This study addresses for the first time, to our knowledge, the function of the cortactin family member HS1 in DCs. In general, hematopoietic lineage cells express HS1, whereas nonhematopoietic cells express cortactin; however, megakaryocytes, platelets, and osteoclasts express both proteins (65
). Thus, an important starting question was whether these cells express HS1, cortactin, or both. Using carefully controlled non-cross-reactive Abs, we find that HS1 is the only cortactin family member present in murine BMDCs. Because cortactin expression has been reported in murine splenic DCs (10
), this raises the possibility that these related, but distinct, cortactin family members are differentially expressed in DC subsets.
Our analysis of BMDCs from HS1−/−
mice revealed three related defects: disorganization of the podosome array, altered lamellipodial dynamics, and diminished directional migration. WASp, which interacts indirectly with HS1, also affects these processes, but our analysis shows that the roles of these two proteins are distinct. It is well established that WASp and its obligate binding partner, WIP, are essential for formation of the actin-rich cores that nucleate podosome biogenesis (10
). Depending on the experimental system, WASp-deficient DCs and macrophages either lack podosomes altogether or show severe reductions in podosome numbers, and our data are consistent with this. In contrast, we find that HS1-deficient DCs and macrophages can form podosomes containing many, if not all, of the characteristic components. However, HS1-deficient cells show disordered podosome array packing and mislocalization of the arrays with respect to the leading edge of the cell. Similar effects were noted in a recent study in which WIP−/−
DCs were reconstituted with a WIP mutant lacking the HS1/cortactin binding site (10
). Thus, we conclude that HS1 is not required for podosome formation, but rather for organization of the podosome array.
The mechanisms through which HS1 controls podosome array organization are unclear. Because HS1 stabilizes branched-actin filaments generated by WASp and other Arp2/3 complex activators, it seems likely that it stabilizes actin filaments within podosomes. Although our FRAP data show that loss of HS1 does not affect actin exchange in mature podosomes, it does delay podosome reformation. Thus, HS1 may stabilize newly formed actin cores, such that the diminished numbers and loose packing of podosomes in HS1-deficient cells could result from stochastic disassembly of some newly formed podosome cores within the array. This interpretation is consistent with the modest decrease in average podosome lifetime in HS1−/−
DCs; this may represent increased instability of a small number of podosomes. Another appealing possibility is that HS1 aids in stabilizing the long actin filaments that form connections between adjacent podosomes (72
). Interestingly, these interconnecting filaments are frequently decorated with clathrin coated endocytic pits (72
), and the HS1 homolog cortactin associates with coated vesicle components (27
). Finally, it should be noted that in addition to directly regulating actin dynamics, HS1 functions as an adaptor molecule and can recruit other signaling molecules to sites of actin polymerization (36
). We show in this study that HS1 is not needed for recruitment of WASp to podosomes, but HS1 could recruit Vav1 or phospholipase Cγ
, proteins that are important regulators of podosome dynamics and directional persistence in DCs (22
In addition to podosomes, HS1 is enriched at lamellipodial edges in DCs. HS1 was sometimes enriched in lamellipodial protrusions in the absence of WASp (see , second row
), but we were unable to quantify the frequency of this localization pattern. The mechanism through which HS1 is localized to lamellipodial protrustions is not known. In addition to binding to the WIP/WASp heterodimer, the SH3 domain of HS1 can interact with other proteins, including Src kinases and Dynamin 2 (Ref. 25
and D.A. Klos Dehring, unpublished observations). In other systems, interaction of phosphotyrosines with Src kinases and other SH2 domain-containing molecules has been shown to mediate plasma membrane targeting (36
). Finally, actin binding could be involved. HS1−/−
DCs show increased protrusion and retraction distance and velocity, indicating that HS1 functions to stabilize lamellipodial dynamics in DCs. This finding is consistent with our previous work showing that HS1 stabilizes lamellipodial protrusions in T cells (36
) and with studies showing that cortactin stabilizes lamellipodial protrusion in nonhematopoietic cells (26
). Function of HS1 in podosomes and at the lamellipodial edge need not be mutually exclusive processes. On the contrary, HS1 is likely to function at both sites, with outcomes that are functionally intertwined via a feedback process. Because forward movement of the DC lamellipodium is closely linked to the cycle of podosome formation and dissolution (12
), erratic leading edge dynamics in HS1-deficient DCs could result in disorganization of the podosome array. Indeed, this seems the likeliest mechanism to create the observed mislocalization of the array with respect to the leading edge of the cell. Conversely, it is thought that the podosome array stabilizes the dominant leading edge of the cell, such that a disordered or misplaced podosome array may lead to lamellipodial instability, and diminished directional persistence during migration.
The importance of HS1 function in podosomes and at the leading edge of the cell is demonstrated by the diminished ability of HS1−/−
DCs to undergo directional chemotaxis. In this study, too, the phenotypes of HS1−/−
DCs are related but distinct. As reported previously (13
), we found that WASp expression is essential for DC migration. WASp−/y
DCs migrating in a chemokine gradient showed a large decrease in velocity, and those cells that did migrate exhibited diminished directional persistence. In contrast, HS1-deficient DCs actually migrated faster than WT cells, but directional persistence was significantly reduced. The defects in directional persistence in HS1−/−
cells may reflect the increased lamellipodial instability in these cells. Alternatively, the defects in directional persistence may reflect the role of podosomes in stabilizing a dominant leading edge (15
). In this scenario, HS1 would function to fine-tune the packing and localization of podosomes formed by WASp to aid the stabilization of the leading edge, promoting efficient directional cell migration. These two possibilities are not mutually exclusive and, in fact, are likely to represent intertwined aspects of HS1 function.
