activation of integrin receptors by extracellular ligand binding mediates the formation of cell - matrix adhesions ( miranti and brugge , 2002 ) . the clustering of activated integrins and integrin - associated proteins locally promotes the activation of downstream signal transduction paths leading to events such as cell migration ( huttenlocher and horwitz , 2011 ) , differentiation ( engler et  al . , 2006 ) , and cancer metastasis ( levental et  al . , 2009 ) . the recruitment of actin - binding proteins , such as talin and vinculin , provides structured linkages between integrins and the actin cytoskeleton ( vogel and sheetz , 2006 ; wehrle - haller , 2012 ) . while the initial clustering of integrin receptors upon binding mobile arg - gly - asp ( rgd ) moieties is independent of traction forces ( yu et  al . , 2011 , 2012a ) , contraction - mediated maturation of integrin clusters results in stable adhesion formation ( moore et  al . , more importantly , the physical characteristics of extracellular matrix ( ecm ) can initiate differential assembly of the actomyosin cytoskeletal network ( geiger et  al . , 2009 ) . for example , fibroblasts on a rigid ecm substrate ( 100  kpa ) are flat , polarized cells with actin stress fibers across the cell body . on softer but chemically identical ecm substrates ( 10  kpa ) , fibroblasts fail to polarize and exhibit fewer and less robust actin stress fibers ( prager - khoutorsky et  al . , 2011 ) . despite numerous studies , the interplay among actin assembly , force generation , and podosomes and focal adhesions are both integrin - mediated multimolecular assemblies for cell adhesion ( calle et  al . , 2006 ; many adherent cells , such as epithelial cells or stromal fibroblasts cultured in  vitro , maintain stable adhesions to the substratum via focal adhesions , adhesion structures interconnected by an actomyosin contractile network ( cai and sheetz , 2009 ; vogel and sheetz , 2009 ) . on the other hand , monocytic - lineage - derived cells such as macrophages utilize an alternative structure known as a podosome as their primary adhesion machinery ( cox et  al . , 2012 ; murphy and courtneidge , 2011 ) . podosomes characteristically contain wasp , cortactin arp2/arp3 , and actin filaments in the center ( podosome core , usually 1  m in diameter and 2  m in height ) , which is surrounded by a ring of integrin and integrin - associated proteins , such as talin , vinculin , and paxillin . alternatively , transformation of fibroblasts by constitutively active src kinase will also drive podosome or invadopodia formation with n - wasp substituting for leukocyte - restricted wasp . n - wasp / wasp and arp2/arp3 are regarded as markers of podosomes as they are not seen at focal adhesions , but otherwise the two adhesive structures share many molecular components , though the spatial organization of these components is very different ( gimona et  al . , 2008 ) . although a wealth of experimental detail is now available , the underlying mechanism of podosome assembly and whether it is force dependent comparable to the situation with focal adhesions are largely unknown . mobile rgd ligands on nanopatterned supported lipid membranes provide a simple means to study force - mediated signal transduction events at the cell membrane and have been widely used in various cell biological investigations , such as studies of the immunological synapse ( mossman et  al . , 2005 ) , ephrin - mediated cancer metastasis ( salaita et  al . , 2010 ) , and force - modulated integrin adhesion ( yu et  al . , 2011 , 2012a ) . previously , we have utilized rgd - tagged lipids in supported membranes ( rgd biotin bound to cascade blue neutravidin bound to biotin lipid ) with or without nanopartitioned lines to trigger integrin activation and to investigate force - dependent and independent functions during early cell spreading ( yu et  al . , 2011 ) . continuous films of rgd membranes generally exhibited long - range lateral mobility ( diffusion coefficient 2  m / s ) and were substrates with infinitesimal elasticity ( evans and yeung , 1994 ; evans and hochmuth , 1978 ) ( zero rigidity / shear modules , equivalent film viscosity 0.1 n - s / m ) . when the long - range mobility of the rgd membrane was locally restricted by fabricating metal lines as nanopartitions within the bilayers ( typically 100  nm line width and 5  nm thick with 14  m line pitch , passivated by bsa or casein ) ( yu