO(4). Throughout the experiment the cultures were agitated constantly on a rotary shaker at 100 rpm. At the selected time points, aliquots of cells were removed and mounted on microscope slides for quantitation using an automatic object analysis program. The residual cells were used for the assessment of neuroglian expression by Western blot analysis. , S2:pRmHa3-nrg; , S2:pRmHa3-nrg; , S2:pRmHa3-nrg; , S2 cells.



Isolation and Analysis of Cellular Membrane Proteins

Transformed cells were induced for 2 days with 0.7 mM CuSO(4) and harvested by centrifugation. A crude membrane preparation was isolated by differential centrifugation, and the fractionation of soluble and peripheral membrane proteins from integral membrane proteins, at pH 10, was performed as described previously(32) . Membrane proteins were fractionated on 7.5% SDS-polyacrylamide gels, followed by overnight transfer to nitrocellulose filters. These Western blots were incubated sequentially with primary antibodies and peroxidase-conjugated secondary antibodies, and then developed with 3,3`-diaminobenzidine (33) .

PI-specific Phospholipase C Treatment

PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis was kindly provided by Dr. M. Low (Columbia University). Treatment of S2 cell membrane preparations with PI-PLC and fractionation of peripheral from integral membrane proteins by pH 10 extraction were performed as described by Hortsch and Goodman(35) .

Prior to PI-PLC treatment of S2 cells, transformed cells were induced at a concentration of 0.5 10^6 cells/ml for 2 days with 0.7 mM CuSO(4) and then gently dissociated into a single cell suspension by pipetting. A small aliquot of cells was incubated for 2 h at 37 °C with 50 units/ml PI-PLC. An equal volume of Schneider's medium containing 12.5% fetal calf serum was added, and cells were incubated at room temperature in a 24-well tissue culture plate. Cell aggregation assays were performed as described previously.


RESULTS

Construction of Neuroglian Expression Vectors and Transfection into S2 Cells

The Drosophila S2 cell expression system was used to determine the ability of Drosophila neuroglian to mediate homophilic cell adhesion. Full-length cDNAs encoding both of the naturally occurring forms of neuroglian were subcloned into the pRmHa3 vector, putting the expression of the neuroglian cDNA inserts under the control of the Drosophila metallothionein promoter(27) . The construction of a novel neuroglian protein form in which the neuroglian transmembrane segment and the cytoplasmic domains were substituted by the GPI anchor signal of Drosophila fasciclin I is outlined in Fig.1. The 5` end of the hybrid open reading frame encodes the first 1121 amino acid residues of the neuroglian protein, which encompasses the starting methionine, the amino-terminal signal sequence, and almost the entire extracellular domain of the mature neuroglian protein. Only the last 18 amino acid residues preceding the neuroglian transmembrane segment are deleted in the new hybrid protein. This neuroglian cDNA fragment was ligated, in frame, to the 53 carboxyl-terminal amino acid residues of the Drosophila fasciclin I open reading frame, which contains a GPI attachment signal. Drosophila fasciclin I is a homophilic CAM that is anchored in the plasma membrane by a carboxyl-terminally attached GPI moiety(34, 35) . This mode of membrane attachment is common among different CAMs including the smallest protein species of NCAM(19) . The transfer of the GPI group onto the carboxyl-terminal amino acid residue of the polypeptide is usually preceded by the proteolytic removal of a hydrophobic carboxyl-terminal signal peptide and a putative cleavage site has been identified in front of the fasciclin I GPI attachment signal(35) . Cleavage at this site would remove the last 26 amino acid residues from the neuroglian-fasciclin I hybrid protein, which would still retain 27 amino acids of fasciclin I protein sequence at its new carboxyl terminus. The adhesion domains of the fasciclin I protein are near its amino terminus, (^3)and the fasciclin I component of the neuroglian protein, therefore, does not contribute to its adhesive properties.

The three neuroglian expression constructs (pRmHa3-nrg, pRmHa3-nrg, and pRmHa3-nrg) were introduced into S2 cells by DNA-calcium phosphate coprecipitation and the expressed proteins were analyzed as described below and under ``Experimental Procedures.''

