This study shows that Cx43 and Cx32 can mediate cell-cell interactions and therefore have adhesive properties. We found that cells expressing high levels of Cx43 aggregated better among themselves and with mock-transfected, low Cx43-expressing cells. In contrast, Cx32+ cells did not adhere to low Cx43-expressing cells and excluded these cells from their aggregates. Thus, adherence between Cx43+ cells probably involves the same docking rules as those required to form a gap-junction channel, as Cx43 and Cx32 do not naturally form gap junctions. Together with the observation that Cx43 enabled adhesion between cells from different tissues and even species, which otherwise would segregate from each other, our data point to the connexins themselves as the proteins directly responsible for aggregation.
Several lines of evidence have suggested that Cx expression increases the adhesive properties of transfected cells and promotes cell migration (Lin et al., 2002
; Elias et al., 2007
). This study explored the adhesion capability of connexins in the context of pure adhesion, excluding other cell functions such as migration and channel formation. Our data provide clear evidence for a direct role of Cx43 and Cx32 in establishing stable cellular contacts.
The extracellular domains of connexons dock with connexons in neighboring cells and thereby link adjacent cells through a mechanism similar to CAMs (Johnson et al., 1974
), but gap junctions have traditionally not been considered as such. The current concept about gap junction formation is that cadherins establish initial cell-cell contacts, and gap junctions only form after cell adhesion has been established. While our study does not question this sequence of events, it suggests the novel concept that docking of connexin hemichannels both directs and stabilizes the contact from the beginning. We observed that cells expressing high levels of Cx drive adhesion with cells expressing low levels of the same Cx. This adhesion capability did not correlate with the functionality of the gap-junction channel because the same cell types do not show significant dye transfer. These observations could be explained if we assume that 1) the probability of interaction with the cell expressing low Cx levels is increased when one of the binding parties expresses high Cx level in the membrane and/or 2) the probability of channel opening does not change with the presence of more connexons in the neighboring cell.
Likely, additional steps are needed to transition to the state that requires higher amounts of connexons recruited in one single area for gap junctions to be formed. Alternatively, it is also possible that the sensitivity of our assay conditions to detect dye transfer is lower than the sensitivity of our adhesion assay to detect cell-cell interactions. In any event, the formation of stable cell-cell contacts was driven by connexin subtypes, rather than by cell-specific differences. The fact that chaotropic treatment with agents like urea is required to split existing gap junctions suggests that the assembly of a gap junction is extraordinarily strong (Manjunath et al., 1984
We must not forget that the occurrence of gap junction channels in the membrane without subsequent opening seems to be very high, at least in cultured cells. Bukauskas et al. (2000)
examined the relationship between clustering of gap-junction channels and electrical coupling, and estimated that only when there are gap junction plaques formed by more than 400 channels electrical coupling is detected. More strikingly, fewer than 2% of these channels are actually open. Thus, adhesion mediated by gap junctions independent of channel opening can be a much more relevant phenomenon than initially considered.
Cx37 can regulate monocyte adhesion in atherosclerotic lesions (Wong et al., 2006
). In this case, adhesion is modulated by the release of ATP via hemichannels. However, we did not see any differential adhesive effect in the presence of apyrase, an enzyme that rapidly degrades ATP (data not shown) supporting the idea that the adhesion effect we report is exclusively mediated by the extracellular domains of connexins. However, we cannot entirely rule out the Cx-mediated regulation of other CAMs at the level of the plasma membrane to help establish stronger adhesive interactions between cells once the initial contact has been formed.
Ca2+ dependence of connexin-mediated adhesion
We have found that Cx-mediated adhesion does not depend on extracellular Ca2+
, as removing CADs in low levels of trypsin and Ca2+
-free medium did not alter the connexins’ ability to mediate adhesion. To promote adherence, connexins must have a docking capability similar to what is required to form intercellular channels, as mutations that affect the docking residues interfere with adhesion (Lin et al., 2002
). Opening hemichannels in nonjunctional membrane stimulates gap junction formation in oocyte pairs (Beahm et al., 2004
), and the opening probability of hemichannels increases as Ca2+
concentration decreases (Gomez-Hernandez et al., 2003
; Thimm et al., 2005). Indeed, the optimal concentration of Ca2+
to detect gap junction channel conductance is low—about 0.1 mM (Dahl et al., 1992
Most of these studies have manipulated Ca2+ to detect functional gap-junction channels. However, there are no studies establishing the optimal Ca2+ conditions to start contacts between two apposing hemichannels, as the available studies have also measured channel conductance and not just physical contact. Here we provide the first evidence that the initial contact between apposing connexins does not require Ca2+, as removing Ca2+ did not alter connexin-mediated adhesion. However, we think that hemichannel docking to form a channel and to mediate adhesion are likely to be similar processes, as they occur in a setting of physiological extracellular Ca2+. Later on, it is possible that the areas where junctions form may contain a low Ca2+ microenvironment that favors the optimal conditions for opening the channel.
