ena / vasp proteins regulate cell migration by promoting actin polymerization at the plasma membrane via antagonizing actin filament capping and acting as processive actin polymerases ( barzik et  al . , 2005 ; bear et  al . , 2002 ; breitsprecher et  al . , 2008 , 2011 ; hansen and mullins , 2010 ; pasic et  al . , 2008 ) .
each family member consists of an n - terminal evh1 domain , a central proline - rich region , and a c - terminal evh2 domain ( bear and gertler , 2009 ) . the evh2 domain , which contains monomeric and f - actin binding sites , is responsible for promoting actin polymerization ( barzik et  al . , 2005 ; breitsprecher et  al .
in contrast , the evh1 domain mediates intracellular targeting of ena / vasp proteins by interacting with a sequence ( d / e)fppppx(d / e)(d / e ) , which is referred to as the  fpppp  motif because these residues are essential for  binding ( bear et  al . , 2000 ; niebuhr et  al .
ena / vasp proteins are recruited to focal adhesions by zyxin , which contains four  fpppp  motifs ( drees et  al . , 2000 ;
the ability of ena / vasp proteins to control cell migration , however , depends on their recruitment to the leading edge ( bear et  al . , 2000 , 2002 ) , by  fpppp
 motif containing mrl proteins ( mig10 , riam , and lamellipodin ; col et  al .
, 2005 ; krause et  al . , 2004 ; lafuente et  al . , 2004 ; quinn et  al . , 2006 ) .
of all the proteins interacting with the evh1 domain of ena / vasp proteins , tes , a focal adhesion protein , stands out as the only one that lacks an  fpppp  motif ( coutts et  al . , 2003 ; garvalov et  al . , 2003 ) .
tes negatively regulates the localization of mena at focal adhesions and also inhibits mena - dependent cell migration ( boda et  al . , 2007 ) .
tes interacts with mena via its c - terminal lim3 domain and is unique in being the only protein that binds a single ena / vasp family member ( boda et  al .
2003 ) . given the interaction of tes with mena , we sought to identify additional atypical evh1 binding partners that also lack 
we found that the evh1 domain interacts directly with abi , a component of the wave regulatory complex ( wrc ) , which plays an essential role in driving cell migration by activating the arp2/3 complex in response to rac signaling ( bisi et  al . , 2013 ) .
our observations demonstrate that the evh1:abi interaction enhances cell migration and the ability of rac - activated wrc to promote arp2/3-mediated actin polymerization as well as the function of wrc in  vivo in drosophila .
to identify ena / vasp binding proteins lacking  fpppp  motifs we performed pull - down assays with gst - tagged evh1 domain of mena on lysates from mv cells , which lack endogenous mena and vasp ( figure  1a ) .
mass spectrometry analysis of the resulting bands identified zyxin and four subunits of the wrc : abi1 , nap1 , pir121 , and wave2 ( figure  1a ) .
western blot analysis confirmed that the mena evh1 domain interacts with the wrc ( figure  1b ) .
furthermore , this interaction depends on its fpppp binding activity , because preincubation of the resin with the fpppp region of zyxin ( evh1-fpppp ) or the lim3 domain of tes ( evh1-lim3 ) inhibited binding ( figure  1b ) .
using bacterially expressed proteins , we found that the evh1 domains of mena , evl , and vasp are all capable of interacting directly with abi1 ( figures 1c and 1d ) .
the evh1 domain of mena can also bind abi2 and abi3 ( figure  1e ) .
human and mouse abi1 lack  fpppp  motifs , but do contain extensive proline rich regions that may contain alternative evh1 binding sites . consistent with this , pull - down assays reveal that a wave complex containing abi1(1 - 159 ) lacking its c - terminal proline rich region and sh3  domain can bind rac1 but not the vasp evh1 domain ( figure  1f ) .
evh1 binding motif was first identified in acta by screening a series of overlapping peptides using a far western approach ( niebuhr et  al . ,
, we found that the mena evh1 domain interacts with a series of peptides covering residues 352394 in the proline rich region of human abi1 ( figure  2a ) .
the evh1 domains of evl and vasp also bound the same region although the peptide binding patterns were not completely identical ( figures s1a and s1b available online ) . probing three overlapping peptide arrays containing systematic amino acid substitutions at each position we found that mutation of proline residues 366368 ( peptide 1 ) and 383385 ( peptide 3 ) as well as phenylalanine 375 ( peptide 2 ) significantly reduced binding ( figure  2b ) .