All Three Neuroglian Protein Forms, Including the GPI-anchored Form, Are Expressed in S2 Cells as Integral Membrane Proteins

The transformed cell lines expressed polypeptides of the expected sizes for the three different neuroglian cDNA constructs (Fig.2). Cellular membranes from untransformed S2 cells did not contain immunoreactive neuroglian proteins. In accordance with previously published results, most of the neuroglian protein found in membranes isolated from Drosophila embryos belonged to the neuroglian protein species(7) . Antibodies specific for either of the two naturally occurring neuroglian forms recognized their respective neuroglian polypeptide epitopes in both the embryonic membrane preparation, as well as in their corresponding transformed cell lines. Neuroglian protein expressed from the pRmHa3-nrg construct was recognized only by the 3F4 mAb, which binds to an epitope in the extracellular domain. The apparent molecular mass of this novel neuroglian form was similar to that of the neuroglian species. However, it appeared to be less focused on SDS-polyacrylamide electrophoresis gels, being visualized as a broader band on Western blots (see Figs. 2-4). We attribute this anomalous electrophoretic behavior to the GPI carbohydrate-lipid moiety of this neuroglian form. Removal of the lipid portion resulted in a significant sharpening of the protein band (Fig.4, lane3).


Figure 2: Different protein forms of neuroglian are present in membrane preparations from embryonic and transformed S2 cells. Western blots of different membrane preparations were stained with various anti-Drosophila neuroglian antibodies. About 3-20 µg of membrane proteins were loaded per lane. Lanes1, a crude membrane preparation from 20-23-h-old Drosophila embryos; lanes2, from untransformed S2 cells; lanes3, from S2:pRmHa3-nrg cells; lanes4, from S2:pRmHa3-nrg cells; lanes5, from S2:pRmHa3-nrg cells. S2 cells were induced for 2 days with 0.7 mM CuSO(4) before a crude membrane preparation was isolated. PanelA was incubated with the mouse anti-neuroglian mAb 3F4, which binds to the extracellular domain of neuroglian and recognizes all three neuroglian protein forms. PanelB was incubated with a rat polyclonal antiserum that recognizes the carboxyl-terminal amino acid residues specific for the neuroglian form. PanelC was incubated with the mouse anti-neuroglian mAb BP-104, which is specific for the neuroglian protein form.




Figure 4: The neuroglian protein form is anchored in the S2 cell membrane by a glycosyl phosphatidylinositol membrane anchor. Membrane preparations isolated from transformed, induced S2 cells (S2:pRmHa3-nrg, lanes 1-4; pRmHa3-nrg, lanes5 and 6) were incubated for 2 h in the presence (lanes 3-6) or absence (lanes1 and 2) of PI-PLC (20 units/ml) and subsequently subjected to an extraction at pH 10 in order to separate soluble (S) from integral membrane (P) proteins. Whereas the neuroglian protein is partially released from the membranes by PI-PLC, the phospholipase has no effect on the membrane association of the neuroglian protein.



A significant portion of the neuroglian protein in embryonic membranes and the membrane preparations from S2 cells expressing the two transmembrane neuroglian forms appeared to be proteolytically processed to a smaller polypeptide species with an apparent molecular mass of about 150 kDa (Fig.2A, indicated by the arrow). Since this degradation product was absent in membranes expressing neuroglian and was not detected by antibodies against either of the neuroglian cytoplasmic domains, we assume that the processing site must be very close to the transmembrane segment, probably on the extracellular aspect of the membrane. Most of this neuroglian degradation product can be released from the membrane by an alkaline pH wash and is therefore not firmly inserted into the lipid bilayer (data not shown). Interestingly, a similar proteolytic processing has been reported for the mouse L1 glycoprotein(36) . This degradation process could constitute an endogenous mechanism to regulate cell adhesion by the proteolytic inactivation of neuroglian protein. Although the preparation of membranes was performed in the presence of a panel of various protease inhibitors, we cannot exclude at the present time that the observed neuroglian degradation might have also occurred during the isolation of the membrane fractions.

