cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion . in most cell types ,
migration - related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant , focal adhesions .
in addition to these adhesive structures , dcs also assemble distinctive adhesions named podosomes that are characteristic of cells of the myeloid lineage including macrophages and osteoclasts [ 4 , 5 ] .
the molecular composition of podosomes is similar to that of focal complexes and focal adhesions .
however , they have a unique organization with actin filaments bundled in discrete foci forming a conical core containing distinctive elements such as gelsolin , cortactin , the actin - nucleating factor arp 2/3 complex , and the scaffolding protein wasp   surrounded by a ring of integrins and integrin - associated proteins [ 4 , 5 ] .
wasp is an essential component of podosomes , where it activates the arp2/3 complex inducing actin polymerization .
wip has also been detected in the core of podosomes in endothelial cells expressing egfp - wip , and although there is evidence indicating that wip plays a major role in regulating the activity of wasp family proteins and the dynamics of actin filaments [ 8 , 9 ] , it remains unknown how wip participates in the assembly of podosomes .
to study the role of wip in podosome formation , the distribution of f - actin and vinculin was examined in wild - type and wip dcs by confocal microscopy .
as expected , podosomes were grouped in clusters in wild - type dcs ( figures 1a and 1c ) .
those few podosomes formed in wip dcs displayed an abnormal structure having less discrete actin cores and disorganized vinculin rings around f - actin ( figure  1d ) .
large focal contacts containing vinculin comprised the most striking adhesion structures in wip dcs ( figures 1b and 1e ) , replacing podosomes as the major points of contact with the substratum
. the critical involvement of wip in dc podosome formation is also supported by the presence of endogenous wip in the core of podosomes in wild - type cells ( figure  1f ) .
our data suggest that the key role of wip in podosome formation is in the organization of f - actin and the clustering of integrins and associated proteins .
the absence of wip had a profound effect on the organization of f - actin within dcs . while the bulk of f - actin in wild - type dcs was associated with the core of podosomes ( table 1 ,
see figure  s1a in the supplemental data available online ) , in wip dcs it distributed among various anomalous structures including actin cables ( table 1 , figure  s1b ) , large fused actin aggregates , commonly with a circular shape ( table 1 , figure  s1c ) , and extensive membrane ruffles ( table 1 , figure  s1d ) .
clustering of podosomal integrins and the integrin - associated proteins paxillin and talin was also affected . in wild - type dcs , 2-integrins formed rings around the f - actin cores of podosomes ( figure  s2a ) as previously described .
in contrast , wip dcs failed to cluster 2-integrins into rings , and instead they formed small integrin - containing focal contacts scattered through the basal surface and around the periphery of the cell ( figure  s2b ) or large plaques associated with anomalous actin - rich aggregates ( figure  s2c ) .
facs analysis showed that wild - type and wip dcs expressed equivalent levels of 1- and 2-integrins ( figure  s3 ) , indicating that wip does not affect integrin expression .
paxillin and talin , which normally array around the podosome core in wild - type dcs   ( figures s2d and s2 g ) , in wip dcs localized to focal adhesions ( figures s2e and s2h ) containing 1 integrins ( data not shown ) or formed amorphous clusters colocalizing with the anomalous actin aggregates ( figures s2f and s2i ) .
the shift of adhesion composition and structure from podosomes ( short half - life in the range of 30 s to 5 min ) to large focal contacts ( long half life in the range of 30 to 60 min ) in wip dcs had a negative impact on adhesion turnover ( figure  1 g ) .
as previously described , interference reflection microscopy ( irm ) analyses showed podosomes in wild - type dcs rapidly assembled and disassembled , giving a high turnover index ( figure  1 g ) . in wip dcs , although multiple transient and unstable lamellar protrusions were developed at the cell periphery , the major adhesion sites in contact with the substratum were highly stable with a significantly lower turnover index compared to wild - type ( figure  1 g ) .
interestingly , we found a decrease in podosome turnover in wild - type dcs plated on fibronectin ( fn ) and icam-1 , indicating that integrin ligands promote podosome stability ( figure  1 g ) .
