Chondroitin sulfate and cytoplasmic domain-dependent membrane targeting of the NG2 proteoglycan promotes retraction fiber formation and cell polarization

William B. Stallcup* and Kimberlee Dahlin-Huppe

The Burnham Institute, La Jolla Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA

*Author for correspondence (e-mail: stallcup{at}burnham.org)

Accepted March 20, 2001


    SUMMARY
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Targeting of the NG2 proteoglycan to cellular retraction fibers was studied by expressing mutant NG2 molecules lacking specific structural elements of the proteoglycan. Both the cytoplasmic domain and the chondroitin sulfate chain of NG2 appear to have roles in sorting NG2 to subcellular microdomains destined to become retraction fibers. Neither of these structural features alone is sufficient to allow optimal targeting of NG2 to retraction fibers, but together they promote efficient localization of the proteoglycan to these sites. This pattern of NG2 sorting seems to be necessary for optimal retraction fiber formation, as cells expressing poorly targeted NG2 mutants are noticeably deficient in their ability to extend retraction fibers. Furthermore, retraction fiber formation correlates strongly with the tendency of cells to assume a polarized morphology with NG2-positive retraction fibers at one pole of the cell and actin-rich lamellipodia at the other. This polarization can be triggered either through engagement of NG2 by the substratum or by exposure to lysophosphatidic acid, a potent activator of the rho GTPase. These results suggest a possible role for NG2 in regulating rho-dependent mechanisms in the trailing processes of motile cells.

Key words: NG2 proteoglycan, Chondroitin sulfate, Cytoplasmic domain, Membrane sorting, Retraction fibers, Cell polarity


    INTRODUCTION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NG2 chondroitin sulfate proteoglycan is expressed by immature progenitor cells in several different developmental lineages, including oligodendrocyte progenitors, chondroblasts, and pericytes/smooth muscle cells (Nishiyama et al., 1991b; 1996a,b; Grako and Stallcup, 1995; Grako et al., 1999; Burg et al., 1999; Schlingemann et al., 1990, 1996). NG2 is also expressed by a number of different types of tumors, including melanomas (Real et al., 1985), glioblastomas (Schrappe et al., 1991), chondrosarcomas (Leger et al., 1994), and myeloid leukemias (Smith et al., 1996). NG2 appears to be important for potentiating cell motility (Burg et al., 1997,1998: Fang et al., 1999; Eisenmann et al., 1999) and for modulating cellular responses to growth factors (Grako and Stallcup, 1995; Grako et al., 1999; Nishiyama et al., 1996b; Goretzki et al., 1999), properties which are critical for the proliferation and migration of both immature progenitor cells and tumor cells.

NG2 is a membrane-spanning protein that interacts with macromolecules on both sides of the plasma membrane. The localization patterns of NG2 on both well-spread and motile cells point to an interaction of the proteoglycan with the actin cytoskeleton (Lin et al., 1996a, b). Furthermore, engagement of NG2 by the substratum triggers cytoskeletal rearrangements that lead to cell spreading and migration. The nature of these cytoskeletal changes suggests the involvement of the rho family GTPases rac and cdc42 in NG2-activated processes (Fang et al., 1999; Eisenmann et al., 1999). We are currently studying the signaling pathways through which NG2 activates these small GTPases. One possible mechanism could involve an interaction of NG2 with PDZ domain-containing scaffolding proteins in the cytoplasm. We have shown that the C-terminus of NG2 interacts with MUPP1, a scaffolding molecule that contains 13 PDZ modules and could therefore provide links to a number of cytoplasmic signaling pathways (Barritt et al., 2000).

On the cell surface, NG2 interacts with a variety of diverse ligands, including extracellular matrix (ECM) components, growth factors and kringle domain proteins. Kringle-dependent binding of NG2 to plasminogen and angiostatin may provide key mechanisms for regulating angiogenesis (Goretzki et al., 2000). The NG2-bound form of plasminogen exhibits an increased rate of conversion to plasmin, whereas binding of NG2 to angiostatin appears to neutralize the inhibitory effect of angiostatin on endothelial cell proliferation. We have tested several growth factors, and among these NG2 was found to selectively bind to platelet-derived growth factor AA (PDGF-AA) and basic fibroblast growth factor (bFGF). NG2 may therefore act as an auxiliary cell surface receptor for these two factors, potentiating their ability to interact with and activate their respective receptor tyrosine kinases (Grako et al., 1999; Goretzki et al., 1999). With respect to NG2-interactive ECM components, the best-studied is type VI collagen, which may interact with NG2 to mediate processes such as cell migration (Burg et al., 1997), cell proliferation (Atkinson et al., 1996; Ruhl et al., 1999a) and protection from apoptosis (Ruhl et al., 1999b). Type VI collagen is thought to play important roles in tissue remodeling, vascular development and wound healing (Rand et al., 1991; Rand et al., 1993; Sage and Vernon, 1994; Oono et al., 1993; Zhang et al., 1994). It is therefore interesting that NG2 is also upregulated in healing wounds and in neovasculature (Schlingemann et al., 1990; Schrappe et al., 1991; Grako and Stallcup, 1995; Burg et al., 1999). The co-localization of NG2 with type VI collagen in some developing tissues (Stallcup et al., 1990; Doane et al., 1998) is consistent with the idea that these molecules interact in vivo.

In the case of heparan sulfate proteoglycans, the heparan sulfate chains play a key role in interactions with ligands. For example, bFGF binding to this class of proteoglycans, along with subsequent activation of bFGF receptors, is highly dependent on the presence of the heparan sulfate chains (Yayon et al., 1991; Rapraeger et al., 1991; Spivack-Kroitzman et al., 1994; Pantoliano et al., 1994). By contrast, we have not been able to demonstrate a role for the NG2 chondroitin sulfate chain in any of the aforementioned paradigms. In studying binding between purified NG2 and its various ligands, we have not found that growth factors, kringle domain proteins or type VI collagen have the ability to discriminate between NG2 with or without chondroitin sulfate chains. In each case the unmodified NG2 core glycoprotein has an affinity for the ligands that matches that of chondroitin sulfate-containing NG2.

These experiments naturally raise the question of why the NG2 core protein carries chondroitin sulfate. In this paper, we show that NG2 is modified with a single chondroitin sulfate chain at Ser999. We also present evidence suggesting that, rather than influencing the binding properties of NG2, this chondroitin sulfate chain may help to dictate the localization of the proteoglycan to specific subcellular microdomains. Our evidence indicates that both the chondroitin sulfate chain and the cytoplasmic domain are involved in targeting NG2 to microdomains destined to become cellular retraction fibers. Expression of NG2 at these sites appears to promote retraction fiber extension and also increases the likelihood that cells will assume a polar morphology resembling that of motile cells.


    MATERIALS AND METHODS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
The B28 rat glioma (Schubert et al., 1974) and the U251 human asyrocytoma (Ponten and Westermark, 1978) were used in these studies. B28 cells transfected with wild-type NG2 and with the chimeric molecules NG2/L1 and NG2/CNTN have been described previously (Nishiyama and Stallcup, 1993; Lin et al., 1996a; Burg et al., 1997; Fang et al., 1999). Cells were grown in Dulbecco’s modified Eagles medium supplemented with 10% fetal calf serum (Tissue Culture Biologicals, Tulare, CA).

Antibodies and reagents
Rabbit polyclonal and mouse monoclonal antibodies against NG2 have been described previously (Nishiyama et al., 1991a; Nishiyama et al., 1995; Tillet et al., 1997). A rabbit antiserum against whole B28 cells has also been described (Lin et al., 1996b; Fang et al., 1999). Monoclonal antibodies against CD44, ß-actin and fascin were purchased from Pharmingen (La Jolla, CA), Sigma (St Louis, MO) and Dako (Carpinteria, CA), respectively.