substitution of prolines 366368 and 383385 with glycine as well as phenylalanine 375 to alanine ( termed abievh1 ) abrogated all peptide interactions with the mena evh1 domain ( figure  2c ) . pull - down assays on lysates from cells expressing gfp - abievh1 confirmed that these mutations disrupt the interaction of abi1 with mena but not the rest of the wave complex ( represented by the pir121 subunit ; figure  2d ) . to investigate the impact of the loss of the interaction between abi1 and ena / vasp proteins on cell migration , we stably expressed gfp - tagged abi or abievh1 in ht1080 cells ( figure  s1c ) .
both gfp - tagged proteins are recruited to the leading edge of migrating cells ( figure  2e ) .
expression of abievh1 but not abi , however , retarded the migration of ht1080 cells into a scratch ( figures 2f and s1d ) .
conversely , in the absence of endogenous abi1 , gfp - tagged abievh1 was less effective at promoting cell migration than the wild - type protein ( figures 2f and s1d ) . given the impact of abievh1 on cell migration , we wondered whether the interaction of evh1 with abi1 modulates the activity of vasp and/or the wrc . to explore this possibility , we performed in  vitro actin polymerization assays using recombinant vasp and wrc ( figures 3 and s2 ) . in the absence of its canonical activator , rac1 , the wrc can not stimulate arp2/3-mediated actin assembly ( figure  3a , red solid curve ) .
in contrast , vasp promotes actin assembly ( figure  3a , black dotted curve ) .
further addition of the wrc produced no further changes in actin assembly ( figure  3a , red dotted curve ) , indicating that vasp does not activate the inhibited state of the wrc , or vice versa . in our assay conditions
( 100  mm kcl ) , the wave1 vca peptide only slightly increases actin assembly by the arp2/3 complex ( figure  3b , green solid curve ) . saturating concentrations of rac1 increase wrc - mediated actin assembly to this same level ( figure  3b , red solid curve ) .
addition of vasp to the rac1-activated wrc significantly increases the extent of actin polymerization ( figure  3b red dotted curve ) in a dose - dependent manner ( figure  3c ) .
this increase is not the result of simple summation of the activity of vasp and the arp2/3 complex , as when the activated wrc is substituted with the vca peptide , the increase in actin polymerization is only additive ( figure  3b , green curves ) . to examine whether direct interaction between vasp and the wrc is responsible for the enhancement in actin assembly
, we tested a variant of the wrc ( wrc - abi1[1159 ] ) , which can not bind the evh1 domain as it lacks the proline rich region of abi1 ( figure  1f ) .
wrc - abi1(1159 ) can be activated by rac1 , but failed to recapitulate the increased activity with vasp seen for the full - length wrc ( figure  3b , compare blue and red dotted curves ) .
we next sought to understand how the vasp - wrc interaction promotes enhanced actin assembly . in principle , this could arise from increased activity of either vasp or the wrc . when the arp2/3 complex is removed from assays containing both vasp and activated wrc , the total activity dropped to the level of vasp alone ( figure  3b , orange curve ) .
therefore , the increased activity depends on the arp2/3 complex , suggesting that vasp increases the activity of wrc , but not vice versa .
because the isolated evh1 domain of vasp can not increase the activity of the wrc ( figure  3d ) , we sought to discover which other regions of vasp are required for this effect .
in addition to the evh1 domain , vasp has a c - terminal evh2 domain , which includes a g - actin binding motif ( gab ) , an f - actin binding motif ( fab ) , and a coiled coil maintaining vasp as a constitutive tetramer ( bear and gertler , 2009 ) .
deletion of the coiled coil , which generates a monomeric form of vasp ( vasp [ 1335 ] ) , fails to recapitulate the increased activity seen with the full - length vasp ( figure  3d ) .
furthermore , the fab motif is also necessary , since its mutation , which completely disables vasp in actin assembly ( breitsprecher et  al .