The covalently attached GPI anchor of the hybrid neuroglian form should provide a strong membrane association, and accordingly, no immunoreactive neuroglian protein was found in cell free tissue culture medium of induced S2:pRmHa3-nrg cells (Fig.3A, lane1). The cells from an equivalent volume of tissue culture medium contained large amounts of neuroglian protein (Fig.3A, lane2). Protein fractionation of membrane preparations at an alkaline pH has been routinely used for the separation of integral membrane proteins from those not embedded in the lipid bilayer(37) , and, like the transmembrane forms of neuroglian, the neuroglian protein was not released from the membrane by an exposure to an alkaline pH of 10 (Fig.3, B and C), demonstrating that the protein is securely associated with the membrane.


Figure 3: The neuroglian form is not secreted into the tissue culture medium and behaves like an integral membrane protein. S2:pRmHa3-nrg cells were induced for 2 days with 0.7 mM CuSO(4) and 20 µl of the tissue culture medium were loaded onto the 7.5% SDS-polyacrylamide electrophoresis gel shown in panelA, lane1. Induced cells from an equivalent volume were loaded in lane2. PanelsB and C show pH 10 extraction experiments; lanes1 contain the starting material before pH 10 extraction, soluble and peripheral membrane proteins are in the soluble fractions (lanes2), and integral membrane proteins are found in the pellet fractions (lanes3). PanelB shows the experiment using S2 cell membranes containing the neuroglian form and panelC the experiment with S2 cell membranes containing the neuroglian form. PanelsA and B are Western blots stained with the mouse anti-neuroglian mAb 3F4, and panelC is a Western blot using the mouse mAb BP-104. Neuroglian is not released from the membrane by pH10 treatment and therefore behaves like an integral membrane protein.



The membrane association of GPI-anchored proteins can be severed by digestion with bacterial PI-PLC. This enzyme catalyzes the hydrolysis and release of a diacylglycerol group from the GPI moiety. Treatment of S2 cell membranes containing neuroglian with PI-PLC led to a loss of membrane association of this neuroglian form during exposure to pH 10 (Fig.4). As expected PI-PLC incubation of membranes containing the transmembrane neuroglian polypeptide had no effect on its membrane attachment (Fig.4, lanes5 and 6). The 53 carboxyl-terminal amino acid residues of the fasciclin I polypeptide are therefore sufficient to direct the attachment of a GPI membrane anchor to the neuroglian hybrid protein.

All Three Neuroglian Forms Induce the Aggregation of Transformed S2 Cells

The low endogenous adhesiveness of the S2 cell line makes it an ideal host for the functional analysis of putative CAMs(26) . Using the Drosophila S2 cell system, we tested the ability of the three different Drosophila neuroglian protein forms to induce cell aggregation. As shown in Fig.5, the two naturally occurring, as well as the neuroglian form, induced a rapid cell aggregation response. The size of the cell aggregates formed by the different neuroglian species varied, with cell lines expressing neuroglian usually giving the largest cellular aggregates (Fig.5D) and cell lines expressing neuroglian giving the smallest (Fig. 5C). Although cell lines expressing different neuroglian protein forms were selected so that lines to be compared expressed maximal amounts of the protein, we observed that cloned cell lines of S2:pRmHa3-nrg seldom expressed intact neuroglian protein at levels as high as the other forms. This may reflect half-life differences between the different forms of the proteins, which may explain the variation in the aggregation results. In addition, there may be also subtle differences in the strength of the cell adhesion reaction that is mediated by the different neuroglian protein forms.


Figure 5: Expression of neuroglian on the surface of S2 cells causes the cells to aggregate. Cells were plated at 1 10^6 cells/ml in 60-mm Petri plates and induced with 0.7 mM CuSO(4) for 24 h. After induction, the cells were gently dissociated by repeated pipetting and cell concentrations were readjusted to 1 10^6 cells/ml. The cells were then shaken at 100 rpm on a rotating platform, at room temperature, for 10-12 h prior to examination. PanelA, untransformed S2 cells; panelB, S2:pRmHa3-nrg; panelC, S2:pRmHa3-nrg; panelD, S2:pRmHa3-nrg. Untransformed S2 cells show no ability to form aggregates (A), whereas cells that express any of the three neuroglian forms show significant aggregation (B-D). Scalebar, 200 µm.



