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Dominant-negative effect on adhesion by myelin Po protein truncated in its cytoplasmic domain

The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic manner (Filbin, M.T., F.S. Walsh, B.D. Trapp, J.A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond.). 344:871-872). In addition, we have shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097). To determine if the presence of these truncated forms of Po could have an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its dominant-negative effect by preventing clustering of full-length Po. We also show that colchicine, which disrupts microtubules, prevents adhesion of cells expressing only the full-length Po. This strengthens our suggestion that an interaction of Po with the cytoskeleton, either directly or indirectly, is required for adhesion to take place.

T HE most abundant protein of PNS myelin, a singledomain Ig-like molecule termed Po, is responsible for compaction at both the extracellular and cytoplasmic membrane surfaces (for review see Filbin and Tennekoon, 1992). Evidence supporting this notion derives from studies both in vitro (D'Urso et al., 1990;Schneider-Schaulies et al., 1990) and in vivo (Giese et al., 1992;Hayasaka et al., 1993;Kulkens et al., 1993). In mice that do not express Po as a result of gene disruption by homologous recombination, the majority of axons are surrounded by loose whorls of uncompacted membranes (Giese et al., 1992). Lack of compaction in these Po -/-mice at both the extracellular and the cyto-plasmic surfaces provides strong evidence that Po is indeed a "double" adhesion molecule, involved in interactions through both its extracellular and cytoplasmic domains. The importance of Po in the compaction and hence the physiological function of PNS myelin is further demonstrated in patients suffering from the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMTIB) t. In all pedigrees of this subclassification of CMT, abnormalities in Po protein have been reported, either mutations or deletions of particular amino acids (Hayasaka et al., 1993;Kulkens et al., 1993;Suet al., 1993;Blanquet-Grossard et al., 1996).
Consistent with observations in vivo, the ability of Po to behave as a homophilic adhesion molecule through inter-actions of its extracellular domains has been clearly shown using in vitro adhesion assays D'Urso et al., 1990;Schneider-Schaulies et al., 1990). In these assays, CHO cells or other fibroblast cell lines, induced to express the Po protein by transfection of the Po cDNA, are at least two orders of magnitude more adhesive than the control cells not expressing Po . In addition, where two Po-expressing cells meet, the protein has been shown to accumulate at the interface of the cells, consistent with a homophilic interaction (D'Urso et al., 1990). These results demonstrate directly that because of its homophilic adhesion, the extracellular domain of Po has the capacity to maintain compaction at the intraperiod line of myelin.
Although the cytoplasmic domain of Po is believed to hold myelin membranes together at the cytoplasmic surfaces, the adhesion of this domain is more difficult to monitor directly in intact cells. However, Ding and Brunden (1994) have shown that the isolated, purified cytoplasmic domain of Po can induce the clustering of lipid vesicles composed of acidic lipids. Furthermore, clustering is influenced by the phosphorylation of Po cytoplasmic domain (Ding and Brunden, 1994). This binding characteristic is in keeping with the suggestion that the cytoplasmic domain of Po interacts with acidic lipids in the opposing membrane to maintain compaction at the major dense line of myelin (Braun, 1984;Lemke et al., 1988).
In addition to its inherent adhesive capabilities, we have recently shown that the cytoplasmic domain of Po can influence the adhesive interactions of the extracellular domain. We showed that although they reach the surface when expressed in CHO cells, Po molecules with either 52 or 59 amino acids deleted from the 69-amino acid-long cytoplasmic domain were not adhesive (Wong and Filbin, 1994). Furthermore, since a portion of the full-length Po expressed in Schwann or CHO cells is insoluble in the nonionic detergent NP-40, an interaction with the cytoskeleton is suggested (Wong and Filbin, 1994). In contrast, only very little of the Po missing 52 amino acids and none of the Po missing the last 59 amino acids is insoluble in this detergent, consistent with a decreased interaction with the cytoskeleton, concomitant with a loss of adhesion. Based on these observations, we suggested a model whereby the cytoplasmic domain of Po influences the adhesion of the extracellular domain by both inducing a conformational change in the extracellular domain and also allowing Po to cluster within the membrane, thereby strengthening the adhesive interactions (Wong and Filbin, 1994). It is proposed that the clustering is brought about by interactions with the cytoskeleton at the early stages of myelination, before cytoplasm is extruded and cytoplasmic surfaces are brought into close apposition. If this model is correct, then the presence in the same membranes of nonadhesive Po proteins with truncated cytoplasmic domains could adversely affect the functioning of the full-length Po. Here we test this hypothesis by coexpressing full-length Po with Po missing either 52 or 59 amino acids from the cytoplasmic domain. We report that in these transfected cell lines, both the full-length and the truncated Po proteins reach the cell surface in quantities that should be sufficient for adhesion to occur. The presence, however, of either of the truncated Po proteins prevents the full-length Po protein from behaving as an adhesion molecule. Because fulllength Po appears to cluster in the membrane when expressed alone but not when coexpressed with truncated Po, we suggest that the dominant-negative effect is brought about by truncated Po, preventing clustering of full-length Po. This shows for the first time that mutated forms of Po can have a dominant-negative effect on the adhesiveness of the wild-type protein. In addition, we show that the microtubule cytoskeleton must be intact for adhesion of the full-length Po to take place.