and groves , 2010 ; yu et  al . , 2010 ) , mobile rgd - integrin  clusters assembled stable adhesions across the adjacent partitions with 1 and 2  m , but not with 4  m , pitch through force generation and adhesion maturation . anionic phospholipids , such as phosphatidylinositol ( 4,5)-bisphosphate ( pip2 ) and phosphatidylinositol ( 3,4,5)-triphosphate ( pip3 ) are dynamically regulated in plasma membranes ( mclaughlin et  al . , 2002 ; xu et  al . , 2003 ) local enrichment of these negatively charged lipids can initiate n - wasp / wasp - mediated actin polymerization at plasma membranes ( papayannopoulos et  al . class ia phosphoinositide 3-kinase ( pi3k ) is composed of two subunits , p85 ( regulatory ) and p110 ( catalytic ) , that phosphorylate pip2 to generate pip3 ( vanhaesebroeck et  al . , 2012 ) , raising local pip3 concentrations . on the other hand , pten dephosphorylates pip3 to pip2 , decreasing pip3 concentrations . while the biochemical interaction of p85beta and focal adhesion kinase ( fak ) has been reported previously ( chen et  al . , 1996 ) , n - wasp and arp2/arp3 actin polymerization complexes are absent at tension - loaded focal adhesions . here , we report that spatiotemporal recruitment of pi3k and local enrichment of pip3 at integrin - mediated adhesion sites on traction - force free rgd membranes play an important role in differential signal transduction leading to podosome formation . although thp1 monocytic cells treated with transforming growth factor 1 have been used as a model system to study podosomes on regular matrix - coated substrates ( monypenny et  al . , 2011 ) , fibroblasts generally do not form podosomes on matrix - coated substrates unless transformed by src ( oikawa et  al . it was therefore surprising to see that nontransformed fibroblasts ( rptp mouse embryonic fibroblasts and ref52 rat fibroblasts ) formed podosome - like adhesions when plated on freely diffusive rgd lipids ( figure  1a ) without artificially elevated src activity . about 70% of ref52 fibroblasts developed podosome - like adhesions after 45  min of initial adhesion ( figure  1b ; total of 321 cells in four experiments ) , while the same cells seeded on immobilized rgd - coated glass consistently formed classic focal adhesions ( figure  1a ) . in parallel , we also observed podosome formation in thp1 cells on rgd - supported bilayers ( figure  1d ; movie s1 ) . to understand the development of podosome - like adhesions in fibroblast cells we found that cells assembled rgd - integrin clusters during the early adhesion process , as we described previously ( yu et  al . similar to the case of focal complex and focal adhesion formation , the activated rgd - integrin receptors promptly recruited various integrin - binding proteins such as talin and paxillin and nucleated micrometer - sized clusters as nascent adhesion structures . however , rgd - integrin clusters on continuous rgd lipid bilayers were not interconnected by actin stress fibers and developed into podosomes after 45  min of initial adhesion ( figure  1c ; movie s2 ) . the formation of podosome - like adhesions on rgd membranes was characterized by actin filament assembly in the podosome core at the center of individual rgd - integrin clusters ( figure  1d ) . integrin - associated proteins , such as talin , paxillin , and vinculin , were consequently repartitioned into the ring structure ( podosome ring ) surrounding the actin core ( figures 1d and 1f ) . to better identify the molecular organizations of podosome - like adhesion on rgd membranes , we rigorously examined more than 20 different molecular components ( table s1 ) that have been identified previously ( linder and kopp , 2005 ) . we found that podosome - like adhesions shared identical components in classic podosomes in macrophages . therefore , we refer to podosome - like adhesions on rgd membranes as  podosomes  in the rest of this article . podosome cores were enriched in f - actin and other characteristic molecular markers , such as arp2/arp3 , wip , and n - wasp , that were absent in classic focal adhesions ( figures 1f and s2a ) . the metalloproteinase , mmp-14 ( mt1-mmp ) , was not enriched at podosomes of nontransformed fibroblasts or thp1 macrophages ( figure  s1b ) . however , src - transformed fibroblasts exhibited a high level of mmp-14 at invadopodia or long - lasting podosomes ( figure  s1b ) , as previously reported ( wu et  al . , 2005 ) . in addition , tks5 , a key adaptor protein in invadopodia formation , was not enriched at podosomes in nontransformed fibroblast on rgd membranes ( figure  s1c ) . intense f - actin polymerization within rgd - integrin clusters as visualized by lifeact was a signature of podosome formation at prepodosomal rgd clusters , and arp2/3 was enriched at podosome cores ( figure  1f ) . the arp2/arp3 inhibitor ck-666 ( nolen et  al . , 2009 ) effectively abolished podosome formation ( 100  m , 28 cells in two experiments ; figure  s1 ) . formins , such as mdia1 , daam1 , and fhod1 , were not enriched at the  podosomes ( figure  s2a ) . smifh2 has been shown to inhibit mdia1- and mdia2-mediated actin polymerization in  vitro and in  vivo ( rizvi et  al . , 2009 ) . however , smifh2 formin inhibitor did not suppress podosome formation in rptp mouse embryonic fibroblasts ( figure  s2b ) . thus , the actin cores of these podosomes were assembled by arp2/arp3-dependent actin polymerization and were not dependent on formin activity . myosin - ii , visualized by myosin regulatory light chain ( mlc ) was not enriched at podosomes . the disassembly of podosomes correlated with sparse recruitment of mlc around the dissociating actin core ( movie s3 ; figure  s4c ) and a return to prepodosomal rgd - integrin clusters . the transition between podosome and adhesion clusters often repeated multiple times . unlike long - lasting invadopodia ( stable for hours ) , each podosome on rgd membranes exhibited a lifespan of 220  min . previously , interference reflection microscopy ( irm ) was used to highlight close - contact zones at the cell - matrix interface ( holt et  al . , 2008 ) , and the podosome core indeed showed close contact to the supporting substrate ( figure  1e ) . the tight contact revealed by destructive interference between two closely apposed interfaces indicated that the podosomes were protrusive ( evans et  al . , 2003 ; monypenny et  al . , 2011 ) . intriguingly , the intensity of rgd fluorescence at the podosome core was diminished and immediately recovered after podosome disassembly ( figure  s3b ) , which indicated that either the cell protrusive force was pushing out the rgd ligands and/or was creating a void in the bilayer . when bilayer continuity was tested with fluorescent lipid probes , such as texas red - labeled 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine ( texas red dhpe ) , doped into the supported bilayer , the fluorophores remained evenly distributed at the podosome core ( figure  s3a ) , suggesting that the podosomes were indeed protrusive . along with arp2/arp3 , cortactin , and wip accumulation ( figure  s2a ) , rgd depletion from the core was another unique phenomenon observed during the transformation from prepodosomal rgd clusters to podosomes . in more than 20 independent experiments with both macrophages and nontransformed fibroblasts , we rigorously examined and confirmed that f - actin polymerization and rgd depletion were always concurrent . while prepodosomal rgd clusters can be in an arbitrary ring shape , we always verified podosome formation by classic podosome markers and rgd depletion inside the rgd ring during live cell imaging ( table s1 ; figure  3b ) . thus , the podosomes in fibroblasts on rgd membranes exhibit the normal protrusive behavior , morphology , and dynamics of podosomes in macrophages . to test whether or not force on adhesions would affect podosome formation , we spread cells on nanopartitioned rgd bilayers where it was previously shown that cells would generate force on adhesions and stabilize them ( yu et  al . , the rgd bilayers were partitioned by nanofabricated metal lines ( typically 100 nm line width and 5 nm in height ) that provided passive resistance to adhesion movement . previously , we demonstrated that the line pitch of partitioning barriers on an rgd membrane was inversely related to cell adhesion area . a smaller line pitch between barriers provided a higher density of barriers to rgd - integrin cluster movement and cells spread over larger areas . cells formed focal adhesions with stress fibers , but they did not form podosomes when plated on nanopartitioned rgd  membranes with a 1  m line pitch ( 10% surface density ) . the immobilized rgd - integrin clusters were linear and were linked by actin fibers ( hollow arrows in figure  2a ) after 60  min of initial adhesion . furthermore , when cells were plated on nanopartitioned rgd membranes