The rho-associated kinase inhibitor Y27632 was obtained from Welfide Corporation, Osaka, Japan. A GST fusion protein for the C3 exoenzyme was a generous gift from Joan Brown (University of California, San Diego, CA).

Immunoblotting
Preparation of NP40 cell extracts was carried out as described by Fang et al. (1999). One half of each cell extract was treated for 1 hour at room temperature with 0.02 units of chondroitinase ABC (ICN Biomedical, Costa Mesa, CA) to remove chondroitin sulfate chains. Samples were then mixed with 2X SDS-PAGE sample buffer and boiled. After fractionation on 3-20% SDS-PAGE gels, samples were electrophoretically transfered to Immobilon P membranes (Millipore, Bedford, MA). Blocking and probing of these membranes were carried out as previously described (Nishiyama et al., 1995; Grako et al., 1999). Immunoreactive bands were visualized using an ECL chemiluminescence kit (Amersham Life Science, Buckinghamshire, UK).

Immunoprecipitation
Immunoprecipitation of detergent-extracted material from [125I]-labeled cells was performed as described previously (Nishiyama et al., 1991a; Dahlin-Huppe et al., 1997), using rabbit anti-NG2 antibodies and protein A-Sepharose (Sigma) to isolate immunoreactive material. After preparation of the immunoprecipitates, half of each sample was treated with 0.02 units of chondroitinase ABC for 1 hour at room temperature. Samples were then boiled in SDS-PAGE sample buffer and fractionated by electrophoresis on 3-20% gradient gels. Gels were dried and analyzed by autoradiography using Kodak X-OMAT AR film.

cDNA constructs and transfections
Mutation of serine to alanine at positions 999 and 1342 in the NG2 core protein (Nishiyama et al., 1991a) was carried out by performing sequential PCR reactions using overlapping primers encoding the mutation (Ausubel et al., 2000). NG2 cDNA in the pcDNAI/amp expression vector was used as the template for the reactions (Nishiyama and Stallcup, 1993; Burg et al., 1997). For the S999A mutation, the two central overlapping primers covered nucleotides 3055-3075 with an AG to GC switch at positions 3064-3065, whereas the 5' and 3' primers covered nucleotides 2188-2208 and 4294-4311, respectively. For the S1342A mutation, the same 5' and 3' primers were used, but the central overlapping primers covered nucleotides 4084-4104 with a T to G switch at position 4093. Final PCR products were cut with DraIII and SplI and ligated into an NG2/pcDNAI/amp plasmid that had been similarly restricted.

We have previously described the use of Lipofectin and LipofectAmine (GIBCO-BRL, Gaithersburg, MD) for expression of wild-type and mutant forms of NG2 in both B28 and U251 cells (Nishiyama and Stallcup, 1993; Lin et al., 1996a; Burg et al., 1997). The S999A and S1342A mutant constructs were expressed in similar fashion in both B28 and U251 cells. Co-transfection with the pSV2neo plasmid (Southern and Berg, 1982) allowed initial selection of positive colonies with G418. These populations were further enriched for NG2-positive cells by immuno-panning of dissociated cells on plates coated with mouse monoclonal antibody D4 against NG2. Panning plates were prepared by incubation of 100 mm Petri dishes with 5 µg purified D4 in 8 ml 0.05 M Tris-Cl, pH 9.5. After a 2 day incubation at 4°C, the plates were blocked for 4 hours with 1% heat-inactivated BSA in PBS. Cells for panning were harvested using enzyme-free cell dissociation buffer (GIBCO-BRL) and plated in the blocked panning dishes in 1% BSA in DMEM. Plates were left undisturbed at 37°C for 15-20 minutes, and then washed thoroughly with four changes of DMEM. Adherent cells were harvested by trypsinization and replated in normal growth medium. In some cases, multiple rounds of panning were required to obtain populations that were sufficiently homogeneous for NG2 expression.

Retraction fiber extension and cell polarization
Cell polarity and the presence of retraction fibers were analyzed in cells plated either on poly L-lysine (PLL)-coated 35 mm tissue culture dishes or on 35 mm petri plates coated with monoclonal antibodies (mAbs) against cell surface components. Preparation of these plates has been described previously (Fang et al., 1999). Cells to be analyzed were serum-starved overnight in DMEM. The following day, cells were harvested using enzyme-free cell dissociation buffer (GIBCO-BRL) and resuspended for 1 hour in DMEM containing 1% heat-inactivated BSA (DMEM/BSA). Assays were initiated by addition of 2x104 cells to each coated dish in 1.5 ml of DMEM containing 1% BSA or other additives such as serum, PDGF (R and D Systems, Minneapolis, MN), or lysophosphatidic acid (Sigma) as described in the figures. Cells were left undisturbed for 8 hours at 37°C before being processed for imaging as described below.

Immunofluorescence
For analysis of cell spreading and retraction fiber formation, cells were fixed on the PLL- or mAb-coated dishes by adding an equal volume of 4% paraformaldehyde to the culture medium. After a 10 minute fixation at room temperature, the cells were washed extensively with PBS and blocked for 30 minutes with DMEM containing 2% FCS. In cases where cells were stained for ß-actin or fascin, fixation was accomplished by a 2 minute incubation at -20°C with absolute methanol, followed by blocking with serum.

Immunofluorescent labeling was performed as described by Dahlin-Huppe et al. (1997). Cells were incubated with primary antibodies for 30 minutes at room temperature, followed by three washes with DMEM containing 2% FCS. Incubation with fluorochrome-coupled goat secondary antibodies against rabbit and mouse immunoglobulins (TAGO, Camarillo, CA) was also carried out for 30 minutes at room temperature. After final washing and fixation with 95% ethanol, specimens were cover-slipped in Immumount (Shandon, Pittsburg, PA) and examined using a Nikon Optiphot microscope equipped for phase contrast and epifluorescence. Photographs were taken with Kodak TMAX 400 film.


    RESULTS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Attachment of chondroitin sulfate to NG2 mutants
On the basis of published information regarding possible consensus sequences for the attachment of chondroitin sulfate chains (Bourdon et al., 1987; Zimmerman and Ruoslahti, 1989), we had previously speculated that serine residues 999 and 1342 represented the most likely sites of chondroitin sulfate attachment to the NG2 core polypeptide (Nishiyama et al., 1991a). To test this directly, we independently mutated each of these two serine residues to alanines and expressed both mutant cDNAs in B28 rat glioma cells and U251 human astrocytoma cells.

The chondroitin sulfate content of NG2 expressed in B28 and U251 cells was assessed biochemically both by immunoblotting and by immunoprecipitation of [125I]-labeled material. Fig. 1 shows immunoblots of wild-type NG2, NG2/S999A and NG2/S1342A extracted from B28 cells. Wild-type NG2 characteristically appears as a mixture of the well-defined 300 kDa core protein devoid of chondroitin sulfate (arrowhead) and a high-molecular-weight smear representing the complete proteoglycan (asterisk). The smear is not always easily visible without over-exposure of the film). Treatment with chondroitinase ABC results in the disappearance of the intact proteoglycan with a concomitant increase in the quantity of the core protein. This same pattern of chondroitinase sensitivity is seen with extracts from two independent clones of B28 cells (clones 42 and 44) expressing the NG2/S1342A mutant, indicating that this mutant is still modified with chondroitin sulfate. By contrast, extracts from two clones of NG2/S999A-expressing B28 cells (clones 51 and 65) do not exhibit sensitivity to chondroitinase ABC. An identical profile of unmodified core protein is seen both before and after chondroitinase digestion, indicating the absence of chondroitin sulfate in this mutant and identifying Ser999 as the attachment site for chondroitin sulfate in NG2. Immunoprecipitation of [125I]-labeled material from this same panel of B28 cell lines also showed that the NG2/S1342A transfectants contained chondroitin sulfate while the NG2/S999A transfectants did not (results not shown).