Truncation of Po cDNA
Po cDNAs missing the nucleotides coding for the last 52 or 59 amino acids were prepared and characterized as previously described (Wong and Filbin, 1994). Briefly, the 1.08-kb ECoRI to XbaI fragment of the Po cDNA (Lemke and Axel, 1985) in bluescriptII (pBSP) was cut at base pairs 599 and 620 by restriction enzymes PstI and NcoI, respectively. The protruding end created by PstI in Po cDNA was filled to create a blunt end with dNTPs and T4 polymerase, whereas the NcoI protruding end was partially filled in by dATP, dCTP, and T4 polymerase, after which the remaining protruding sequence was cut by Mungbean nuclease to make it blunt ended. The truncated, blunt-ended Po cDNA was religated into pBSP vector that had been cut by Smal restriction enzyme, which results in a blunt end. A new stop codon and four new amino acids (Arg, Gly, lie, and His) were brought in at the end of each truncated Po cDNA as a result of a frame shift. This was confirmed by sequencing the 3' end of each truncated Po cDNA (Sanger et al., 1977). For the PstI and NcoI truncated Po cDNA, nucleotides coding for the last 59 and 52 amino acids, respectively, of Po's cytoplasmic domain were missing.

Ligation of Truncated Po cDNAs into a Suitable Plasmid
The plasmid used for the expression of truncated Po cDNAs has been described previously (Lee and Nathans, 1988;. Briefly, after attachment of the appropriate linkers, the truncated Po cDNAs were ligated into the pSJL plasmid at a unique Xhol cloning site downstream from the mouse metallothionein promoter and upstream from the poly (A) tail of the SV-40 t-antigen gene. The plasmid also contained the mini genes for G418 resistance and dihydrofotate reductase (dhfr). The orientation of the Po cDNA in the plasmid was confirmed by restriction enzyme digestion.

Transfection
CHO cells were transfected with 1-2 txg of DNA per 10-cm plate by calcium phosphate precipitation (Graham and van der Eb, 1973) followed by a glycerol shock (Frost and Williams, 1978). A molar ratio of 1:1 of pSJL containing full-length Po cDNA/pSJL containing TPo52 or TPo59 cDNAs was used. The cells were passed, 1:2, the following day, and 3 d after transfection, 400 Ixg/ml of active G418 was added to the culture medium. Colonies appeared after ~3 wk and a number were picked, expanded, and single-cell cloned by limiting dilution. Several of these single cell clones shown by Western blot analysis to be positive for expression of full-length Po, as well as either of the truncated Po proteins, were used for gene anaplification. cells in increasing concentrations (0.05-2.0 ~M) of methotrexate (MTX). Cells were plated at 5 x 105 cells per 10-cm dish, and those surviving after 2-3 wk at each concentration of MTX were allowed to multiply before being replated on the higher concentration of MTX. At different stages in the gene amplification procedure, cells were monitored for Po expression by Western blot analysis and again single cell cloned.

Deglycosylation of Po with Endoglycosidase F (Endo F)
Cells, 80-90% confluent, were lysated in 50 mM Tris, pH 7.2, containing 1% NP-40 and the following antiproteases: 1 ug/ml leupeptin, 2 ug/ml antipain, 10 ug/ml benzamidine, l0 U/ml aprotinin, 1 ug/ml chemostatin, 1 ug/ml pepstatin, and 0.1 mM PMSF. The lysate from one 10-cm dish was divided in half. To one half. 0.4 U of Endo F was added (Boehringer-Mannheim Corp., Indianapolis, IN). Samples, with and without Endo F, were incubated at 37°C for 16 h, after which the proteins were precipitated with 8 vol of ice-cold acetone. Samples were then subjected to SDS-PAGE, followed by tranfer to nitrocellulose and immunodetection for Po.

Biotinylation of Cell Surface Proteins
Cells at ~90% confluency in 10-cm culture dishes were washed with PBS three times and then incubated with 0.5 mg/ml of sulfo-NHS-biotin for 30 rain at room temperature, according to the manufacturer's instructions (Pierce, Rockford, IL). The labeling solution was removed and the cells were incubated one more time with the same concentration of sulfo-NHSbiotin for another 30 rain. The cells were then washed with PBS three times and Po was immunoprecipitated from the surface biotin-labeled cells.