with a 4  m line pitch that did not support force generation , podosome formation was restored . at the beginning , cells nucleated rgd clusters adjacent to nanopatterned lines with a 4  m pitch , but there were no actin stress fibers between rgd clusters and cells had a smaller spread area . after 60  min of adhesion , these rgd clusters were also converted to podosomes ( white arrowheads in figure  2a ) . interestingly , when rgd membranes were partitioned by dot arrays ( 300   300  nm metal areas with 1  m pitch , also 9% of surface density ) that provided no spatial confinement of rgd ligands , cells failed to develop force - stabilized rgd - integrin clusters and formed podosomes ( figure  2a ) . with increasing distance between the membrane partitions , fewer force - stabilized adhesion sites were nucleated , and podosome formation consequently increased ( figure  2c ; total of 47 cells in three experiments ) . more surprisingly , when a single cell adhered to both a continuous and a partitioned rgd membrane , podosomes formed only on the continuous region and did not form at the partitioning lines ( figure  2b ; movie s4 ) . thus , we suggest that force generation by contraction to the lines produced a local signal that suppressed podosome formation within spatially restricted regions . to determine what factors might be involved in stimulating podosome formation in the absence of force , we looked at src kinase activity and pip3 formation . after inhibition of src by pp2 ( 1020  m , 2  hr ) , podosomes still formed , but at only 40% of the frequency of control cells ( figure  s6a ; total of 104 cells in three experiments ) . in the case of pip3 formation , we found the localized recruitment of class 1a pi3k regulatory subunit p85beta at prepodosomal rgd clusters preceded actin assembly and appeared to trigger podosome formation ( figures 3a and 3b , arrow ) . during the initial phase of podosome formation , eyfp - p85beta was initially recruited to a subset of preexisting integrin - rgd clusters ( figure  s4a ) and then it expanded to the podosome rings ( figure  3b ; movie s5 ) . in parallel , we monitored the time - dependence of pip3 production by measuring the level of akt - ph binding and we measured a marked increase in akt - ph binding during the transition from rgd clusters to podosomes ( figure  3c ) . in addition , n - wasp was recruited at podosome cores ( figure  3d ) . to determine if local enrichment of pip3 triggered podosome formation , we inhibited pi3k activity by wortmannin ( 100  nm ) . fibroblasts can still develop initial rgd clusters after pi3k inhibition , but podosome formation on rgd membranes was blocked ( figures 4c and s6c ; total of 108 cells in four experiments ) . while p85beta was also found at regular focal adhesions ( figure  s5a ) , there were no significant changes in local pip3 levels at the adhesions ( figure  s5b ) , and n - wasp was not recruited at focal adhesions ( figure  s5c ) . thus , we suggest the recruitment of class ia pi3k caused the rise in pip3 levels that led to f - actin assembly in the transformation of prepodosomal rgd - integrin clusters to podosomes on traction - force - free rgd membranes . in terms of the degradation of pip3 and podosome disassembly , it was often found above the focal plane of rgd - integrin clusters and decreased as f - actin disassembled ( figure  s4b ) . in addition , the level of pip3 decreased with the disassembly of the f - actin core . this suggests that a sustained level of pip3 is necessary for maintenance of the f - actin core . as p85beta is known to bind to substrates with phosphotyrosines via sh2 domains ( songyang et  al . , 1993 ) classical pi3k activation often involved autophosphorylation of receptor tyrosine kinase ( rtk ) , as well as focal adhesion kinase ( fak ) ( chen et  al . , 1996 ) . after testing a number of rtk inhibitors that did not block podosome formation , we tested the dual fak and pyk2 kinase inhibitor pf-562271 ( roberts et  al . , 2008 ) and found that it efficiently suppressed podosome formation on rgd membranes . we found that fak was recruited to rgd - integrin clusters ( figure  4a ) and y397 of fak was autophosphorylated and colocalized with eyfp - p85beta at podosomes ( figure  4b ) . when fak / pyk2 autophosphorylation was inhibited by pf-562271 ( 10  m , 4  hr pretreated ) , fibroblasts can still form initial rgd clusters , but p85beta recruitment was suppressed . only 