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Fig. 1. Evaluation of chondroitin sulfate content by immunoblotting. Immunoblot analysis with rabbit anti-NG2 antibody was performed on detergent extracts of B28 cells transfected with wild-type and mutant NG2. Half of each sample was treated with chondroitinase ABC to remove chondroitin sulfate chains (+), while the other half was left untreated (-). In the case of B28/NG2.6 cells, which express wild-type NG2 (c6/wt), this chondroitinase treatment converts the high-molecular-weight smear representing the mature proteoglycan (asterisk) into the 300 kDa core glycoprotein (arrowhead). A similar increase in the quantity of the core is seen with extracts of cells expressing the NG2/S1342A mutant (clones 42 and 44), indicating that this form of NG2 still contains chondroitin sulfate. By contrast, chondroitinase treatment has no apparent effect on extracts of cells expressing the NG2/S999A mutant (clones 51 and 65), suggesting that this form of NG2 is not modified with chondroitin sulfate.

 
An identical set of results was obtained with immunoblots and immunoprecipitations of material extracted from U251 transfectants (results not shown), suggesting that different cell types utilize the same attachment site on the NG2 core protein.

NG2 localization to retraction fibers
Retraction fibers are often seen in association with cells that have rounded up in preparation for cell division (Mitchison, 1992). Similarly, we have previously shown that a large percentage of the B28 cells on a PLL-coated dish can be forced to round up by treating them with colchicine, and that under these circumstances the cells exhibit dense arrays of retraction fibers (Lin et al., 1996a). Fig. 2 (a,b) shows the array of retraction fibers associated with colchicine-treated B28 cells that are transfected with NG2. These protrusions are vividly stained both by poly-specific antibodies against whole B28 cells (a) and by NG2 antibodies (b). By contrast, colchicine treatment of B28 cells transfected with the chondroitin sulfate-free mutant NG2/S999A yields a dense array of retraction fibers (e) that are only poorly stained by the NG2 antibody (f). The sparse, punctate NG2 staining pattern for this mutant is quite distinct from the strong, continuous staining of retraction fibers seen in wild-type transfectants. The serine to alanine mutant NG2/S1342A, which does not affect chondroitin sulfate attachment, behaves like wild-type NG2 in that it is highly expressed in retraction fibers (c,d). To determine whether the low NG2 content of the NG2/S999A retraction fibers is caused by the absence of chondroitin sulfate, we chondroitinase-treated wild-type NG2 transfectants and treated them with colchicine. These cells were then analyzed by double staining with anti-NG2 and anti-B28 antibodies. Fig. 2 (g,h) shows that the retraction fibers of these chondroitinase-treated cells exhibit the same type of reduced, punctate NG2 staining seen in the NG2/S999A transfectants, suggesting that the presence of the chondroitin sulfate chain helps to determine localization of NG2 to the retraction fibers.



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Fig. 2. Chondroitin sulfate affects targeting of NG2 to retraction fibers. B28 cells expressing wild-type NG2 (clone 6, a,b,g,h), NG2/S1342A (clone 42, c,d), NG2/S999A (clone 51, e,f), NG2/L1 (clone 5, i,j) and NG2/CNTN (clone M, k,l) were grown on poly L-lysine-coated dishes overnight. In panels g and h, cells expressing the wild-type NG2 were chondroitinase treated for 1 hour at 37°C. All sets of cells were then colchicine treated (10-5 M) for 30 minutes at 37°C and stained with monoclonal anti-NG2 antibody (b,d,f,h,j,l) and poly-specific rabbit anti-B28 antibody (a,c,e,g,i,k). After colchicine treatment, all cells exhibit dense arrays of retraction fibers as shown by the staining with poly-specific antibody. However, NG2 staining in the NG2/S999A cells is much weaker than in the wild-type or NG2/S1342A cells. Removal of chondroitin sulfate from cells expressing wild-type NG2 also results in diminished localization of NG2 to the retraction fibers (g,h). Diminished, punctate NG2 staining in retraction fibers is also seen in the NG2/CNTN transfectants, whereas no retraction fiber staining for NG2 is observed for NG2/L1. Bar, 10 µm (j).

 
To determine whether chondroitin sulfate is the only factor that determines NG2 localization to retraction fibers, we also examined B28 cells expressing chimeric NG2 molecules lacking the NG2 transmembrane and cytoplasmic domain. In the NG2/L1 chimera, the transmembrane and cytoplasmic domains of NG2 are replaced by the corresponding domains of the neuronal cell adhesion molecule L1 (Lin et al., 1996a,b). In the NG2/CNTN chimera, the NG2 ectodomain is anchored to the membrane by the GPI linkage motif of contactin (Dahlin-Huppe et al., 1997). Both of these chimeras still contain the NG2 chondroitin sulfate chain (Fang et al., 1999). Fig. 2 (k,l) shows that the retraction fibers of colchicine-treated NG2/CNTN transfectants also exhibit the reduced, punctate pattern of NG2 localization seen with NG2/S999A transfectants. Even more dramatically, the NG2/L1 chimera is completely absent from retraction fibers (i,j). These results show that chondroitin sulfate alone does not produce efficient NG2 targeting to retraction fibers, but that the NG2 cytoplasmic domain also plays a role in this process.

Role of NG2 in retraction fiber formation
Fig. 2 shows examples of retraction fiber extensions that remain associated with the culture surface when the cell cytoplasm contracts during cell rounding. Under some circumstances, however, even well-spread cells can extend retraction fibers. For example, when grown overnight on PLL-coated plates, NG2-transfected B28 cells have a strong tendency to develop retraction fibers that are positive for NG2, even when they have not been treated with colchicine and are still well spread (Lin et al., 1996b). Fig. 3 (a,b) shows an example of the NG2-positive retraction fibers extended by B28 cells transfected with wild-type NG2. As in the case of the colchicine-treated cells of Fig. 2, these retraction fibers are brightly labeled both with the poly-specific B28 antiserum and with NG2 antibody. NG2/S1342A-transfected B28 cells (c,d) exhibit a pattern of NG2-positive retraction fiber formation that is indistinguishable from that of the wild-type NG2 transfectants. By contrast, parental cells (g,h) and NG2/S999A transfectants (e,f) appear to extend a much smaller number of retraction fibers. The few retraction fibers present on the NG2/S999A transfectants exhibit the same faint punctate pattern of NG2 staining (f) seen in the colchicine-treated cells in Fig. 2 (e,f). Parental B28 cells are of course negative for NG2 (h).



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Fig. 3. Localization of NG2 to cellular retraction fibers. Parental B28 cells (g,h) and B28 cells expressing wild-type NG2 (clone 6, a,b), NG2/S1342A (clone 42, c,d), and NG2/S999A (clone 51, e,f) were grown overnight on poly L-lysine-coated plates, fixed with 2% paraformaldehyde, and then double-labeled with monoclonal anti-NG2 antibody (b,d,f,,h) and rabbit antibody against whole B28 cells (a,c,e,g). The paraformaldehyde fixation is important because antibody incubations and repeated washing of living cells tend to induce cell ‘rounding’, accompanied by retraction fiber formation. Thus, the fixed cells give a more accurate picture of cell morphology under actual culture conditions. In the case of cells expressing wild-type NG2 and NG2/S1342A, abundant retraction fibers visible by staining with the poly-specific B28 antiserum are also heavily labeled by the anti-NG2 antibody. B28 cells and transfected cells expressing NG2/S999A have many fewer retraction fibers. In the NG2/S999A transfectants these fibers are not well stained by the anti-NG2 antibody, as seen previously in Fig. 2. B28 cells are negative for NG2. Bar, 10 µm (h).