Immunoprecipitation of Po
Sulfo-NHS-biotin-labeled cells in a 10-cm dish were lysed with 300 ixl of 250 mM Tris, pH 7.4, containing 2% SDS, 4 mM EDTA, and 150 mM NaCl. The cell lysate was collected by centrifugation at 14,000 rpm for 10 rain. The supernatant fraction was removed to a fresh tube. A 200qxl aliquot of this fraction was diluted to 1 ml in 250 mM Tris, pH 7.5, containing 4 mM EDTA, 150 mM NaC1, and 2.5% Triton. Then 15 Ixl of Po polyclonal antibody was added and the mixture was incubated at 4°C overnight, with gentle rocking. 100 Ixl of 10% protein A-Sepharose was added to the immunoprecipitated sample, and the mixture was incubated for an additional 2 h at 4°C with gentle rocking. The incubation mixture was centrifuged at 14,000 rpm for 10 min and the pellet was washed three times with the dilution buffer described above. Where indicated, the samples were deglycosylated with Endo F, as described above, before being eluted from the protein A-Sepharose. After the final wash, 50 Ixl of l X SDS sample buffer and 4 Ixl of 13-mercaptoethanol were added to the pellet, which was incubated for 3 rain at 98°C and then centrifuged for 10 min at 14,000 rpm. The supernatant fraction was collected, subjected to SDS-PAGE (Laemmli, 1970), and biotinylated Po was detected by immunodetection for biotin after Western blotting using streptavidin-HRP (1:7,000) and enhanced chemiluminescence (ECL), according to the manufacturers instructions (Amersham, Corp., Arlington Heights, IL).
To detect Po that may be shed from transfected cells, cells were grown in 3 ml of media containing 2% serum per 10-cm dish. After 24 h, media from 3 x l~cm dishes, each with cells coexpressing full-length and truncated Po at ~80°/o confluency, was collected and combined. The media was immunoprecipitated for Po as described above except that the initial volume of Po antibody used was increased to 90 Ixl. As controls, media from cells expressing only Po or control-transfected cells was used.

Quantitation of Po Expressed at the Cell Surface
An ELISA was carried out as previously described by Doherty et al, (1990), modified as follows. Between 2,000 and 3,000 cells per well were plated in a 96-well ELISA plate and allowed to attach for 2 d. The cells were rinsed twice with PBS, fixed for 30 min with 4% paraformaldehyde, and then rinsed with PBS. The cells were blocked for 30 rain with 3% normal goat serum in MEM and then incubated overnight at 4°C with a rabbit Po peptide antibody directed against sequences in the extracellular domain of Po (1:1,500) in MEM containing 1% normal goat serum. The cells were rinsed, blocked again, and then incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (1:250) (Sigma Chemical Co.). The cells were rinsed with PBS and then water. Color was developed by the addition of 50 gl of 0.2% (wt/vol) O-phenylenediamine (Sigma Chemical Co.) and 0.02% H20~ in citrate buffer (pH 5.0) to each well. The reaction was stopped after 15 rain by the addition of 50 ~xl of 4.5 M HzSO4 and the optical density at 490 nm was determined with a 96-well plate reader. All incubations were at room temperature unless stated otherwise. Controls consisted of control transfected cells incubated with Po antibodies. For each experiment, 40 wells were assayed at least three times for each cell line. The average number of cells per well was estimated by removing them with trypsin and counting them with a Coulter counter. Cells from five separate wells were counted for each 96-well plate. Results were standardized to absorbance units per cell.

Adhesion Assay
The adhesion assay was carried out as previously described  with the following modification. Cells at 80-90% confluence were washed with PBS and incubated with 5 U/ml trypsin (GIBCO BRL, Gaithersburg, MD) for 2-3 min at room temperature, washed twice with MEM, and finally resuspended in MEM containing 10% FCS by three passages through an 18-gauge syringe. Suspensions, containing a minimum of 95% single cells, were diluted to a final concentration of 1.5-2 × l06 cells per ml and allowed to aggregate at 37°C in 5% CO2 with gentle rocking at 5 rpm. Before sampling, the tubes were gently inverted and aliquots were removed at intervals, examined under the microscope, and the total particle number was determined with a Coulter counter. At least three separate incubations were performed for each experiment, and duplicate samples were withdrawn at each time point and counted three times each.

Mixed Adhesion Assay
The mixed adhesion assay was carried out as previously described (Filbin and Tennekoon. 1993) with the following modifications. A single-cell suspension of cells expressing only full-length Po was mixed at a 50:50 ratio with cells coexpressing Po and Po truncated by 59 amino acids (TPo59) to give a final cell concentration of 2-3 x 106 cells per ml. Aggregation was then allowed to proceed as described above. To distinguish the two different populations of cells, in each experiment one cell population or other was vitally labeled by incubation with 10 IxM of the dye calcein AM (Molecular Probes, Eugene, OR), for 15 min at 37°C and washed with PBS before mixing. Samples were withdrawn at different times and examined under both phase and fluorescent microscopes. At least three separate incubations were performed for each experiment, and duplicate samples were withdrawn at each time point.