25% of fibroblasts were able to form podosomes on rgd membranes ( figures 4c and s6d ; total of 139 cells in three experiments ) . thus , we suggest autophosphorylation of fak ( y397 ) and pyk2 ( y407 ) recruited p85beta that triggered local enrichment of pip3 at prepodosomal rgd clusters . since artificially upregulated rhoa - gtp levels and cellular contractility abolished podosome formation ( schramp et  al . , 2008 ; van helden et  al . , 2008 ) , we decided to determine if reduced rhoa - gtp was also correlated with podosome formation . inhibiting rho - associated protein kinase ( rock ) activity ( y-27632 , 10  m ) or downregulating myosin - ii - mediated contractility ( blebbistatin , 50  m ) did not affect podosome formation on rgd membranes ( figure  s6b ; total of 60 and 59 cells examined in four experiments , respectively ) . in contrast , artificially upregulating cellular contractility by the rhoa agonist lysophosphatidic acid ( lpa , 40  m ) or expressing a constitutively active rhoa - q63l mutant effectively inhibited podosome formation ( figure  s6b ; total of 62 and 87 cells examined in four experiments , respectively ) . furthermore , we utilized a fluorescence resonance energy transfer ( fret)-based rhoa biosensor ( pertz et  al . , 2006 ) to measure rhoa activity when cells adhered to different substrates ( figure  5b ) . ref52 cells plated on the mobile rgd membrane had a significantly lower fret efficiency ( 0.45  0.01 sem , n  = 30 ; figure  5c ) than on rgd - coated glass ( 0.73  0.02 sem , n  = 25 ; figure  5c ; p value  < 0.0001 , two - sample t test , two - tailed ) . thus , low levels of rhoa - gtp seem to be important for podosome formation . to further investigate rhoa regulation , we tested two rhoa gtpase - activating proteins ( gaps ) , dlc1 and arap3 , which localized to podosomes . while dlc1 was recruited at both focal adhesion and podosome rings through tensin ( schramp et  al . , 2008 ) , arap3 has been shown to bind to pip3 at plasma membranes ( krugmann et  al . , 2002 , 2004 ) . indeed , we found that arap3 was recruited at pip3-enriched podosome cores after  f - actin core formation ( figure  5a ; movie s6 ) . we tested whether dlc1 or arap3 played important roles in podosome formation by transiently overexpressing catalytic - dead rhoa gap mutants dlc1-r677e and arap3-r982a , respectively . podosome formation was suppressed to 25% in the case of arap3-r982a ( figure  5d ) , but not significantly disrupted in the case of dlc1-r677e ( total of 149 and 114 cells in three experiments , respectively ) . when pi3k was inhibited , the fret efficiency of the rhoa biosensor increased significantly ( 0.85  0.01 sem , n  = 29 ) , even when cells adhered to the mobile rgd membrane ( figure  5c ) , and podosome formation was suppressed . these observations indicate that the rhoa - gtp level  is inversely correlated with podosome formation and that recruitment of arap3 and possibly other rhoa - gaps by local enrichment of pip3 provided a positive feedback to downregulate cellular rhoa level in podosome - forming cells . in these studies , we have demonstrated for that plating cells onto rgd ligands linked to fluid lipid bilayers caused the formation of integrin - based podosome - like adhesions . surprisingly , such a response was characteristic not only of cells that produce podosomes under normal culture conditions , such as the macrophage line used here , but also of fibroblasts , which do not produce podosomes when plated onto although large traction forces mediated by rhoa are important for focal adhesion maturation in fibroblasts , there is no evidence that they are major determinants in podosome formation . notably , macrophages do not develop large traction forces on matrix - coated substrates ( frol et  al . , 2009 ) or mature focal adhesions , the implications are that the components needed to form podosomes are present in fibroblasts and immune cells , but the combination of cell contractility and matrix mechanics plays the critical role in determining which type of adhesion is formed . the podosome - like adhesions that form in the absence of force in nontransformed fibroblasts are indistinguishable from podosomes in macrophages in terms of morphology , components ( table s1 ) , and protrusive dynamics ( figure  1e ) . however , their physiological functions , such as chemotaxis and endocytosis / exocytosis , need to be further examined . spatial depletion of