 
A similar set of observations is obtained when cells are allowed to attach and spread overnight on plates coated with a monoclonal antibody (mAb D120) that recognizes an epitope near the type VI collagen-binding domain of NG2 (Fang et al., 1999). Wild-type and NG2/S1342A transfectants are still superior to NG2/S999A transfectants in their ability to extend retraction fibers (results not shown). Thus, the differences in the tendency of these cell types to form retraction fibers are observed whether the cells are anchored by attachment to poly L-lysine or by engagement of cell-surface NG2 by an antibody-coated substratum.

Fig. 4 quantifies the formation of retraction fibers in two ways. Part A presents the percentage of cells of each type that have any detectable retraction fibers. Part B compares the number of retraction fibers per cell for each of the cell types. These data not only illustrate the greater tendency of wild-type and NG2/S1342A transfectants to extend retraction fibers, but also show that even when parental cells and NG2/S999A transfectants exhibit retraction fibers, they display fewer protrusions per cell. Both sets of data therefore emphasize the difference between the two classes of cells and suggest that the chondroitin sulfate-dependent targeting of NG2 to retraction fibers may actually be important for fiber formation.



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Fig. 4. Quantitation of retraction fiber formation. (A) Using cultures like those described in Fig. 3, we examined at least 200 cells to determine the percentage of cells that displayed retraction fibers, as determined by staining with the poly-specific rabbit antibody against B28 cells. The figure compares parental B28 cells (P) and B28 cells transfected with wild-type NG2 (clone 6), NG2/S1342A (clones 42 and c44), NG2/S999A (clones 51 and c65), NG2/L1 (clone 5) and NG2/CNTN (clone M). Mean and standard error values were calculated from data obtained with three replicate plates of each cell type. (B) Using the same sets of cells described in A, we counted the number of retraction fibers per cell. Values for at least 13 cells are plotted for each cell type in the figure. Horizontal bars show the mean value for each cell type. Cells with small numbers of retraction fibers are the same ones identified as low responders in A.

 
Fig. 4 also presents data for B28 cells expressing the two chimeric proteins NG2/L1 and NG2/CNTN. Both of these chimeric transfectants are similar to the NG2/S999A transfectants in their diminished ability to extend retraction fibers. The importance of the NG2 cytoplasmic domain in retraction fiber formation is consistent with the finding that, along with the chondroitin sulfate chain, the cytoplasmic tail also plays a role in targeting NG2 to retraction fibers.

Role of NG2 in determining cell polarity
In determining the relative tendencies of various cell types to form retraction fibers, it became apparent to us that NG2-transfected B28 cells plated on either PLL-coated dishes or mAb D120-coated dishes have a tendency to develop polarized morphologies. This phenomenon was not addressed in the quantitation of retraction fibers shown in Figs 3 and 4, but it seemed worthy of further attention. Fig. 5 shows examples of these polarized cells double stained for NG2 and actin (a,b) and for poly-specific B28 determinants and the actin-binding protein fascin (c,d). The retraction fibers on one pole of these cells are rich in NG2, whereas lamellipodia on the opposite pole are rich in actin and fascin. Short NG2-positive spikes are also present on the lamellipodia of these cells. Previously, we showed that actin and fascin-rich lamellipodia represent the leading edges of motile B28 cells, whereas retraction fibers represent the trailing edges (Lin et al., 1996b). Thus the polarized cells seen in our current experiments have morphologies that one would expect of motile cells.



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Fig. 5. NG2-mediated cell polarization. B28 cells transfected with wild-type NG2 were serum-starved overnight, resuspended in DMEM/BSA for 1 hour, and then allowed to spread for 8 hours in DMEM/FCS on plates coated with mAb D120. Cells were then fixed and double-stained with rabbit anti-NG2 (a) and mouse monoclonal anti-ß-actin (b), or with rabbit anti-B28 (c) and mouse monoclonal anti-fascin (d). Retraction fibers at one pole of the cell are strongly NG2-positive, whereas actin and fascin are localized to lamellipodia at the opposite pole. Bar, 10 µm (a).

 
We noticed that the frequency of cell polarization on PLL-coated plates was increased in serum-containing medium, but was much more rare under serum-free conditions. To determine what component of serum might be responsible for the increased polarization, we supplemented the serum-free medium with PDGF, which stimulates membrane ruffling by activation of the rac GTPase (Ridley et al., 1992), and with lysophosphatidic acid (LPA), which is known to activate the rho GTPase (Ridley and Hall, 1992). Fig. 6 shows that LPA doubles the number of polarized cells seen on PLL plates, whereas PDGF is not active in this regard. This indicates that activation of rho is important for initiating cell polarization. This conclusion is reinforced by the finding that the response to LPA can be inhibited both by the C3 exoenzyme, a specific inactivator of rho (Sekine et al., 1989), and by Y27632, an inhibitor of the rho-associated kinase (Uehata et al., 1997).



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Fig. 6. Involvement of the Rho GTPase in cell polarization. NG2-transfected B28 cells were serum-starved overnight, resuspended in DMEM/BSA for 1 hour, then allowed to spread for 8 hours on PLL- or mAb-coated plates. Cells were then fixed with paraformaldehyde and stained with poly-specific rabbit antibody against B28 cells. The percentage of morphologically polarized cells was determined under a variety of conditions. On PLL plates the control condition was 1% BSA in DMEM, to which were added either 10 ng/ml of PDGF-AA and PDGF-BB or 1 µg/ml LPA. For mAb-coated plates the control condition is mAb CD44 coating, whereas the stimulatory condition is mAb D120 coating. When used, the C3 exoenzyme (3 µg/ml) and the Y27632 (Y) rho-associated kinase inhibitor (20 µM) were added to cells during the 1 hour suspension period and maintained in the cultures throughout the spreading phase of the experiment. Mean and standard error values for each condition were calculated from data obtained from three replicate plates.

 
The effect of mAb D120-coated dishes on cell polarization can be seen by comparing this surface to dishes coated with a monoclonal antibody against another cell surface proteoglycan, CD44 (Fig. 6). The mAb D120 surface is several-fold more active in promoting polarization than the mAb CD44 surface. Moreover, addition of LPA to cells on mAb D120 plates does not further stimulate polarization. This suggests that engagement of NG2 may trigger rho activation, rendering LPA treatment superfluous. The inhibition of mAb D120-induced polarization by both C3 and Y27632 supports this interpretation.

Because chondroitin sulfate-containing NG2 seems to be important for stimulating retraction fiber formation, we wondered if the proteoglycan might also be important for determining the ability of cells to polarize. Fig. 7 shows that on both PLL- and mAb D120-coated plates, NG2/S1342A transfectants (b,e) assume polarized morphologies indistinguishable from those of wild-type NG2 transfectants (a,d). By contrast, NG2/S999A transfectants are much less likely to extend retraction fibers and polarize (c,f). A quantitative assessment of these phenomena is presented in Fig. 8. In part A, we compare the ability of LPA to stimulate cell polarization on PLL-coated plates. Two clones of NG2/S999A-transfected B28 cells closely resemble parental B28 cells in their failure to polarize in response to LPA. By contrast, two clones of NG2/S1342A transfectants are stimulated in a fashion similar to that seen with wild-type transfectants. In part B, we compare polarization of this same set of cell types on dishes coated with mAb D120 and mAb CD44. Cells expressing chondroitin sulfate-containing NG2 are stimulated to polarize on the mAb D120 surfaces, whereas cells expressing chondroitin sulfate-negative NG2 do not discriminate between the CD44 and D120 plates. This set of results indicates that the chondroitin sulfate-dependent sorting of NG2 to retraction fibers is important for the ability of cells to polarize.