Chemical Cross-linking of Surface Po
Cells (80-90% confluent), surface labeled with sulfo-NHS-biotin while as a monolayer in a 10-cm dish, were removed by incubation with ice-cold calcium, magnesium-free PBS (CMF PBS). Cells were collected by centrifugation in a microfuge and resuspended in 2 ml of CMF PBS. The sample was divided in half. To one half, 10 ~tl of 0.5 mM 3,3' dithiobis(sulfosuccinimidyl propionate) (DTSSP) in DMSO was added, and to the other, 10 Ixl of DMSO was added. Both samples were rocked gently for 45 rain at room temperature. The reaction was stopped by the addition of 20 Ixl of 1 M Tris, pH 7.5, followed by a further 15 rain incubation while rocking. The cells were then washed three times with CMF PBS and lysed in immuno-precipitation buffer. Samples were immunoprecipitated with Po antibody and deglycosylated as described above. Samples were separated by SDS-PAGE, with and without 13-mercaptoethanol, as indicated, and biotinylated proteins were detected by chemiluminescence as described above.

Treatment of Cells with Agents That Disrupt the Cytoskeleton
A single cell suspension of cells expressing full length Po or control cells was incubated with either 100 p.M colchicine or 35 ~g/ml cytochalasin for 30 min at 37°C while gently rocking. The cells were then washed and resuspended in MEM with or without either colchicine or cytochalsin. The ability of the cells to exclude 0.4% trypan blue was used as assessment of their viability. The adhesion assay was carried out in the presence or absence of either colchicine or cytochalasin.

Coexpression of Full-Length Po and Truncated Po Proteins in CHO Cells
We have already established (a) that full-length Po can behave as a homophilic adhesion molecule via interactions of its extracellular domain  and (b) that the cytoplasmic domain must be intact for extracellular domain adhesion to take place. We demonstrated that Po proteins missing either 52 or 59 amino acids from the cytoplasmic sequences are not adhesive (Wong and Filbin, 1994). To test if the presence of the cytoplasmic domaintruncated Po proteins could influence the adhesive functioning of the full-length Po molecules, full-length Po (designated Po) and Po missing either 52 (TPo52) or 59 amino acids (TPo59) from their cytoplasmic sequences were coexpressed in CHO cells. The adhesiveness of these coexpressors was then compared to the adhesiveness of the cells expressing only the full-length Po.
Plasmids containing the cDNA for Po were transfected into CHO cells at a 1:1 molar ratio with ptasmids containing the cDNA for either TPo52 or TPo59 (Fig. 1). After selection in G418, amplification in methotrexate , and single cell cloning by limiting dilution, a number of clones were shown by Western blotting to express both the full-length and the truncated Po proteins. Fig. 2 shows the results from two such clones, one coexpressing Po and TPo52, designated Po/TPo52 (Fig. 2, lanes g and h) and the other coexpressing Po and TPo59, designated Po/TPo59 (Fig. 2, lanes i and j). Sugar residues, which represent approximately 6% by weight of the molecule, are attached to Po protein via a single N-linkage at asparagine 93 (Everly et al., 1973;Kitamura et al., 1976;Lemke and Axel, 1985;Sakamoto et al., 1987). The Po proteins expressed by Po/TPo52 and Po/TPo59 cell lines are of the same approximate molecular weights as when they are expressed individually in CHO cells, indicating that they are all glycosylated (Fig. 2, compare lanes g and i with a for the Po, lane c with g for the TPo52, and lane e with i for the TPo59). This is confirmed by the decrease in size for all Po species after treatment with Endo F, which removes all N-linked sugars. As the gels represented in Fig. 2 are of low percentage acrylamide, the glycosylation pattern appears as multiple minor components. This can be attributed to microheterogeneity of the glycosylation pattern, as only sharp bands are observed after deglycosylation with Endo F. Occasionally a band of about 20 kD is apparent. It is well documented that Po is very susceptible to proteolysis (Cammer et al., 1981) despite the presence of antiproteases when the cells are lysed. As this proteolysis is likely to occur only when the cells are lysed, it would not affect the adhesion of the intact cells. This view is supported by the fact that this proteolytic form of Po is sometimes apparent in lysates from cells expressing only the full-length Po protein which are adhesive (Fig. 2, lane a). It can also be seen from Fig. 2, and more clearly when the proteins are deglycosylated, that each of the two cell lines, PoFFPo52 and PoFFPo59, express amounts of fulllength Po, per 30 Ixg of total protein, at least equivalent to that expressed by the CHO cell line expressing only fulllength Po, already shown by us to be adhesive (Fig. 2, lanes a and b; Tennekoon, 1991, 1993;Wong and Filbin, 1994). Hence, these cell lines are expressing sufficient full-length protein to measure the adhesiveness of Po. In addition, it is apparent from Fig. 2 that these coexpressors are expressing approximately equal amounts of the full-length Po and the truncated Po, either TPo52 or TPo59. Therefore, these two cells lines, Po/TPo52 and Po/TPo59, are suitable to assess if the truncated Po proteins affect the adhesiveness of the full-length protein. However, before these experiments were carried out, we first ensured not only that Po was reaching the cell surface in these coexpressors but that both the full-length and the truncated Po proteins are reaching the cell surface in approximately equal amounts. Surface expression of Po in the coexpressors was (a) quantitated by an ELISA and compared to the surface expression of cells expressing only full-length Po, and (b) surface expression of Po and TPo in each coexpressor was compared by labeling of surface proteins, followed by immunoprecipitation of Po, as described below.