mobile rgd ligands and destructive interference at podosomes by irm indicated that there was active protrusion of the podosome core ( figures 1e and s3b ) . while ligands on supported membranes were freely diffusive , the observed spatial exclusion of rgd - neutravidin ( 5.4  5.8  nm footprint in the x - y dimension ; hendrickson et  al . , 1989 ) at the podosome core could not be explained by a simple repartitioning effect from ligation with integrin receptor ( 8  12  nm footprint in the x - y dimension ; nermut et  al . , 1988 ) . nevertheless , supported lipid membrane remains evenly distributed at the podosome core ( figure  s3a ) . the vertical force required to physically penetrate a lipid membrane via biomimetic stealth probes ( 200  nm in diameter ) we conclude that the protrusive force at podosomes was less than needed to penetrate the membrane but sufficient to block diffusion of rgd - neutravidin into the contact region . invadopodia or long - lived stable podosomes ( more than 30  min ) have been widely investigated in src - transformed cells ( induction of constitutively activated src kinase ) and invasive cancer cell lines ( huveneers et  al . . however , podosome - like adhesions on rgd membranes and invadopodia in src - transformed cells are different in both dynamics and molecular components ( table s1 ) . constitutively activated src causes hyperactivation of various downstream targets , such as arhgef5 rhoa - gef ( kuroiwa et  al . , 2011 ) , phosphorylation of tks5/grb2 complexes ( oikawa et  al . , 2008 ) , and mmp-14 secretion ( poincloux et  al . , 2009 ; notably , tks5 ( figure  s1c ) is not enriched at podosomes in nontransformed fibroblast on rgd membranes . in addition , we have examined the potential recruitment of mmp-14 and found that it was present at invadopodia in invasive cancer cells and src - transformed fibroblasts . however , in nontransformed fibroblasts , most of the mmp-14 remained in endocytic vesicles and there was only a weak recruitment of mmp-14 around the podosomes ( figure  s1b ) . this is all consistent with the hypothesis that podosomes formed in the absence of force are aided by but do not require src activity . likewise , diaphanous - related formins are required for invadopodia formation in invasive mda - mb-231 breast adenocarcinoma cells ( lizrraga et  al . , 2009 ) . however , when we treated nontransformed fibroblasts and thp1-differentated macrophages with the formin inhibitor smifh2 , we still observed podosome formation at a similar density to control cells . we suggest that podosomes on rgd membranes indeed differ from long - lived invadopodia in src - transformed cells or invasive cancer cells in their lifespan , formin involvement , tks5 recruitment , and mmp-14 secretion . our data suggest that conventional traction force development and myosin - ii activities are dispensable in podosome formation . mobile rgd membranes with nanopartitioning lines provide a unique platform to examine the force - regulated adhesion structure transformation ( figure  2a ) . as cells adhere to rgd membranes , initial integrin activation results in rgd clustering without traction force . with dense line - partitioned rgd membranes , such as with 1 or 2  m line pitch , forces can be generated on rgd - liganded integrins to form traction - force loaded adhesions ( figure  2a ) after activation of initial spreading . because the local contraction units are unable to span the 4  m spacing ( ghassemi et  al . , 2012 ; yu et  al . , 2011 ) , the podosomes form as well on the larger line spacing as on continuous bilayers . in addition , myosin - ii is not recruited during podosome formation on rgd membrane ( figure  s4c ) . thus , our data indicate that podosome formation requires minimum traction force development and that the local force generation between rgd - integrin adhesion clusters at dense line - partitioned rgd membranes inhibits podosome formation . when the same cell covers both the 2  m pitch lines and a continuous membrane ( figure  2b ) , there is a remarkably local formation of focal adhesions at the lines while podosomes form over continuous membrane regions , indicating that the effect of contractility is local and may involve spatial contact signals . although we do not fully know how local contractions are translated into inhibition of podosome formation , we find that peak production of pip3 is the key upstream event to trigger the transformation