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Fig. 7. Effect of NG2 chondroitin sulfate chain on cell polarization. Cell polarization was examined after 8 hours on PLL plates in the presence of 1 µg/ml LPA (d-f) and on mAb D120 plates in DMEM/BSA (a-c). Under both sets of conditions, B28 cells transfected with wild-type NG2 (a,d) and with NG2/S1342A (b,e) exhibited a high tendency to polarize. By contrast, many fewer instances of polarization were observed for NG2/S999A-transfected cells (c,f). Bar, 10 µm (f).

 


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Fig. 8. Quantitation of cell polarization. B28 cells transfected with wild-type NG2 (clone 6) and with NG2/S1342A (clones 42 and 44) and NG2/S999A (clones 51 and 65) were compared with parental B28 cells (P) for their ability to polarize after 8 hours of spreading on PLL-coated (A) and mAb-coated dishes (B). After spreading, the cells were fixed with paraformaldehyde and stained with poly-specific anti-B28 antibody as shown in Fig. 7 to allow determination of the percentage of polarized cells. Mean and standard error values were calculated from data obtained with three replicate plates for each cell type. On PLL dishes the addition of LPA to the control BSA-containing medium stimulated polarization in wild-type and NG2/S1342A transfectants, but not in parental cells or NG2/S999A transfectants. On D120-coated dishes, wild-type and NG2/S1342A transfectants had much a greater tendency to polarize than on mAb CD44-coated dishes, whereas NG2/S999A transfectants did not distinguish significantly between the two surfaces.

 

    DISCUSSION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One immediate result of our experiments is the ability to assign Ser999 as the principal site of chondroitin sulfate attachment to the NG2 polypeptide. On the basis of the apparent difference in molecular size between the NG2 core glycoprotein (300 kDa) and the intact proteoglycan (400-600 kDa smear) on SDS-PAGE, we had speculated that there might be several chondroitin sulfate chains associated with the core. The presence of at least two good consensus sequences for chondroitin sulfate attachment at Ser999 (EGSGD) and Ser1342 (SGLG, Nishiyama et al., 1991a) seemed consistent with this prediction. However, our current experiments with point mutants have shown that NG2/S1342A still contains chondroitin sulfate, whereas NG2/S999A is devoid of glycosaminoglycan, indicating that Ser1342 is not used as an attachment site. Thus, at least in this case, the E/DGSGE/D motif (Zimmermann and Ruoslahti, 1989) is utilized, whereas the SGXG motif (Bourdon et al., 1987) is not. This usage may highlight the importance of the precise placement of acidic residues flanking the serine-glycine pair in the attachment site. This motif is abundant in versican (Zimmermann and Ruoslahti, 1989), and is also found in syndecan and in type IX collagen (Saunders et al., 1989; Huber et al., 1988). An EGSG motif is also utilized as an attachment site for heparan sulfate in the perlecan domain V (Friedrich et al., 1999).

In several previous reports from our lab, we were not able to demonstrate a functional role for the chondroitin sulfate attached to the NG2 core glycoprotein. The single chondroitin sulfate chain of NG2 does not have an apparent effect on the ability of the proteoglycan to bind the growth factors PDGF-AA and bFGF (Goretzki et al., 1999) or the kringle-domain-containing proteins plasminogen and angiostatin (Goretzki et al., 2000). Our experiments with type VI collagen also suggest that its interaction with NG2 does not require the presence of chondroitin sulfate on the NG2 core (Stallcup et al., 1990; Burg et al., 1996; Tillet et al., 1997).

Insight into the functional importance of the chondroitin sulfate chain has been provided by our current work with the chondroitin sulfate-free NG2/S999A mutant. These findings indicate that the chondroitin sulfate chain of NG2 is important for localization of the proteoglycan to retraction fibers. Compared to wild-type NG2 and the chondroitin sulfate-containing mutant NG2/S1342A, NG2/S999A is present in greatly reduced amounts in cellular retraction fibers. Chondroitinase treatment of cells expressing wild-type NG2 results in this same type of reduced NG2 expression in retraction fibers, consistent with a role for the NG2 chondroitin sulfate chain in localizing the proteoglycan to these structures.

There are a few precedents for the role of carbohydrate moieties in targeting of proteins to specific subcellular domains. N- and O-linked glycans have been implicated in the transport of proteins to the cell surface and in targeting proteins to the apical surfaces of polarized cells (Guan et al., 1985; Scheiffele et al., 1995; Yeaman et al., 1997; Gut et al., 1998; Benting et al., 1999). In some cases the presence of these glycans seems to be more important for apical sorting than the GPI anchor, which has received much attention in this regard (Benting et al., 1999). There is also a report implicating heparan sulfate in the sorting of glypican to the apical and basolateral surfaces of polarized epithelial cells (Mertens et al., 1996). In the absence of heparan sulfate chains, glypican is specifically targeted to apical surfaces by virtue of its GPI anchor. Addition of heparan sulfate appears to antagonize this mechanism, resulting in sorting of the proteoglycan to basolateral surfaces.

In the case of NG2 we do not yet have sufficient data to determine whether the chondroitin sulfate chain plays an active or inhibitory role in protein sorting. For example, it is possible to imagine that the glycosaminoglycan chain could act as a specific tag that directs NG2 to membrane microdomains that are destined to become retraction fibers upon NG2 expression. It is also possible that unmodified NG2 contains sorting signals that actively direct it to subcellular sites other than retraction fibers. Masking of these signals by the chondroitin sulfate chain might allow NG2 to occupy sites in retraction fiber microdomains that would otherwise be off limits to this protein. Further work will be required to evaluate the validity of these models.

Chondroitin sulfate is not the only factor that affects localization of NG2 to retraction fibers. As suggested by a previous study (Lin et al., 1996a), the NG2 cytoplasmic domain also plays a role in this phenomenon. Chimeric molecules lacking the NG2 cytoplasmic domain are poorly localized to retraction fibers, even though they contain the intact NG2 ectodomain including the chondroitin sulfate chain. For example, the chimeric molecule NG2/CNTN, in which the NG2 ectodomain is anchored in the plasma membrane by a GPI linkage, shows low levels of localization to retraction fibers. The NG2/L1 chimera, in which the NG2 ectodomain is anchored to the membrane by the transmembrane and cytoplasmic domains of the L1 neuronal cell adhesion molecule, is undetectable in retraction fibers. The existence of these types of differences in the incorporation of various membrane proteins into retraction fibers was previously suggested by our observation that intact L1 and other molecules such as the neural cell adhesion molecule (NCAM) and the CD44 proteoglycan are absent from retraction fibers, whereas integrins and NG2 are present in these protrusions (Lin et al., 1996a,b). The absence of NG2/L1 from retraction fibers may indicate that the L1 cytoplasmic domain is actively involved in sorting the protein to sites other than retraction fibers. In this context, a role for the cytoplasmic domain has been noted in the sorting of several membrane-spanning proteins to the basolateral surfaces of polarized epithelial cells (Hopkins, 1991; Miettienen et al., 1994).