Untransformed S2 cells (Fig.5A and 6) or S2 cells transformed with neuroglian cDNAs in the antisense orientation do not aggregate. Furthermore, the neuroglian-induced cell aggregation is calcium-independent (data not shown).

Our initial attempts to quantify cell aggregation involved preinduction with CuSO(4), followed by dissociation of aggregates and assessment of reaggregation over the next 2-4 h. This approach was complicated by the fact that after 24 h of induction many of the neuroglian-expressing cells are so tightly bound that they could not be dissociated quantitatively without destroying the cells. For this reason we chose to begin the quantification experiments at the point of induction and to assess aggregation at time points over 3 days of induction. Therefore, data for these experiments represent a combination of the kinetics of protein induction and the kinetics of cell aggregation.

None of the cell lines showed any tendency to aggregate prior to induction. Within 12 h after induction, cell lines expressing any of the three neuroglian forms show significant levels of aggregation as compared to untransfected S2 cells (Fig.6). At this time point, expression levels of the various neuroglian proteins as measured by Western blot analysis, were less than 20% of the eventual maximal levels (data not shown). Maximal levels of protein expression for all three neuroglian forms were attained by 36-48 h after induction (data not shown) with maximal levels of cell aggregation occurring at 48-60 h.

At maximal aggregation the nrg- and nrg-expressing S2 cell lines attained greater than 80% of cells incorporated into multicellular aggregates. Antibody staining demonstrated that the unincorporated cells were generally cells that no longer expressed high levels of the protein. Both the nrg and nrg cell lines tended to form large aggregates, as shown in Fig.5.

The nrg cells also showed significant aggregation as compared to untransfected S2 cells but less than that observed with the other neuroglian forms. Despite efforts to isolate and compare cell lines that express similar levels of the different neuroglian forms, we have been unable to isolate a line that consistently retains high levels of expression of the 167-kDa form of neuroglian. At maximal induction, the nrg line expressed about 50% of the protein expressed by the nrg and nrg lines (data not shown). This lower level of neuroglian protein expression may account for the generally lower level of total aggregation in the quantitation experiments and also for the smaller size of the nrg aggregates (Fig.5). As with the nrg and nrg lines, the cells that did not participate in cellular aggregation tended to be cells that had lost the ability to express high levels of neuroglian protein. The experiments shown in Fig.5and Fig. 6demonstrate that all three neuroglian protein forms induce a robust aggregation response upon cell surface expression in S2 cells.

The preincubation of induced S2:pRmHa3-nrg cells with PI-PLC completely abolished the formation of cell aggregates (Fig.7), but PI-PLC treatment had no influence on the aggregation of S2:pRmHa3-nrg cells. A strong membrane attachment is therefore required for neuroglian-induced cell aggregation.


Figure 7: PI-PLC treatment abolishes neuroglian induced cell aggregation. Induced S2:pRmHa3-nrg (panelsA and B) or S2:pRmHa3-nrg cells (panels C and D) were incubated in the presence (panelsA and C) or in the absence (panelsB and D) of 50 units/ml PI-PLC. Cells were incubated with PI-PLC for 2 h at 37 °C, dispersed into single cells, and then allowed to aggregate on a shaking platform at room temperature. A representative field of each cell population is shown. Removal of neuroglian from the cell surface abolishes the ability of S2:pRmHa3-nrg cells to aggregate. Scalebar, 200 µm.