Surface Expression of Po Protein
To quantitate the level of expression of Po at the cell surface in the coexprcssors, an ELISA assay was carried out on fixed, unpermeabilized cells. Cells expressing either Po/ TPo59 or Po/TPo52 (Fig. 3, columns 1 and 2) expressed at least equivalent levels of Po at the cell surface as cells expressing only full-length Po (Fig. 3, column 3), which we had already shown to be adhesive Tennekoon, 1991;Wong and Filbin, 1994). In fact, the Po/TPo59 cells are expressing significantly more Po at the surface than the cells expressing only fulllength Po. Only background staining was apparent for control-transfected cells when the Po antibody was used (Fig. 3, column 4) and when the ELISA was carried out with an antibody raised to the cytoplasmic domain of Po (results not shown). This demonstrates that the majority of 0 t, Q,. cells are intact and that under these conditions of staining, only surface Po is being measured. To confirm that both Po proteins, the full-length and the truncated proteins, were reaching the cell surface in the coexpressors, surface proteins were first labeled with biotin, and Po was then immunoprecipitated from the total cell lysate and deglycosylated to aid in detection and quantitation. The biotinylated proteins in the precipitate were identified on Western blots with streptavidin-HRP and a chemiluminescence detection system. Fig. 4 shows that for the cotransfected cells, both full-length and truncated Po arc biotinylated for both the Po/TPo52 and Po/TPo59 cell lines, demonstrating that for each cell line, both proteins are reaching the cell surface (Fig. 4, lanes c and d). For each of the coexpressors, approximately equal amounts of the TPo52 or TPo59 and Po appear to be biotinylated, suggesting that they appear at the surface in equal amounts (Fig. 4, lane c and d). Furthermore, in both Po/TPo52 and Po/TPo59 there appears to be approximately the same amount of the full-length Po as when it is expressed alone (Fig. 4, lane b, c, and d), again suggesting that there is sufficient full-length Po at the surface in these coexpressors to be adhesive. These three lines were then used to compare the effect of coexpression of Po proteins truncated in their cytoplasmic domains on the adhesiveness of the fulllength Po proteins.

Adhesion of Cells Coexpressing Full-Length and Truncated Po Proteins
To assess the adhesiveness of the CHO cells simultaneously expressing full-length and truncated Po proteins, relative to cells expressing only the full-length Po protein, we carried out a reaggregation assay as we have used previously to monitor the adhesiveness of Po protein under various conditions Tennekoon, 1991, 1993;Wong and Filbin, 1994;Zhang and Filbin, 1994). In this assay, a single cell suspension is incubated and aggregation is monitored both by microscopic examination and by counting the total particle number at various times. This permits both a qualitative (microscopic examination) and quantitative (counting) analysis since a  and examined under the microscope (a-d results after 60 min aggregation), and the total particle number was counted in a Coulter counter. The total particle number ± SE was plotted against time (e). decrease in total particle number correlates with aggregate formation, and the rate of aggregate formation is an indication of the strength of adhesion.
By 60 min, only the cells expressing the full-length Po alone had formed large aggregates (Fig. 5 c). As we have previously reported Filbin and Tennekoon, 1991;Wong and Filbin, 1994;Zhang and Filbin, 1994), these aggregates consist of up to 300 cells per aggregate. In sharp contrast, the cells coexpressing truncated Po proteins with the full-length Po had not formed aggregates within the same time (Fig. 5, a and b). They were predominantly single cells, doublets, and triplets and were indistinguishable from the appearance of the controltransfected cells (Fig. 5 d). Consistent with the microscopic observations, the total particle number for the cells expressing only the full-length Po had dropped to ~20% of the starting number by 60 min. By contrast, the total particle number for the cells coexpressing full-length and truncated Po proteins (Po/TPo52 and Po/TPo59) dropped to about only 80% in the same time, a value similar to that for the control cells (Fig. 5 e).
It is possible that, because of the short cytoplasmic domain, truncated Po is being shed from the coexpressing cells and, in a soluble form, blocking Po-Po interactions between cells, consequently preventing aggregation. This does not appear to be the case for two reasons. First, after immunoprecipitation with Po antibody of conditioned media from Po-expressing cells (combined media from 3 × 10-cm dishes of semiconfluent cells, conditioned 24 h), no Po was detected in the precipitate. Second, when a singlecell suspension of a 50:50 mix of cells expressing only fulllength Po and cells coexpressing full-length and truncated Po were allowed to aggregate, with one cell type vitally labeled with a fluorescent dye, only the cells expressing fulllength Po alone formed aggregates. The cells coexpressing full-length and truncated Po remained as mostly single cells with very few joining the aggregates. The same result was obtained regardless of which cell population was vitally labeled (Fig. 6, A and B). Therefore, the presence of cells coexpressing full-length and truncated Po has no effect on the aggregation properties of cells expressing only full-length Po.
Taken together, these results strongly suggest that, even though expression of the full-length Po protein should be sufficient to increase the adhesiveness of these cells, adhesion of full-length Po is prevented by the presence of truncated Po in the same membrane. Truncated Po, therefore, has a dominant-negative effect on adhesion.