In addition to the importance of both the chondroitin sulfate chain and the cytoplasmic domain for sorting of NG2 to retraction fibers, we also find that expression of NG2 with these two structural features is important for the actual formation of the retraction fibers in well-spread cells. Although colchicine-induced cell rounding seems to allow NG2-independent formation of retraction fibers in all cell types examined, efficient retraction fiber extension by well-spread cells is highly dependent on the presence of NG2 containing both the chondroitin sulfate chain and the cytoplasmic domain. Thus, well-spread B28 cells expressing wild-type NG2 or the NG2/S1342A mutant are significantly more active in retraction fiber formation than parental B28 cells or cells expressing the chondroitin sulfate-free NG2/S999A mutant. Cells expressing the NG2/L1 and NG2/CNTN chimeric molecules also have only low tendencies to develop retraction fibers.

At present, we do not fully understand the distinction between colchicine-induced and NG2-dependent retraction fiber formation. From the results presented in this and earlier papers (Lin et al., 1996a; Lin et al., 1996b), it appears that the molecular composition of the two types of retraction fibers may be very similar, and that the requirements for NG2 targeting are the same in both cases. However, it seems possible that the colchicine-mediated process represents a passive, NG2-independent mechanism in which cellular extensions merely remain attached to the substratum as the cell cytoplasm retracts. But, as discussed more fully below, the NG2-dependent process does not require cell rounding, and may represent an active process in which cellular protrusions are formed as a result of transmembrane signaling and cytoskeletal rearrangement.

Our experiments suggest that NG2 is not merely inserted into pre-existing retraction fibers, as these structures seem to have a low probability of forming on well-spread cells in the absence of NG2. Instead, the data are more consistent with a model in which retraction fiber formation on well-spread cells is actively enhanced at sites where NG2 is inserted into the membrane. This leads to the question of how these insertion sites are selected. Because wild-type NG2, NG2/S999A, NG2/L1 and NG2/CNTN are all expressed on the cell surface at high density, one possibility is that NG2 molecules are inserted randomly into the cell membrane, and that retraction fiber formation is efficiently stimulated only by NG2 molecules containing both the chondroitin sulfate chain and an intact cytoplasmic domain. Arguing against the idea of random cell surface expression is the observation that NG2/S999A and the two chimeric molecules are expressed at much lower densities in retraction fibers than wild-type NG2. This suggests that NG2 is preferentially targeted to certain membrane microdomains and that the proteoglycan plays a role in converting these sites into retraction fibers.

One implication of these results is that, although the chondroitin sulfate chain of NG2 seems to have little effect on the binding of NG2 to extracellular matrix ligands such as type VI collagen (Burg et al., 1996; Tillet et al., 1997), it may nevertheless have a dramatic effect on the sub-cellular sites at which NG2 interacts with matrix components. In other words, the interaction of type VI collagen with chondroitin sulfate-containing NG2 localized to retraction fibers may have a different effect on the cell than its interaction with chondroitin sulfate-free NG2 localized elsewhere on the cell surface. The evidence presented here suggests that when NG2 is localized to retraction fibers, interaction of the proteoglycan with the substratum leads to the development of cell polarity.

This apparent effect of NG2-positive retraction fibers on cell polarization is the most striking result of our experiments on the mechanisms that control NG2 sorting to specific cellular microdomains. Cells that are able to extend NG2-positive retraction fibers have a greatly increased tendency to assume a polarized morphology resembling that of motile cells. These cells exhibit ‘leading’ edges with actin-rich lamellipodia and ‘trailing’ edges with NG2-positive retraction fibers. Presumably because of their inability to extend NG2-positive retraction fibers, NG2-negative cells or cells expressing NG2 without either the chondroitin sulfate chain or the cytoplasmic domain exhibit only a low tendency to polarize.

In addition to retraction fiber extension, the morphological polarization of B28 cells also seems to require participation of the rho GTPase, as evidenced by the ability of LPA to stimulate polarization on PLL plates. Inhibition of the LPA response by the specific rho inactivator C3 and by the rho-associated kinase inhibitor Y27632 further demonstrate the involvement of rho in this phenomenon. Stimulation of rho alone is not sufficient to induce polarization, as neither parental B28 cells nor cells transfected with the NG2/S999A mutant exhibit an increased tendency to polarize in response to LPA. Apparently, both NG2-positive retraction fiber extension and rho activation are required for morphological polarization. Interestingly, when NG2 is engaged on surfaces coated with mAb D120, the requirement for LPA in cell polarization disappears. This suggests that NG2 engagement may trigger the activation of rho, an idea that is supported by the ability of both C3 and Y27632 to inhibit cell polarization on mAb D120. This involvement of rho is reminiscent of previous results from our lab and others indicating that NG2 engagement triggers rapid activation of the rho family GTPases rac and cdc42, leading to later activation of rho itself (Fang et al., 1999; Eisenmann et al., 1999).

Several groups have demonstrated a connection between rho activation and microtubule depolymerization (Enomoto, 1996; Liu et al., 1998; Zhang et al., 1997; Cook et al., 1998), whereas others have found a correlation between microtubule lattice formation and rac activation (Best et al., 1996; Ren et al., 1998). Because microtubule growth takes place at the leading edges of motile cells and microtubule shortening occurs at the trailing edges, it has been proposed that elongation stimulates rac-dependent membrane ruffling in lamellipodia, whereas shortening is responsible for rho-dependent contractility and membrane consolidation in the tail (Waterman-Storer and Salmon, 1999; Ballestrem et al., 2000). Our results suggest that NG2 could be an important factor in regulating rho activation at the trailing edges of motile cells. Because retraction fibers may represent a specialized mechanism for facilitating release of the trailing edge from the substratum (Chen, 1981; Mitchison, 1992; Sheetz, 1994), the role of NG2 in the formation and behavior of these protrusions may be a crucial factor in determining cell motility.

It remains to be demonstrated whether cells in our studies that display a higher tendency to polarize also exhibit higher levels of motility: that is, whether B28 cells expressing wild-type NG2 are more highly motile than cells expressing NG2 mutants such as NG2/S999A or NG2/CNTN. This will require quantification of cell motility on surfaces coated with mAb D120 or on PLL-coated surfaces in the presence of LPA. We have previously noted differences in the motility of NG2-positive and negative cells in response to the NG2 ligand type VI collagen (Burg et al., 1997). It will therefore be of interest to examine cell polarization on this surface. If we can demonstrate a correlation between NG2-dependent polarization and cell motility, this will be extremely relevant to the possible function of NG2 in vivo. NG2 is expressed on a number of different immature cell types, including oligodendrocyte progenitors (Nishiyama et al., 1996a,b), chondroblasts (Nishiyama et al., 1991b), smooth muscle cells and vascular pericytes (Grako and Stallcup, 1995; Grako et al., 1999; Burg et al., 1999; Schlingemann et al., 1990), and could contribute to the critical ability of these cells to migrate during the course of tissue development. NG2 is also highly expressed in some types of tumors, including melanomas (Real et al., 1985), and has been implicated in the metastatic potential of these tumor cells (Burg et al., 1998). The role of NG2 in cell migration is therefore of importance during both normal and neoplastic development.


    ACKNOWLEDGMENTS
 
This work was supported by NIH grants RO1 AR44400, RO1 NS21990, and PO1 HD25938 to WBS. We thank Dr Joan Brown for her gift of the C3 exoenzyme, and the Welfide Corporation for their gift of the Y27632 inhibitor.


    REFERENCES
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Atkinson, J., Ruhl, M., Becker, J., Ackermann, R. and Schuppan, D. (1996). Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro. Exp. Cell Res. 228, 283-291.[Medline]

Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K. (2000). Current Protocols in Molecular Biology. John Wiley & Sons, New York.