Our data point to a hierarchical relationship between WASp and HS1 in controlling DC actin dynamics. HS1 interaction with WASp is indirect and involves binding of the HS1 SH3 domain to WIP. Using add-back experiments, we found that WASp can localize to podosomes independently of HS1, but HS1 is not recruited efficiently to podosomes in the absence of WASp or if its WIP-binding SH3 domain is mutated. This indicates that HS1 is recruited to podosomes through SH3-domain-dependent interactions with the WIP/WASp complex. These results are consistent with a recent study showing that cortactin fails to localize to podosomes in WIP−/−
DCs and that the proline rich region of WIP responsible for binding to cortactin’s SH3 domain is important for podosome architechture (10
). Indeed, WIP seems to play a key role in podosome assembly, because WASp also fails to localize to podosomes in WIP−/−
). Given our data showing that HS1 binds directly to WIP rather than to WASp, it would be interesting to ask whether WASP−/Y
DCs fail to correctly localize WIP.
Our functional studies also support the view that the WASp/WIP heterodimer serves a central role in podosome biogenesis, whereas HS1 fine-tunes podosome array organization. In cells lacking both WASp and HS1, the defects in both cell morphology and chemotaxis were indistinguishable from cells deficient for WASp alone. One finding, however, suggests that HS1 function may be at least partially independent of WASp. In analyzing the 30–40% of DCs that generated podosomes in the absence of WASp, we observed loose packing of podosome arrays if HS1 was also absent, but tight packing of podosome arrays if HS1 was expressed (either WASp single knockout cells or DKO cells transduced with HS1). This result is particularly surprising given our finding that HS1 fails to localize to podosomes efficiently in the absence of WASp. This apparent discrepancy may reflect WASp-independent HS1 function at the leading edge. Alternatively, it may reflect the ability of HS1 to interact weakly with podosomes by binding to F-actin. Evidence that such binding occurs is shown by the higher frequency of podosome localization of the HS1 SH3 domain mutant in WASp-sufficient cells as compared with WT HS1 in WASp−/y DCs (see ).
The mild podosome phenotype we observe in HS1−/−
DCs is somewhat surprising given that cortactin is essential for formation of invadopodia in metastatic tumor cells (77
). Although we cannot exclude the possibility that HS1−/−
mice undergo compensatory developmental changes that blunt the DC phenotype, we deem this unlikely because we found no upregulation of WASp or cortactin, and because similar defects were observed with HS1 shRNA in a macrophage cell line that also lacks cortactin. A more likely possibility is that HS1 and cortactin are functionally distinct. Because HS1 is expressed in hematopoietic cells that generate podosomes, whereas cortactin is typically expressed in nonhematopoietic cells that generate invadopodia, it will be interesting to explore the differential role of these proteins in generating podosomes versus invadopodia. Interestingly, cortactin is required for formation of podosomes in osteoclasts, hematopoietic lineage cells that express both HS1 and cortactin (66
). Although this may represent an exception to the podosome/invadopodium distinction, osteoclast podosomes resemble invadopodia in that they are key sites of matrix degradation. This points to another interesting difference between HS1 and cortactin. Although cortactin is required for matrix metalloproteinase release at invadopodia in nonhematopoietic cells (79
) and at podosomes in splenic DCs (10
), we found no requirement for HS1 in metalloproteinase release in BMDCs. It will be interesting to explore these distinctions by asking whether it is possible to rescue HS1−/−
cells with cortactin and vice versa.
The relationship between HS1 and WASp defined in this study is also somewhat different from the relationship between cortactin and N-WASp in other cell types. Although we find that WASp recruits HS1 to podosomes, cortactin has been shown to recruit and promote N-WASp activity at sites of actin polymerization (80
). Moreover, we find that the WIP-binding SH3 domain of HS1 is needed for recruitment to podosomes in DCs, but this domain of cortactin has been shown to be dispensible for podosome targeting in osteoclasts (66
). Phosphorylation of cortactin has been shown to play an important role in its ability to regulate N-WASP (38
). Phosphorylation of HS1 is important for its actin-regulatory function in T cells and NK cells (36
), but its role in DCs remains to be explored.
An important open question in this field is the extent to which podosomes are important for DC function in vivo. WASP−/y
DCs have significant migration defects in vivo, but it is unclear to what extent this reflects a requirement for podosome formation. It has long been assumed that podosomes are sites for integrin-dependent adhesion to the extracellular matrix, but the importance of integrins in regulating DC migration is complex and highly dependent on environmental cues (4
). In this context, an appealing possibility is that these structures are important as mechanosensors, to allow DCs to adapt to movement along variable surfaces (9
). Finally, because podosomes are most prominent in immature DCs, these structures may a play an important role in maintaining cell anchorage and/or dynamics of dendritic processes in peripheral tissues. By identifying and characterizing individual proteins that control distinct aspects of podosome function, we will have a better understanding of whether and how these structures contribute to the regulation of DC movements during an in vivo immune response.