The requirement of Ca2+
for gap-junction channel formation in epidermal cell lines is primarily due to the Ca2+
dependence of CADs (Jongen et al., 1991
). In that study, a direct correlation between connexins and E-cadherin expression was found. However, Xu et al. (2001)
have shown that N-cadherin-deficient neural crest cells still contain numerous Cx-positive membrane contacts despite the fact that dye coupling is virtually eliminated. These results are consistent with our observation that Cx-mediated adhesion is independent of the presence of extracellular Ca2+
. Indeed, the fact that it is possible to dissociate adhesion from coupling (Oliveira et al., 2005
) suggests that additional domains may have to be engaged in order to establish a functional gap-junction channel.
What is the relevance of connexins as CAMs?
The presence of connexins has been observed in a broad array of states, from development to disease. Connexins are detected early in the embryo, and their capability to function as CAMs fits well with a role during early stages of development.
In the developing nervous system, proliferating cells in the rostral migratory stream (RMS) may provide an example of connexins’ dual function in adhesion and communication. In the RMS, neuroblasts from the subventricular zone migrate towards the olfactory bulb while they are still proliferating. The existence of neurogenic areas of the RMS that are highly coupled has been reported (Menezes et al., 2000
). These cells are apposed to cells of the astrocytic lineage and, in this interphase, there might be a dynamic interplay of adhesion-separation in which connexins might simultaneously subserve adhesion and communication functions.
A similar scenario takes place in the developing heart, where cardiac neural crest cells
migrate for tissue remodeling in the conotruncal cardiac region (Huang et al., 1998
). These cells migrate as groups of cells organized in sheets and streams. Both Cx43 expression and gap-junction communication are abundant in this migratory route. When N-cadherin is downregulated, there are still abundant Cx43-mediated contacts at the cell surface despite the fact that gap junction communication is markedly reduced. Only when the levels of Cx43 decrease significantly, as in mice lacking Cx43, the rate of migration is highly reduced, resulting in fewer cells in the outflow tract towards the heart (Xu et al., 2001
). These studies highlight the importance of Cx43 contacts during group migration, evidencing again the dissociation between adhesion and channel function in another cell population. They also evidence the separation between the roles of N-cadherin and Cx43 in neural crest cells, even though both molecules have the ability to regulate migration.
A recent study on the mechanisms of radial neuronal migration in the developing neocortex has confirmed these observations. Radial glial cells provide a scaffold for the movement of neuronal precursors to their final cortical destinations. The downregulation of Cx26 or Cx43 promoted a reduction in the number of neurons reaching their final targets (Elias et al., 2007
). In accordance with our previous data (Lin et al., 2002
), only the residues involved in cell-cell contact via connexins, but not those required for the formation of a gap-junction channel, were important for migration to occur. Interestingly, Elias et al. did not find a change in the expression pattern of other cell-cell and cell-matrix adhesion molecules like N-cadherin, zona occludens-1 or β1-integrin, supporting a role for connexins as the sole mediators of the adhesion effect.
High coupling and expression of connexins is prevalent in adult astrocytes. Although other connexins have been reported, Cx43 is the major astrocytic Cx. So, this Cx can be a critical CAM for the maintenance of the astrocytic syncitium. In addition, glioma cells expressing high levels of Cx43 can interact with host astrocytes and invade the brain parenchyma (Lin et al., 2002
), contributing to the dissemination of astrocytomas. Oliveira et al. (2005)
have confirmed that Cx43 expression enables glioma cells to migrate from the tumor core and invade the adjacent parenchyma. In this case, the migrating ability depends on the capacity of Cx43 to form functional gap-junction channels and not just cell-cell contacts. In contrast, the same study shows that chemical inhibition of gap junction coupling prevented migration but enhanced the adhesive interactions between homotypic cells. This observation points once more to the dissociation of these two cellular functions—channel formation (in this case, allowing migration) and adhesion (restricting movement). It also confirms previous data on the involvement of different domains to mediate each particular function.
Unfortunately, it has not been possible to accurately measure the number of gap-junction channels that are open in vivo because most of the studies refer of functional coupling in terms of dye-transfer capability or measuring electrical conductance without stating the actual percentage of opened gap junctions involved. It is therefore likely that, in physiological conditions, ‘adhesive’ gap junctions are more relevant than initially thought. At this point, however, it is not possible to firmly establish the biological importance of Cx-mediated adhesion.
In summary, our results provide evidence that Cx expression is sufficient to permit adhesion between glial cell lines. We have shown that, even in the absence of other CAMS, Cx expression may be sufficient to establish adhesive interactions without intervention of a functional gap-junction channel.