Clustering of Po and Truncated Po Proteins
To determine if Po clusters in the membrane before interacting with a Po molecule in an opposing cell, a single-cell suspension of cells expressing only full-length Po was treated with the reducible cross-linking agent DTSSP. Before exposure to cross-linker, the cells were surface biotinylated when growing as a monolayer. After incubation with cross-linking agent, the cells were lysated, immunoprecipiated with Po antibody, and, to aid in detection, degtycosylated. The biotinylated proteins in the precipitate were identified on Western blots with streptavidin-HRP and a chemiluminescence detection system. It was found that if cells expressing only full-length Po are treated with cross-linking agent and not reduced, little or no Po is detected in the immunoprecipitates (Fig. 7, lane b). Under the same conditions, but without cross-linker, an abundance of Po is precipitated under nonreducing conditions, apparent at a molecular weight consistent with unreduced Po (Fig. 7, lane a). Similarly, if the cells are exposed to cross-linker and the lysate immunoprecipitated in the absence of reducing agent, but 13-mercapoethanol is added to the sample immediately before separation by SDS-PAGE, an abundance of Po is apparent, equivalent to samples that were not cross-linked (Fig. 7, lanes c and d). These results suggest that when cross-linked, full-length Po expressed alone by CHO cells, after immunoprecipitation, is in very large aggregates that do not enter a 12% polyacrylamide gel and are therefore not detected; Po in these samples is only detected if the aggregates are disrupted by a reducing agent before loading on the gel. In contrast, if truncated Po is coexpressed with full-length Po in the same cell, approximately the same amount of each Po protein is apparent under nonreducing conditions, regardless of whether or not the samples were treated with cross-linking agent, suggesting that there are no large aggregates (Fig. 7, lanes   e-h). It should be noted that because of the differences in degree of biotinylation for each cell preparation, only samples in lanes a-d, e and f, and g and h, can be compared quantitatively with each other. However, although the overall level of biotinylation appears less for the cells coexpressing Po and TP59 (Fig. 7, lanes g and h) than for the other cells, it is still obvious that there is approximately the same amount of Po with and without cross-linking; there are no large aggregates. Together, these results suggest that when expressed alone, full-length Po clusters within the membrane even before coming in contact with a Po protein in an opposing cell. However, the presence of truncated Po protein not only does not cluster itself but also prevents clustering of the full-length Po protein.

The Effect of Colchicine and Cytochalasin on the Adhesiveness of Full-Length Po Protein
We have already shown that a percentage of full-length Po is insoluble in the detergent NP-40, while only very little TPo52 is insoluble and TPo59 is completely soluble (Wong and Filbin, 1994). Insolubility in this nonionic detergent suggests that a protein is interacting with the cytoskeleton.
To determine if indeed disruption of the cytoskeleton affects the adhesion of Po expressed in C H O cells, two drugs were used: cytochalasin, which disrupts microfilaments, and colchicine, which disrupts microtubules. Drug concentrations and conditions of incubation were as used by others to successfully disrupt the cytoskeleton (Cheung and Juliano, 1984;Murray and Jensen, 1992). Pretreatment of the cells and inclusion of cytochalasin in the incubation had no effect on the adhesiveness of the Po-expressing ceils (results not shown). On the other hand, treatment of the cells with 100 txM colchicine prevented aggregate formation completely; by 60 rain the total particle number for the colchicine treated cells expressing full-length Po and the control cells dropped to only 80% (Fig. 8 a). There was no difference in the surface expression of Po on cells treated or not treated with colchicine, as assessed by an ELISA (Fig. 8 b). Colchicine had no effect on the behavior of the control transfected cells (Fig. 8, a and b). These results suggest that the adhesiveness of the extracellular domain of full-length Po is dependent on an interaction of the cytoplasmic domain, either directly or indirectly, with microtubules.