Ballestrem, C., Wehrle-Haller, B., Hinz, B. and Imhof, B. (2000). Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control directed cell migration. Mol. Biol. Cell 11, 2999-3012.[Abstract/Free Full Text]

Barritt, D., Pearn, M., Zisch, A., Lee, S., Javier, R., Pasquale, E. and Stallcup, W. (2000). The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2. J. Cell. Biochem. 79, 213-224.[Medline]

Benting, J., Rietveld, A. and Simons, K. (1999). N-glycans mediate the apical sorting of a GPI-anchored, raft-associated protein in Madin-Darby canine kidney cells. J. Cell Biol. 146, 313-320.[Abstract/Free Full Text]

Best, A., Ahmed, S., Kozma, R. and Lim, L. (1996). The ras-related GTPase rac1 binds tubulin. J. Biol. Chem. 7, 3756-3762.

Bourdon, M., Krusius, T., Campbell, S., Schwartz, N. and Ruoslahti, E. (1987). Identification and synthesis of a recognition signal for the attachment of glycosaminoglycans to proteins. Proc. Natl. Acad. Sci. USA 84, 3194-3198.[Abstract]

Burg, M., Tillet, E., Timpl, R. and Stallcup, W. (1996). Binding of the NG2 proteoglycan to type VI collagen and other extracellular matrix molecules. J. Biol. Chem. 271, 26110-26116.[Abstract/Free Full Text]

Burg, M., Nishiyama, A. and Stallcup, W. (1997). A central segment of the NG2 proteoglycan is critical for the ability of glioma cells to bind and migrate toward type VI collagen. Exp. Cell Res. 235, 254-264.[Medline]

Burg, M., Grako, K. and Stallcup, W. (1998). Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells. J. Cell Physiol. 177, 299-312.[Medline]

Burg, M., Pasqualini, R., Arap, W., Ruoslahti, E. and Stallcup, W. (1999). NG2 proteoglycan-binding peptides target tumor neovasculature. Cancer Res. 59, 2869-2874.[Abstract/Free Full Text]

Chen, W. (1981). Surface changes during retraction-induced spreading of fibroblasts. J. Cell Sci. 49, 11-13.

Cook, T., Nagasaki, T. and Gundersen, G. (1998). Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophasphatidic acid. J. Cell Biol. 141, 175-185.[Abstract/Free Full Text]

Dahlin-Huppe, K., Berglund, E., Ranscht, B. and Stallcup, W. (1997). Mutational analysis of the L1 neuronal cell adhesion molecule identifies membrane-proximal amino acids of the cytoplasmic domain that are required for cytoskeletal anchorage. Mol. Cell. Neurosci. 9, 144-156.[Medline]

Doane, K., Howell, S. and Birk, D. (1998). Identification and functional characterization of two type VI collagen receptors, {alpha}3ß1 integrin and NG2, during avain corneal stromal development. Invest. Ophthalmol. Vis. Sci. 39, 263-276.[Abstract]

Eisenmann, K., McCarthy, J., Simpson, M., Keely, P., Guan, J., Tachibana, K., Lim, L., Manser, E., Furcht, L. and Iida, J. (1999). Melanoma chondroitin sulfate proteoglycan regulates cell spreading through Cdc42, Ack-1, and p130cas. Nat. Cell Biol. 1, 507-513.[Medline]

Enomoto, T. (1996). Microtubule disruption induces the formation of actin stress fibers and focal adhesions in cultured cells: possible involvement of the rho signal cascade. Cell Struct. Funct. 21, 317-326.[Medline]

Fang, X., Burg, M., Barritt, D., Dahlin-Huppe, K., Nishiyama, A. and Stallcup, W. (1999). Cytoskeletal reorganization induced by engagement of the NG2 proteoglycan leads to cell spreading and migration. Mol. Biol. Cell. 10, 3373-3387.[Abstract/Free Full Text]

Friedrich, M., Gohring, W., Morgelin, M., Brancaccio, A., David, G. and Timpl, R. (1999). Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V. J. Mol. Biol. 294, 259-270.[Medline]

Goretzki, L., Burg, M., Grako, K. and Stallcup, W. (1999). High affinity binding of bFGF and PDGF-AA to the core protein of the NG2 proteoglycan. J. Biol. Chem. 274, 16831-16837.[Abstract/Free Full Text]

Goretzki, L., Lombardo, C. and Stallcup, W. (2000). Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen). J. Biol. Chem. 275, 28625-28633.[Abstract/Free Full Text]

Grako, K. A. and Stallcup, W. (1995). Participation of the NG2 proteoglycan in rat aortic smooth muscle cell responses to platelet-derived growth factor. Exp. Cell Res. 221, 231-240.[Medline]

Grako, K., Ochiya, T., Barritt, D., Nishiyama, A. and Stallcup, W. (1999). PDGF {alpha}-receptor is unresponsive to PDGF-AA in aortic smooth muscle cells from the NG2 knockout mouse. J. Cell Sci. 112, 905-915.[Abstract/Free Full Text]

Guan, J., Machamer, C. and Rose, J. (1985). Glycosylation allows cell surface transport of an anchored secretory protein. Cell 42, 489-496.[Medline]

Gut, A., Kappeler, N., Hyka, N., Balda, M., Hauri, H. and Matter, K. (1998). Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins. EMBO J. 17, 1919-1929.[Free Full Text]

Hopkins, C. (1991). Polarity signals. Cell 68, 827-829.

Huber, S., Winterhalter, K. and Vaughn, L. (1988). Isolation and sequence analysis of the glycosaminoglycan attachment site of type IX collagen. J. Biol. Chem. 263, 752-756.[Abstract/Free Full Text]

Leger, O., Johnson-Leger, P., Jackson, E., Coles, B. and Dean, C. (1994). The chondroitin sulfate proteoglycan NG2 is a tumour specific antigen on the chemically-induced rat sarcoma HSN. Int. J. Cancer 58, 700-705.[Medline]

Lin, X., Dahlin-Huppe, K. and Stallcup, W. (1996a). Interaction of the NG2 proteoglycan with the actin cytoskeleton. J. Cell. Biochem. 63, 463-477.[Medline]

Lin, X., Grako, K., Burg, M. and Stallcup, W. (1996b). NG2 proteoglycan and the actin-binding protein fascin define separate populations of actin-containing filopodia and lamellipodia during cell spreading and migration. Mol. Biol. Cell 7, 1977-1993.[Abstract]

Liu, B., Chrzanowski-Wodnicka, M. and Burridge, K. (1998). Microtubule depolymerization induces stress fibers, focal adhesions, and DNA synthesis via the GTP-binding protein rho. Cell Adhes. Commun. 5, 249-255.[Medline]

Mertens, G., Van der Schueren, B., van den Berghe, H. and David, G. (1996). Heparan sulfate expression in polarized epithelial cells: the apical sorting of glypican (GPI-anchored proteoglycan) is inversely related to its heparan sulfate content. J. Cell Biol. 132, 487-497.[Abstract]

Miettienen, H., Edwards, S. and Jalkenen, M. (1994). Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions. Mol. Biol. Cell 5, 1325-1339.[Abstract]

Mitchison, T. (1992). Actin-based motility on retraction fibers in mitotic PtK2 cells. Cell Motil. Cytoskeleton 22, 135-151.[Medline]

Nishiyama, A., Dahlin, K., Prince, J., Johnstone, S. and Stallcup, W. (1991a). The primary structure of NG2, a novel membrane-spanning proteoglycan. J. Cell Biol. 114, 359-371.[Abstract]

Nishiyama, A., Dahlin, K. and Stallcup, W. (1991b). The expression of NG2 proteoglycan in the developing rat limb. Development 111, 933-944.[Abstract]