this stability of adhesion on integrin ligands correlates with an increase in the percentage of wild - type dcs with well - defined podosomes and small focal contacts ( figure  1a ) and accumulation of podosomal f - actin , integrins , and associated proteins ( data not shown ) .
in contrast , no significant differences were detected in turnover index of adhesions between wip dcs plated on different substrata ( figure  1 g ) .
the presence of integrin ligands failed to induce major changes in the composition of adhesions of wip  dcs ( figure  1b ) except for an increase in the  size of the focal adhesions . taken together , our data indicate that wip plays a key role in regulating dc adhesion structure and dynamics on biologically relevant substrata , processes crucial for the migratory capacity of cells .
stabilization of the leading edge in the direction of dc movement is provided by podosomes [ 13 , 14 ] .
migrating wild - type dcs clearly polarized , forming a patent leading edge , with the rear of the cell retracting at a similar rate , allowing net translocation of the cell body .
in contrast , wip dcs failed to develop a major leading front and instead formed multiple simultaneous and unstable lateral lamellae and ruffles ( figures 2a2e ; movies s1 and s2 ) .
cortactin is an arp2/3 activator that plays a major role in the formation of podosomes [ 15 , 16 ] , establishment of cell polarization , and also generation of cellular cortical protrusions .
it has been proposed that cortactin synergises with the wasp family of proteins to induce actin polymerization , and it is thought that the actin filament meshwork triggered by the n - wasp / cortactin pair may have a different organization from actin meshworks triggered by n - wasp or cortactin alone .
importantly , wip has been shown to interact with cortactin and induce cortactin - mediated actin polymerization .
our results show that in the absence of wip , cortactin is displaced from podosome cores where it normally colocalizes with f - actin ( figure  2f ) and is instead associated with random lamellar protrusions and ruffles over the cell periphery ( figure  2 g ) .
in addition , we found there was a higher proportion of cortactin associated with the cytoskeletal fraction ( triton insoluble ) in adherent wip dcs compared to wild - type dcs ( figure  2h ) . taken together ,
these results show that in the absence of wip , association of cortactin with the cytoskeleton is deregulated and associated with the formation of random cortical protrusions and loss of dc polarity .
this suggests that the wasp / wip complex may couple with cortactin to induce a type of meshwork of actin filaments and associated proteins that allow the clustering of 2-integrins during the assembly of podosomes and dc polarization . in the absence of wip
, cortactin can not be recruited to form a complex with wasp , discrete actin foci in podosomes fail to form , and instead , cortactin codistributes with actin filaments at the cell margin , resulting in the random protrusions and reduced motility ( data not shown ) observed in wip dcs .
we have previously shown that wasp dcs display equivalent anomalies in 2-integrin clustering   and focal contact formation to that observed in wip dcs .
also we find here that like wasp dcs , wip dcs fail to polarize and form and stabilize consistent leading edges .
recently , it has been proposed that wip contributes to the stabilization of wasp expression and the regulation of wasp - mediated actin rearrangement [ 8 , 22 ] .
we find here that wip dcs express low levels of wasp at the protein level ( figure  3a ) , as recently described for wip t - lymphocytes .
levels of wip expression were not affected by the lack of wasp in wasp dcs ( figure  3b ) . since mrna levels of wasp in wip dcs were comparable to those of wild - type dcs ( figure  3c )
the levels of expression of the wip binding partners cortactin , nck , and crkl was not affected in wip dcs ( figure  s4 ) .
it has been predicted that wip may prevent the exposure of wasp family proteins to proteases , because the 25-residue wasp binding motif from wip wraps around the wip binding domain of n - wasp .
wasp , but not n - wasp , is a substrate for the protease calpain in platelets , and we have recently found that calpain also cleaves wasp in migrating dcs .
our present results show that wip prevents wasp cleavage by calpain in dcs since inhibition of this protease in wip dcs resulted in major recovery of wasp expression ( figures 3d and 3e ) .