Discussion
It is widely accepted that the cytoplasmic domain of Po is responsible for compaction at the major dense line of myelin and, most likely, that this is a result of an interaction with acidic lipids in the opposing membrane (for reviews see Braun, 1984;Lemke et al., 1988;Filbin and Tennekoon, 1992;Giese et al., 1992;Ding and Brunden, 1994). In addition, we have demonstrated that an intact cytoplasmic domain is required for the adhesion of the extracellular domain of Po to take place and that full-length, but not truncated Po, is likely to interact with the cytoskeleton (Wong and Filbin, 1994). Based on these observations, we proposed a model to explain the adhesion of Po whereby the cytoplasmic domain can influence the adhesion of the extracellular domain, perhaps by inducing a conformational change. In addition, we suggest that the molecule must cluster within the membrane, probably after an interaction of the cytoplasmic domain with the cytoskeleton, for stable membrane-membrane adhesion to occur (Wong and Filbin, 1994). Here we strengthen this model. We show that Po truncated in its cytoplasmic sequences can have a dominant-negative effect on the adhesion of the extracellular domain. When the full-length Po is expressed in the same membrane as the truncated Po, adhesion does not occur. Therefore, the model we propose can be extended to suggest that not only must Po cluster in the membrane, but there must also be a critical number of functional Po molecules within the cluster for stable adhesion to take place (Fig. 9). The cross-linking studies indicate that when expressed alone, full-length Po does indeed cluster, but when coexpressed with truncated Po, no clusters form. In keeping with the model, we suggest that the truncated Po molecules are having a dominant-negative effect on adhesion by preventing the clustering of fulllength Po (Fig. 9). Furthermore, the ability of cholchicine to disrupt the adhesion of the full-length Po strengthens our suggestion that an interaction of Po with the cytoskeleton is crucial for adhesion to take place.
This model contributes to the emerging picture of how Po functions in compact myelin. The idea of clustering and increased adhesion allows for a single, small molecule to result in strong adhesion, as is found in compact myelin. To our knowledge, this is the first time a dominant-negative effect has been reported for an Ig-like adhesion molecule. The ability of Po to function in a dominant-negative manner may have relevance to the phenotype of the demyelinating disease CMT1B. Point mutations or single amino acid deletions in Po protein have been reported for all patients suffering from CMT1B (for reviews see Chance and Pleasure, 1993;Chance and Fischbeck, 1994;Patel and Lupski, 1994). However, these patients are all heterozygotes for the mutated gene and so produce 50% of the usual levels of normal protein. In some instances of CMT1B, a reduction in functional protein could account for the disease, particularly in those patients with a mild phenotype. This is supported by the observation that in mice heterozygote for the Po null mutation, myelination and myelin maintenance is normal until the mice are about 4-too old. At this later stage of development, demyelination is apparent in a majority of the nerves examined from these older mice (Martini et al., 1995). This suggests that half the amount of Po protein is sufficient for formation of normal myelin initially but not to sustain compact myelin throughout the lifetime of the animal. On the other hand, gene dose cannot alone explain the phenotype of all pedigrees of CMT1B (for reviews see Chance and Pleasure, 1993;Chance and Fischbeck, 1994;Patel and Lupski, 1994). For those patients with a more severe phenotype and an earlier onset of the disease, the presence of the mutated Po protein is likely to contribute directly to the manifestation of the disease. As half the Po protein in these patients is also normal, the presence of the mutated protein, as we report here for truncated Po, could be having a dominant-negative effect on the functioning of the wild- were preincubated with 100 IxM colchine for 30 rain and then allowed to aggregate in the presence of 100 IzM colchicine, Po(+) and C(+), or cells were not treated with colchicine, Po(-). Samples were withdrawn at intervals and the total particle number counted. The total particle number + SE was plotted against time. type protein. Although these mutations are for the most part in the extracellular sequences of Po, the model we present and the notion of a critical number of functional Po molecules being necessary for effective membranemembrane adhesion still applies to these mutations. That is to say, the Po molecules mutated in their extracellular sequences are not adhesive and so dilute the number of functional Po molecules in each cluster, diminishing the sum total of adhesion affinity per cluster to the point where membranes are no longer held together. In those patients with a late onset and mild phenotype, it is possible that some forms of mutated Po never reach myelin and so have no dominant-negative effect. Consequently demyelination in these patients is a result, solely, of gene dose. As we have suggested before, because there are no cytoskeletal elements in compact myelin, it is probable that the interaction of Po with the cytoskeleton takes place at the early stages of myelination, before cytoplasm is extruded from the leaflets ( Fig. 9; Wong and Filbin, 1994). A portion of Po expressed in Schwann cells is also insoluble in NP-40, which shows that the putative cytoskeleton interaction is not an artifact of CHO cells (Filbin and Wong, 