from prepodosomal integrin - rgd clusters to podosomes ( figure  6 ) . class ia pi3k regulatory subunit p85beta is first recruited at activated integrins through binding to autophosphorylated fak and possibly pyk2 , and local production of pip3 is observed by an increased level of akt - ph - gfp . . indeed , autophosphorylation site y397 of fak can bind p85beta ( chen et  al . , 1996 ) , and pyk2 has the same sequence ( y - a - e - i ) at its tyrosine autophosphorylation site , y402 . in the case of fibroblasts , the activation of pyk2 appears to be best correlated with podosome formation on rgd lipids , since fak cells and cells treated with a fak - specific inhibitor form podosomes normally ( c .- h.y . and m.p.s . , unpublished data ) . pyk2 also plays an important role in podosome formation in osteoclasts ( gil - henn et  al . , 2007 ) , but we can not rule out another kinase because there could be off - target inhibition of other kinases by pf-562271 . in addition , fak can phosphorylate n - wasp and promote actin polymerization , and inhibition of fak kinase activity suppresses n - wasp activity ( tang et  al . , however , n - wasp can still be phosphorylated by other kinases , such as src family kinases ( dovas and cox , 2010 ) or abl kinase ( burton et  al . , 2005 ) . while n - wasp could be activated by other kinases , n - wasp may fail to be recruited at rgd clusters without local enrichment of pip3 . we suggest that inhibition of fak and pyk2 autophosphorylation provides a mechanism to abolish local production of pip3 by perturbing p85/pi3k association . as expected , inhibition of pi3k also suppresses podosome formation ( figure  4c ) . another protein that binds to pip3 is pten , and it dephosphorylates pip3 , thereby causing the loss of actin polymerizing proteins . it has previously been shown that pten is present in and regulates podosome / invadopodia formation ( hoshino et  al . , 2012 ; poon et  al . , 2010 ) . pten associates with podosomes after the actin core is formed and contributes to the disassembly of the podosome f - actin core . thus , it seems that the cycle of podosome formation and disassembly is primarily dependent upon the local levels of pip3 on plasma membranes . this can explain the regional differences in podosome formation we see in single cells ( figure  2b ; movie s4 ) through slower two - dimensional diffusion of pip3 lipids rather than fast diffusive cytosolic signals . a factor that could contribute indirectly to the formation of podosomes is rhoa activity . using the fret - based rhoa biosensor , lower rhoa - gtp levels are observed when cells develop podosomes , and pharmaceutically activating rhoa - mediated contractility using lpa is seen to abolish podosome formation on rgd membranes , in agreement with previous reports ( schramp et  al . , 2008 ; van helden et  al . , more than 70 rho gaps have been identified in eukaryotes ( tcherkezian and lamarche - vane , 2007 ) , and it remains unclear how rhoa activities are differentially regulated during adhesion formation . while dlc1 is linked to downregulation of rhoa in src - transformed cells ( schramp et  al . , 2008 ) , we find that the pip3-binding protein arap3 is another rhoa - regulating factor recruited at podosome cores . arap3 contains both rhoa gap and arf6 gap domains , and the rhoa gap function of arap3 is activated by rap - gtp ( krugmann et  al . arap3 s arf6 gap function in  vivo , however , is still under investigation ( gambardella et  al . , 2011 ) . inhibition of pi3k upregulates rhoa - gtp and cellular contractility ( krugmann et  al . , 2004 ; our rhoa biosensor measurements also agree with previous findings ( figure  5c ) . using overexpressed catalytic - dead rhoa gap mutants , we find arap3-r982a moderately suppresses podosome formation , while dlc1-r677e has no significant effect . thus , our results indicate that manipulations of traction force development at integrin - matrix clusters can serve  as a mechanical signal to modulate adhesion phenotype switching and rhoa activities . in conclusion , we suggest that the development of podosomes as adhesion structures implicates the absence of traction forces between integrin receptors and matrix ligands . lack of traction forces at activated rgd - integrin clusters results in  spatial - temporal recruitment of p85beta and local enrichment of pip3 , which is not observed in force - loaded focal adhesions . this pip3-dependent pathway