this suggests that wip may play a crucial role in regulating the accessibility of wasp for calpain cleavage , which takes place during dissolution of podosomes . a role for wip in the maintenance of wasp levels
is also suggested by recent findings showing that in was patients , mutations that prevent wasp binding to wip result in diminished expression of wasp [ 24 , 25 ] .
we suggest that the low levels of wasp in these was patients may be the result of extensive calpain - mediated cleavage because of failure of wasp - wip interaction .
surprisingly , the recovery of wasp levels in wip dcs by treatment with calpain inhibitors was not sufficient to induce podosome formation , and large focal contacts remained the major adhesion structures ( figure  4a ) .
in addition , forced expression of egfp - wasp in wip dcs treated with calpain inhibitors alone or in conjunction with proteasome inhibitors   failed to restore podosome formation , and egfp - wasp remained diffusely distributed in the cytoplasm or associated with anomalous f - actin aggregates that could contain vinculin forming abnormal plaques colocalizing with f - actin ( figure  4b , top and middle ) .
this contrasts with the observed recovery of podosome formation and dynamics in wasp dcs ( where wip levels are normal ) after the expression of egfp - wasp ( figure  4b , bottom ) [ 11 , 13 ] .
we excluded the possibility that wip binding is essential for the intrinsic actin polymerizing activity of wasp because specific wasp mutants ( wasp a134 t and wasp r138p ) that interrupt wip binding [ 24 , 27 ] showed an actin polymerizing activity equivalent to wild - type wasp ( figure  4d ) .
these results indicate that wip not only protects wasp from calpain cleavage but also facilitates the localization of wasp to sites of actin polymerization and the organization of integrins , vinculin , and other integrin - associated proteins in circular clusters . the mechanisms by which wip regulates wasp activity to allow podosome formation are yet to be fully elucidated .
it is possible that wasp / wip interactions may directly facilitate the recruitment of wasp to cell - surface receptors .
alternatively , wip may facilitate the bridging of wasp to these receptors by mediating the interaction with other adaptor proteins   or may regulate the function of regulatory proteins such as kinases , known to affect wasp activity [ 30 , 31 ] .
our data show that wip by itself is not sufficient to promote podosome assembly because wasp dcs , which express normal levels of wip , also lack podosomes and assemble large focal contacts and disorganized actin clumps instead .
it is tempting to speculate that the cellular responses ( of dc ) to external soluble ligands such as chemoattractants would recruit wasp / wip complexes coupled to integrin - associated proteins   to trigger the assembly of podosomes at appropriate sites in the developing leading edge of the cell .
these complexes would provide a cytoskeletal platform that regulates the accurate clustering and activation of 2-integrins , facilitating their function .
taken together , our results suggest that wasp and wip work as a functional unit in a cellular context as has been previously proposed [ 8 , 9 ] to regulate podosome formation and dynamics during the establishment of cell polarity as seen in the chemotactic response to extracellular stimuli . in summary , we have shown that in dcs wip is essential for podosome formation and cell polarity by preventing the extensive degradation of wasp by calpain and by facilitating the recruitment of wasp to discrete foci to form the core of podosomes .
the tight coupling between wasp and wip is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of dcs .
 summarythe wiskott - aldrich syndrome protein ( wasp ) is an adaptor protein that is essential for podosome formation in hematopoietic cells [ 1 ] . given that 80% of identified wiskott - aldrich syndrome patients result from mutations in the binding site for wasp - interacting - protein ( wip ) [ 2 ] , we examined the possible role of wip in the regulation of podosome architecture and cell motility in dendritic cells ( dcs ) . 
 our results show that wip is essential both for the formation of actin cores containing wasp and cortactin and for the organization of integrin and integrin - associated proteins in circular arrays , specific characteristics of podosome structure . 
 we also found that wip is essential for the maintenance of the high turnover of adhesions and polarity in dcs . 
 wip exerts these functions by regulating calpain - mediated cleavage of wasp and by facilitating the localization of wasp to sites of actin polymerization at podosomes . 
 taken together , our results indicate that wip is critical for the regulation of both the stability and localization of wasp in migrating dcs and suggest that wasp and wip operate as a functional unit to control dc motility in response to changes in the extracellular environment . 
Results and Discussion