1994). The ability of colchicine to abolish adhesion of fulllength Po greatly strengthens the idea that an interaction with the cytoskeleton is necessary for adhesion of Po to take place. Drugs that disrupt the cytoskeleton have been shown to prevent the adhesion of other molecules (Cheung and Juliano, 1984;Carpen et al., 1992;Murray and Jensen, 1992;Kinch et al., 1993). Although disruption of the microtubule network has an effect on the functioning of a number of adhesion molecules (Cheung and .luliano, 1984;Domnina et al., 1985;Kinch et al., 1993;Timar et al., 1995), more often the actin network has been implicated (for review see Pavalko and Otey, 1994). It should be noted, however, that the precise and perhaps overlapping roles of the different cytoskeletal networks has not been fully characterized. Microtubules are known to be involved in vesicular transport (Hugon et al., 1987;Achier et al., 1989;Gilbert et al., 1991). Indeed in Schwann cells treated with colchicine in vivo, Po-containing vesicles have been shown to accumulate in the perinuclear region of the cell. Consequently, Po does not reach myelin (Trapp et al., 1995). In the Po-expressing CHO cells used in the adhesion assay here, there is no difference in the surface expression of Po on cells treated or not treated with colchicine. It is possible that microtubules not only transport the Po-containing vesicles to myelin but also participate in Po clustering before disengaging.
The model we propose for Po to function as an adhesion molecule in membranes/myelin (Fig. 9) is similar to the models proposed for other types of adhesion molecules, namely the cadherins and the integrins. Cadherins are known to interact via their cytoplasmic domains with molecules termed catenins, which in turn interact with the cytoskeleton (Nagafuchi and Takeichi, 1988;Hirano et al., 1992). As we suggest for Po, an interaction of cadherins with the cytoskeleton and concurrent with this, an intact cytoplasmic domain, is necessary for the homophilic adhesion of cadherins to take place (Nagafuchi and Takeichi, 1988;Nagafuchi et al., 1991;Kintner, 1992;Levine et al., A. Full-length Po The cytoskeleton reorganizes and pulls back, but the formation of clusters is prevented by the presence of the truncated Po molecules. The overall adhesiveness is decreased because of the presence of the nonadhesive, truncated Po molecules. Adhesion is not strong enough to hold two membranes together. Funayama et al., 1995). For both types of molecule, this involves an interaction with the cytoskeleton, and for integrins, clustering is required. There are no known signaling motifs in the cytoplasmic domain of Po. However, the possibility that Po plays a role in signal transduction is suggested by two observations. First, in the Po -/-mice there is a misregulation of other myelin proteins, suggesting that Po can influence, either directly or indirectly, their expression (Giese et al., 1992). Second, when Hela cells (a cervical carcinoma cell line of epithelial origin) are induced to express Po by transfection, they revert to the morphology of a nontransformed phenotype (Doyle et al., 1995). In addition, these Po-expressing Hela cells up-regulate the expression of the adhesion molecule N-cadherin and a number of junction-associated proteins. The up-regulation of these molecules and the consequent loss of the transformed morphology could result from direct signaling via Po. In summary, we have shown that Po missing either 52 or 59 amino acids from the cytoplasmic domain when coexpressed in the same membrane prevents the adhesion of the full-length Po protein. We suggest, therefore, that the truncated Po molecule can have a dominant-negative effect on the adhesion of Po. Furthermore, the ability of cholchicine to disrupt adhesion of the full-length Po protein is a strong indication that an interaction with the cytoskeleton is essential for adhesion of the extracellular domain of Po to take place. Finally, the ability of mutated forms of Po to behave in a dominant-negative fashion may have relevance to the manifestation of the phenotype in the demyelinating disease, CMT1B, in which patients have a mutation in the Po gene.
We would like to thank Dr. Roger Persell for critically reading this manuscript and Dr. Yamina Merazga and Marat Bakman for their expert technical skills. This work was supported by a grant from the National Institutes of Health (NIH) NS 26242 and a core facility grant from the Research Center for Minorities Institute, NIH. M.T. Filbin is the recipient of an Established Investigator award from the American Heart Association, New York chapter.
Received for publication 8 February 1996 and in revised form 26 June 19%, 1994). Integrins have been shown to cluster in the membrane and in so doing cluster other proteins located at the cytoplasmic surface (for reviews see Hynes, 1992;Gumbiner, 1993). Mutated forms of both cadherins (Kintner, 1992;Fujimori and Takeichi, 1993;Levine et al., 1994;) and integrins (Balzac et al., 1994;Lukashev et al., 1994;Smilenov et al., 1994), like Po, have been shown to have dominant-negative effects on the adhesion of their respective wild-type counterpart. These mutations consist of truncations of either the cytoplasmic or extracellular domain, suggesting that, as we suggest for Po, there is an interdependence in the functioning of the two regions of the molecule, despite the fact that a lipid membrane separates the two.
Recently, although neither carries a recognizable signaling motif in the cytoplasmic domain, both cadherins and integrins have been implicated in signal transduction (Akiyama et al., 1994;Huang et al., 1993;Heasman et al., 1994;