Neuroglian-induced Aggregation of S2 Cells Occurs by a Homophilic Mechanism, and the Three Neuroglian Protein Forms Interact with Each Other

Cell mixing experiments were performed to demonstrate that the observed cell aggregation response is due to neuroglian-mediated homophilic cell adhesion rather than an interaction between neuroglian and a heterophilic receptor endogenous to S2 cells. Untransformed S2 cells were marked by labeling with the vital fluorescent membrane dye DiI. Labeled S2 cells were then mixed with an equal number of unlabeled S2:pRmHa3-nrg cells and the cells were induced together and assayed for aggregation. Fig.8(A and B) shows that the fluorescent S2 cells do not aggregate and that the unlabeled S2:pRmHa3-nrg cells form aggregates that exclude the labeled S2 cells. To demonstrate the appearance of aggregates in which labeled and unlabeled cells interact, S2:pRmHa3-nrg cells were labeled with DiI and then mixed and aggregated with an equal number of unlabeled cells of the same type. Large aggregates form that contain both labeled and unlabeled cells (Fig.8, C and D). The large proportion of labeled cells in these aggregates is in contrast to the virtual absence of labeled S2 cells from the aggregates in Fig.8B. The results of these experiments demonstrate that cell aggregation in these assays is due to neuroglian-mediated homophilic cell adhesion. Cell mixing experiments were also used to determine whether the different forms of neuroglian interact in a form-specific manner or whether the different protein forms can interact with each other. S2:pRmHa3-nrg, S2:pRmHa3-nrg, and S2:pRmHa3-nrg cells were mixed in various combinations and the ability of the various cell lines to interact with each other in cell aggregation was assessed. In each experiment one cell type was labeled with DiI and one cell type was unlabeled. All combinations of cell lines showed significant mixing of both labeled and unlabeled cells in the cell aggregates (Fig.9), indicating that the different forms of neuroglian can interact with each other during cell aggregation. These mixing experiments also demonstrate that the adhesive properties of the S2:pRmHa3-nrg cells are not due to the 27 fasciclin I amino acid residues at its carboxyl terminus.


Figure 8: Neuroglian-expressing cells do not interact with untransformed S2 cells. PanelsA and B, untransformed S2 cells were labeled with DiI, and the labeled cells were then mixed with an equal number of unlabeled S2:pRmHa3-nrg cells. The cells were induced together and assayed for aggregation as described. PanelA shows all cells under Nomarski optics, and panelB shows the same field of view with only the labeled S2 cells visualized using rhodamine epifluorescence. PanelsC and D, an experiment similar to that described for panelsA and B, except that S2:pRmHa3-nrg cells were used as both the labeled and unlabeled cell types. Neuroglian-expressing cells form aggregates only with other neuroglian-expressing cells and do not interact with untransformed S2 cells, demonstrating that neuroglian is not binding to a heterophilic receptor that is constitutively expressed by S2 cells. Scalebar, 200 µm.




Figure 9: The three neuroglian protein forms interact with each other. Cell lines expressing different forms of the neuroglian protein were mixed to determine the extent of the interaction between the different forms. In each experiment a cell line expressing one form of neuroglian was marked by labeling with DiI, and the labeled cells were then mixed with an equal number of unlabeled cells expressing a different form of neuroglian. The cells were induced together and assayed for aggregation as described. PanelsA and B, C and D, and E and F represent the same fields of view showing all cells under Nomarski optics (A, C, and E) or only the labeled cell type visualized using rhodamine epifluorescence (B, D, and F). PanelsA and B show pRmHa3-nrg mixed with DiI-labeled pRmHa3-nrg. PanelsC and D show pRmHa3-nrg mixed with DiI-labeled pRmHa3-nrg. PanelsE and F show pRmHa3-nrg mixed with DiI labeled pRmHa3-nrg. Each neuroglian-expressing cell line formed mixed aggregates with all other neuroglian-expressing lines regardless of the specific form of the neuroglian protein, demonstrating that the specificity of neuroglian-mediated homophilic adhesion is not affected by the cytoplasmic domains. Scalebar, 200 µm.




DISCUSSION

The structural and sequence analysis of Drosophila neuroglian indicates a strong homology to other members of the L1-CAM gene family(6) . The data presented here confirm that this similarity also extends to some functional aspects. Like its vertebrate ``siblings'' L1, Nr-CAM, NILE, and Ng-CAM(4, 38, 39, 40) , Drosophila neuroglian functions as a calcium-independent, homophilic CAM.