of podosome formation does not  require the induction of constitutively activated src kinase and is further aided by the inactivation of rhoa by pip3-mediated recruitment of arap3 . we suggest that local contractions may directly inhibit podosome formation while facilitating focal adhesion formation through a block of the pip3-dependent pathway . the transformation between prepodosomal rgd - integrin  clusters and podosomes is a remarkable example of mechanosensing through cell - adhesion processes . the reorganization of adhesion structures triggered by changing microenvironments has become an emerging theme of adaptive regulation in cellular signaling . force and matrix ligand and integrin composition are all critical factors regulating adhesion phenotype and turnover . nontransformed rptp mouse embryonic fibroblasts ( su et  al . , 1999 ) , rat embryonic fibroblast ( ref52 ) , and thp-1 ( human monocytic leukemia cells ) were used in this study . detailed information regarding cell culture , transfection protocol , the plasmids of fluorescent fusion proteins , and microscopy methods can be found in the supplemental experimental procedures . 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) and 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-(cap biotinyl ) ( 16:0 biotinyl - cap - pe ) were purchased from avanti polar lipids . the lipids ( 0.2 mol% of biotinyl - cap - pe and 99.8 mol% of dopc ) were mixed with an equal volume of 1 pbs and then pipetted onto cleaned glass substrates for the self - assembly processes . a total of 0.1  g / ml of cascade blue neutravidin ( life technologies ) or dylight 680 neutravidin ( thermo fisher scientific ) was added onto supported lipid membranes , followed by 1  g / ml of biotinylated rgd , cyclo ( arg - gly - asp - d - phe - lys[biotin - peg - peg ] ; peptides international ) . detailed information regarding lipid preparation and membrane functionalization can be found in the supplemental experimental procedures . nanoimprint lithography was utilized to fabricate the physical barriers on glass substrates , and detailed preparation methods were previously described ( yu et  al . , 2011 ) . in brief , a silicon - based imprint mold was fabricated by electron - beam lithography and anisotropic etching processes . first , coverglasses ( warner instruments ) were cleaned by piranha solution ( sulfuric acid and hydrogen peroxide , mixed in 3:1 ratio ) for 15  min , rinsed with deionized water , and then spin - coated with uv - curable imprint polymers . patterns were then transferred from the mold to the glass by high - pressure stamping the imprint mold onto the polymer - coated coverglass and curing the polymer by uv exposure . after demolding , oxygen plasma etching was used to extend imprinted trenches vertically to the surface of the coverglass . a thin chromium metal layer was deposited onto the exposed glass surface by thermal evaporation . typically , metal lines were 100  nm in width and 5  nm in height with a gap distance ranging from 1 to 4  m .
summarymatrix - activated integrins can form different adhesion structures . 
 we report that nontransformed fibroblasts develop podosome - like adhesions when spread on fluid arg - gly - asp peptide ( rgd)-lipid surfaces , whereas they habitually form focal adhesions on rigid rgd glass surfaces . 
 similar to classic macrophage podosomes , the podosome - like adhesions are protrusive and characterized by doughnut - shaped rgd rings that surround characteristic core components including f - actin , n - wasp , and arp2/arp3 . 
 furthermore , there are 18 podosome markers in these adhesions , though they lack matrix metalloproteinases that characterize invadopodia and podosomes of src - transformed cells . 
 when nontransformed cells develop force on integrin - rgd clusters by pulling rgd lipids to prefabricated rigid barriers ( metal lines spaced by 12  m ) , these podosomes fail to form and instead form focal adhesions . 
 the formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase ( pi3k ) and the production of phosphatidylinositol-(3,4,5)-triphosphate ( pip3 ) in a fak / pyk2-dependent manner . 
 enrichment of pip3 precedes n - wasp activation and the recruitment of rhoa - gap arap3 . 
 we propose that adhesion structures can be modulated by traction force development and that production of pip3 stimulates podosome formation and subsequent rhoa downregulation in the absence of traction force .