Nishiyama, A. and Stallcup, W. (1993). Expression of the NG2 proteoglycan causes retention of type VI collagen on the cell surface. Mol. Biol. Cell 4, 1097-1108.[Abstract]

Nishiyama, A., Lin, X. and Stallcup, W. (1995). Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis. Mol. Biol. Cell 6, 1819-1832.[Abstract]

Nishiyama, A., Lin, X., Giese, N., Heldin, C. and Stallcup, W. (1996a). Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain. J. Neurosci. Res. 43, 299-314.[Medline]

Nishiyama, A., Lin, X., Giese, N., Heldin, C. and Stallcup, W. (1996b). Interaction between NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells is required for optimal response to PDGF. J. Neurosci. Res. 43, 315-330.[Medline]

Oono, T., Specks, U., Eckes, B., Majewski, S., Hunzelmann, N., Timpl, R. and Krieg, T. (1993). Expression of type XI collagen mRNA during wound healing. J. Invest. Dermatol. 100, 329-334.[Abstract]

Pantoliano, M., Horlick, R., Springer, B., Dyk, D., Tobery, T., Wetmore, D., Lear, J., Nahapetian, A., Bradley, J. and Sisk, W. (1994). Multivalent ligand-receptor binding interactions in the fibroblast growth factor system produce a cooperative growth factor and heparan mechanism for receptor dimerization. Biochemistry 33, 10229-10248.[Medline]

Ponten, J. and Westermark, B. (1978). Properties of human malignant glioma cells in vitro. Med. Biol. 56, 184-193.[Medline]

Rand, J., Patel, N., Schwartz, E., Zhou, S. and Potter, B. (1991). 150-kD von Willebrand binding protein extracted from human vascular subendothelium is type VI collagen. J. Clin. Invest. 88, 253-259.[Medline]

Rand, J., Wu, X., Potter, B., Uson, R. and Gordon, R. (1993). Co-localization of von Willebrand factor and type VI collagen in human vascular subendothelium. Am. J. Pathol. 142, 843-850.[Abstract]

Rapraeger, A., Krufka, A. and Olwin, B. (1991). Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 252, 1705-1708.[Medline]

Real, F., Houghton, A., Albino, A., Cordon-Cardo, C., Melamed, M., Oettgen, H. and Old, L. (1985). Surface antigens of melanomas and melanocytes defined by mouse monoclonal antibodies: specificity analysis and comparison of antigen expression in cultured cells and tissues. Cancer Res. 45, 4401-4411.[Abstract]

Ren, Y., Li, R., Zheng, Y. and Busch, H. (1998). Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for rac and rho GTPases. J. Biol. Chem. 273, 34954-34960.[Abstract/Free Full Text]

Ridley, A. and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389-399.[Medline]

Ridley, A., Paterson, H., Johnston, C., Diekmann, D. and Hall, A. (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401-410.[Medline]

Ruhl, M., Johannsen, M., Atkinson, J., Manski, D., Sahin, E. and Schuppan, D. (1999a). Soluble collagen VI induces tyrosine phosphorylation of paxillin, and focal adhesion kinase and activates the MAP kinase erk2 in fibroblasts. Exp. Cell Res. 250, 548-557.[Medline]

Ruhl, M., Sahin, E., Johannsen, M., Somasundaram, R., Manski, D., Riecken, E. and Schuppan, D. (1999b). Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax. J. Biol. Chem. 274, 34361-34368.[Abstract/Free Full Text]

Sage, E. and Vernon, R. (1994). Regulation of angiogenesis by extracellular matrix: the growth and the glue. J. Hypertens. (Suppl.) 12, S145-S152.[Medline]

Saunders, S., Jalkanen, M., O’Farrell, S. and Bernfield, M. (1989). Molecular cloning of syndecan, an integral membrane proteoglycan. J. Cell Biol. 108, 1547-1556.[Abstract]

Scheiffele, P., Peranen, J. and Simons, K. (1995). N-glycans as apical sorting signals in epithelial cells. Nature 378, 96-98.[Medline]

Schlingemann, R., Rietveld, F., de Waal, R., Ferrone, S. and Ruiter, D. (1990). Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am. J. Pathol. 136, 1393-1405.[Abstract]

Schlingemann, R., Oosterwijk, E., Wesseling, P., Rietveld, F. and Ruiter, D. (1996). Aminopeptidase A is a constituent of activated pericytes in angiogenesis. J. Pathol. 179, 436-442.[Medline]

Schrappe, M., Klier, F., Spiro, R., Waltz, T., Reisfeld, R. and Gladson, C. (1991). Correlation of chondroitin sulfate proteoglycan expression on proliferating brain capillary endothelial cells with the malignant phenotype of astroglial cells. Cancer Res. 51, 4986-4993.[Abstract]

Schubert, D., Heinemann, S., Carlisle, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J., Culp, W. and Brandt, B. (1974). Clonal cell lines from the rat central nervous system. Nature 249, 224-227.[Medline]

Sekine, A., Fujiwara, M. and Narumiya, S. (1989). Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyl transferase. J. Biol. Chem. 264, 8602-8605.[Abstract/Free Full Text]

Sheetz, M. (1994). Cell migration by graded attachment by graded attachment to substrates and contraction. Semin. Cell Biol. 5, 149-155.[Medline]

Smith, F. O., Rauch, C., Williams, D. E., March, C. J., Arthur, D., Hilden, J., Lampkin, B. C., Buckley, J. D., Buckley, C. V., Woods, W. G. et al. (1996). The human homologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromosome band 11q23. Blood 87, 1123-1133.[Abstract/Free Full Text]

Southern, P. and Berg, P. (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, 327-341.[Medline]

Spivack-Kroitzman, T., Lemmon, M., Dikic, I., Ladbury, J., Pichasi, D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J. and Lax, I. (1994). Heparan-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell prolferation. Cell 79, 1015-1024.[Medline]

Stallcup, W., Dahlin, K. and Healy, P. (1990). Interaction of the NG2 chondroitin sulfate proteoglycan with type VI collagen. J. Cell Biol. 111, 3177-3188.[Abstract]

Tillet, E., Ruggiero, F., Nishiyama, A. and Stallcup, W. (1997). The membrane-spanning proteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein. J. Biol. Chem. 272, 10769-10776.[Abstract/Free Full Text]

Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M. et al. (1997). Calcium sensitization of smooth muscle mediated by a rho-associated protein kinase in hypertension. Nature 389, 990-994.[Medline]

Yayon, A., Klagsbrun, M., Esko, J., Leder, P. and Ornitz, D. (1991). Cellsurface heparan-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 87, 833-844.

Yeaman, C., Le Gall, A., Baldwin, A., Monlauzeur, L., Le Bivic, A. and Rodriguez-Boulan, E. (1997). The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J. Cell. Biol. 139, 929-940.[Abstract/Free Full Text]

Waterman-Storer, C. and Salmon, E. (1999). Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr. Opin. Cell Biol. 11, 61-67.[Medline]

Zhang, L., Laato, M., Muona, P., Kalimo, H. and Peltonen, J. (1994). Normal and hypertrophic scars: quantification and localization of messenger RNAs for type I, III, and VI collagens. Br. J. Dermatol. 130, 453-459.[Medline]

Zhang, Q., Magnusson, M. and Mosher, D. (1997). Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through rho-dependent actin stress fiber formation and cell contraction. Mol. Biol. Cell 8, 1415-1425.[Abstract]

Zimmermann, D. and Ruoslahti, E. (1989). Multiple domains of the large fibroblast proteoglycan versican. EMBO J. 8, 2975-2981.[Abstract]