Two different neuroglian protein forms are generated in vivo by alternative splicing(7) . The two neuroglian polypeptides differ only in their cytoplasmic domains and are expressed in a tissue-specific fashion during development. When transfected into and expressed in Drosophila S2 cells, both of these neuroglian species mediate homophilic cell adhesion. A third artificial form of neuroglian, in which the transmembrane segment and cytoplasmic domains are replaced with a GPI moiety, retains this ability to induce cell adhesion and can interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that the neuroglian cytoplasmic domains are not essential for neuroglian-mediated homophilic cell adhesion activity.

Members from all major groups of substrate and cell adhesion molecules have been found to be associated with different components of the cellular cytoskeleton. This includes several members of the immunoglobulin domain superfamily. The 180-kDa form of NCAM and members of the L1-CAM family either bind either directly or via an ankyrin linker to the cellular, submembranous actin-spectrin network(18, 41, 42) . We recently obtained experimental evidence that both alternative cytoplasmic domains are capable of interacting with the membrane cytoskeleton protein ankyrin. (^4)Such interactions of CAMs with cytoskeletal elements might be of great functional importance for the recruitment of CAMs or cytoskeletal components to regions of cell-cell contact, for the regulation of cell migration and cell shape, for the mediation of CAM-induced signaling events, or for the regulation of adhesive specificity and strength. From the data presented here, it appears that the binding of neuroglian protein to elements of the cytoskeleton is not required for its homophilic recognition ability. However, it is still possible that such interactions might have a modulating effect on extracellular neuroglian-mediated homophilic binding.

Besides these homophilic contacts, some L1-type CAMs are also engaged in heterophilic recognition events(38, 40, 43) . Mouse L1-CAM has been shown to interact with NCAM(44) , and chicken Ng-CAM binds to axonin-1/TAG-1 as well as to neurocan, a brain chondroitin sulfate proteoglycan(45, 46, 47) . Both chicken Ng-CAM as well as Nr-CAM/Bravo, both members of the L1 family, interact with the immunoglobulin domain F11 molecule(43, 48) . The heterophilic recognition properties of CAMs appear to be more sensitive to deletions in their cytoplasmic domains than their homophilic. In the case of PECAM-1, partial deletions of its cytoplasmic domain inactivates its heterophilic binding to heparin but uncovers a calcium-independent homophilic interaction(22) . However, in contrast to neuroglian the total deletion of its cytoplasmic domain renders the PECAM-1 molecule completely adhesion-incompetent. It would be of interest to determine whether hetero- and homophilic adhesion processes of other CAMs are regulated differently by their cytoplasmic domains. Since no potential heterophilic binding partner for Drosophila neuroglian has been identified so far, we are currently unable to address this aspect for the neuroglian molecule.


FOOTNOTES

*
The work presented in this manuscript was supported in part by grants from the University of Michigan Cancer Center (Institutional Grant from the American Cancer Society) and the National Institutes of Health (Grant HD29388 to M. H.) and by grants from the National Science Foundation (Grant IBN-9120981), the American Cancer Society (Grant IRG IN-17), the Purdue Research Foundation, and the Showalter Foundation (to A. B.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Michigan, Ann Arbor, MI 48109-0616. Tel.: 313-747-2720; Fax: 313-763-1166.

^1
The abbreviations used are: CAM, cell adhesion molecule; DiI, 1,1`-dioctadecyl-3,3,3`-tetramethylindocarbocyanine perchlorate; GPI, glycosyl phosphatidylinositol; PI-PLC, phosphatidylinositol phospholipase C; PCR, polymerase chain reaction; PECAM, platelet/endothelial cell adhesion molecule; NCAM, neural cell adhesion molecule; Nr-CAM, Ng-CAM related cell adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule.

^2
M. Hortsch, unpublished results.

^3
M. Seeger, personal communication.

^4
R. R. Dubreuil, G. MacVicar, S. Dissanayake, C. Liu, D. Homer, and M. Hortsch, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Margit Burmeister for comments on the manuscript.


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