Within the family of mammalian syndecans, syndecan-1 has been linked to the modulation of cell proliferation, migration, and the regulation of the actin cytoskeleton. Thus, syndecan-1 has completely distinct activities from syndecan-4, which cooperates in the assembly of contractile focal adhesions (reviewed by Couchman, 2003
), or syndecan-2, which is involved in the assembly of dendritic spines or the ECM (Ethell et al., 2001
; reviewed by Tkachenko et al., 2005
). Although mechanisms of signaling by syndecan-2 and -4 are known to depend on their V regions, there is no knowledge of the specific roles of syndecan-1 V region. In this study, we establish for the first time that critical determinants for transducing cytoskeletal organization in response to antibody ligation or physiological ECM ligands lie within aa 294–307 of the unique V region of syndecan-1. By four criteria (deletion mutants, point mutants, NCAD chimeras, and use of TAT-V peptide), the C2 region is not essential for this activity. We demonstrate that upon ligation and clustering either by antibody or ECM ligands, cell-surface wild-type syndecan-1 enters a detergent-resistant state and that this state depends on the V region. We have also identified a role of the V region in 3-D cell migration.
Our analysis of syndecan-1 deletion mutants focused attention on aa 297–307 as critical for cell spreading and actin bundling (). The analysis of point mutations within the V region then revealed a complex functionality of the region. Of the 12 single or double point mutations prepared, 7 resulted in partial impairment of cell spreading and 5 of these also significantly decreased the number of fascin-and-actin bundles (). More severe phenotypes were obtained when the most inhibitory mutations were combined together, resulting in similar levels of inhibition of cell spreading and fascin-and-actin bundle assembly. The function-perturbing mutations corresponded to a subset of the amino acids that are conserved across all syndecan V regions and to the unique GG motif within the syndecan-1 V region hypervariable domain (). Further experiments will be needed to establish if these residues are important for the structure of syndecan-1 cytoplasmic domain or for functional protein-protein interactions or both. K189 and K194 of syndecan-4, which align with K293 and K302 of syndecan-1, are important for the ionic interactions by which syndecan-4 cytoplasmic domains form obligate dimers (Shin et al., 2001
). Syndecan-1 and -2 cytoplasmic domains do not dimerize in solution (Oh et al., 1997
); however, it is likely that all syndecan cytoplasmic domains oligomerize in vivo by lateral interactions as a result of activation and self-association of their extracellular and transmembrane domains (reviewed by Couchman, 2003
). Ionic interactions of the conserved positively charged residues could also be important in this process.
The mutation of Y300 to glutamic acid impaired actin organization activity. Constitutive tyrosine phosphorylation of syndecan-1 occurs in lymphoma cell lines, but the residues phosphorylated and functional significance are unknown (Ott and Rapraeger, 1998
). Although the Y300E mutation clearly impaired function in our experimental system, the Y300A did not (). It is also the case that the surrounding amino acids do not conform to a consensus tyrosine kinase phosphorylation motif (established by search of PROSITE). Thus, the effect of the Y300E mutation is more likely due to the change of charge and side group than to phosphomimetic activity.
Interestingly, the activity of syndecan-1 in lamellipodial spreading and actin bundling correlated with incorporation of syndecan-1 into a detergent-insoluble fraction. This process was specific, because upon cell adhesion to fibronectin that involves a different state of cytoskeletal organization, overexpression of syndecan-1 does not result in alterations to cytoskeletal organization and syndecan-1 is detergent-extractable (). Detergent-insolubility of membrane proteins has been related to their incorporation into cholesterol-rich membrane microdomains and to signaling across the plasma membrane (Simon and Ikonen, 1997
). Indeed, we have found that cholesterol-depletion by β-methyl cyclodextrin renders cell-surface syndecan-1 constitutively detergent-extractable and results in build-up of intracellular syndecan-1 (unpublished observations). Thus, signaling by syndecan-1 to the cytoskeleton appears to depend on its incorporation into lipid rafts.
An important outcome of our studies was the distinction revealed between effects of syndecan-1 on cell-spreading, the number of fascin-and-actin bundles per cell and the length of any residual bundles. The independent approaches taken to perturb wild-type syndecan-1 function: point mutation of syndecan-1 or use of TAT-S1V peptide consistently had significant effects on cell spreading and the numbers of fascin-and-actin bundles, yet the length of any remaining bundles was consistently less significantly altered (Figures and ). Both cell spreading and the assembly of fascin-and-actin bundles depend initially on actin nucleation and polymerization. In the spread lamellipodium, actin filaments are assembled as a branched network, in general by the action of Arp2/3 complex (Pollard and Borisy, 2003
). Correlated immunofluorescence and electron microscopic analysis has uncovered that fascin and F-actin bundles in the lamellipodia of melanoma cells are initiated from preexisting actin microfilaments within the lamellipodium that become clustered at cell margins by an activity of vasodilator-stimulated phosphoprotein, (VASP), and then elongated into parallel bundles by the cross-linking activity of fascin (Svitkina et al., 2003
). The quantitatively distinct effects of syndecan-1 perturbation on cell area and bundle number versus the length of residual bundles in our study strongly imply that syndecan-1 specifically regulates F-actin organization at the level of cell spreading and bundle initiation, with bundle elongation under separate downstream regulation. Thus, any bundles that do initiate under the inhibitory conditions continue to elongate to the same length as in control cells. With regard to the initiation process, we envision that activation of syndecan-1 may recruit and/or activate components necessary for actin nucleation or filament assembly and that the recruitment of fascin to prebundle sites can be controlled separately from its F-actin cross-linking activity. Indeed, we previously placed activation of the known regulators of actin nucleation, Cdc42 and Rac, upstream of TSP-1-induced cell spreading and fascin-and-actin bundle assembly (Adams and Schwartz, 2000
). Understanding how actin is assembled and bundles initiate as a result of syndecan-1 signaling will require identification of binding partners of the syndecan-1 V region.
Our data also demonstrate that the activity of syndecan-1 V region is necessary in the physiological responses of cells to ECM ligands and the regulation of cell migration. In vivo, down-regulation of syndecan-1 on invasive carcinoma cells and up-regulation on tumor-associated fibroblasts are both associated with activation of tumor cell migration (Timar et al., 2002
). Either gene knockout or overexpression of syndecan-1 result in reduced cell migration during wound-healing (Stepp et al.,
2001; Elenius et al., 2004
). Thus, as established for many proteins that function in cell migration (reviewed by Webb et al., 2003
), a certain balance of syndecan-1 expression appears to be necessary to support optimal cell migration. We found that the endogenous syndecan-1 of C2C12 cells becomes localized in leading edge ruffles after wounding of a cell monolayer, suggestive of a role in directional cell migration. In agreement with other reports, overexpression of syndecan-1 inhibited 3-D cell migration, probably due to stabilization of cell protrusions leading to decreased adhesion dynamics. We established the role of the V region in this activity by two criteria: specific inhibition of migration involving endogenous syndecan-1 by TAT-V peptide and the much reduced activity of the S1MUT5Y300E protein. We note that the mutant did not completely restore migration to control levels, which suggests that other domains of syndecan-1, for example, the transmembrane and extracellular domain, might contribute to its inhibitory activity (Langford et al., 2005
). In summary, we have identified a mechanism for syndecan-1 signaling to specific states of cytoskeletal organization that is dependent on the molecular nature of the V region and on incorporation of syndecan-1 into a detergent-insoluble form.