, 2008 , 2011 ; hansen and mullins , 2010 ; pasic et  al . , 2008 ) , no longer allows vasp to enhance actin assembly by the wrc even at a concentration of 5  m ( figure  3e ) .
in contrast , mutation of the gab motif alone , which significantly decreases the activity of vasp in actin polymerization , still allows vasp to stimulate wrc activity ( but only at high concentrations of 15  m range ; figure  3f ) .
taken together , our data suggest that disrupting the actin polymerization activity of vasp impairs its ability to enhance wrc activity . to facilitate further analysis of the physiological role of the interaction between ena / vasp proteins and the wrc
, we switched to drosophila because it only has a single isoform for each protein .
drosophila abi ( dabi ) and the evh1 domain of ena have 38% and 72% sequence identity to their respective human counterparts ( figure  4a ) .
drosophila abi also lacks the evh1 binding motif we identified in human abi1 ( figure  4a ) .
nevertheless , the evh1 domain of ena still retains the wave complex from drosophila s2 cell lysates ( figure  4b ) . using a far western approach
, we found that the evh1 domain of ena bound two sets of dabi peptides containing an 
fpppp  motifs ( ball et  al . , 2000 ; niebuhr et  al .
, 1997 ; peterson and volkman , 2009 ) . far western analysis of peptide arrays containing systematic substitutions at each position confirmed that both lpppp motifs are important for evh1 binding ( figure  4e ) . to establish if these motifs are important in the context of dabi , we performed pull - down assays on wild - type and mutants in which the two  lpppp  motifs were mutated to agggg , either alone or in combination ( figure  5a ) .
substitution of residues 311315 ( mut1 ) substantially weakened but did not completely abolish the interaction of dabi with the ena evh1 domain .
in contrast , mutation of residues 374378 ( mut2 ) resulted in a loss of binding ( figure  5a ) .
motifs did not , however , disrupt the ability of dabi ( dabiena ) to colocalize with wave at the plasma membrane of drosophila s2 cells ( figure  5b ) .
the dabiena mutant was also as effective as the wild - type protein at rescuing the  spiky  morphology of drosophila s2 cells induced by rnai - mediated loss of endogenous dabi ( figure  5c ; kunda et  al . , 2003 ; rogers et  al . ,
2003 ) . to examine whether the interaction between ena and dabi helps to stabilize the wrc at the plasma membrane , we performed fluorescence recovery after photobleaching ( frap ) experiments on drosophila s2 cells treated with rnai targeting the 3 utr of endogenous dabi and expressing gfp - tagged abi or abiena .
we found that the loss of ena binding results in a statistically significant 1.38-fold increase in the exchange rate of dabi at the plasma membrane ( figure  5d ) .
importantly , pull - down assays on s2 lysates demonstrate that the loss of the interaction with ena did not disrupt the ability of dabi to incorporate into the wave complex ( figure  5e ) . to investigate the physiological significance of the interaction between ena and abi in regulating wrc functions in  vivo
, we took advantage of abi mutant flies ( stephan et  al . , 2011 ) .
we first examined the consequences of the loss of the abi : ena interaction in drosophila macrophages .
structured - illumination microscopy analysis of spreading wild - type macrophages reveals a highly polarized actin cytoskeleton with a broad lamellipodial cell front ( figures 6a and 6b ) .
macrophage - specific knockdown of wave in larval macrophages using the hemolectin - gal4 driver completely disrupts lamellipodia formation ( figure  6a ; sander et  al . , 2013 ) .
the abi mutant macrophages also had a similar  spiky  morphology ( figures 6a and 6b ) .
these defects in cell morphology were substantially rescued by ubiquitous reexpression of wild - type abi but not by the mutant lacking both ena binding motifs ( abiena ) from the same genomic locus ( figures 6a and 6b ) . to further analyze differences in lamellipodia protrusions and cell shape of rescued macrophages
we found that cells expressing abi exhibit more stable and periodic membrane protrusions , whereas the abiena population are significantly less circular ( more spiky ) and have a reduced rate of membrane protrusion , ( figure  6c ; movie s1 ) .
furthermore , in contrast to wild - type macrophages , ena is no longer at the leading edge of lamellipodial protrusions but becomes relocalized to the tips of filopodia - like protrusions and along stress fiber - like actin bundles when it can not interact with abi ( figure  6d ) . in  vivo , abi and a functional wrc
are required in the drosophila larval visual system for the correct axonal targeting of photoreceptor neurons ( r - cells ) to their respective optic ganglions in the fly brain ( stephan et  al . , 2011 ) .
remarkably , we found that the loss of the ability of abi to interact with ena resulted in a similar defect in r - cell targeting as the complete loss of abi ( figures 7a and 7b ) .
we also examined the impact of an abi mutant lacking its ena binding sites and the c - terminal sh3 domain ( abienash3 ) because an abi transgene lacking its c - terminal sh3 domain is able to substantially rescue abi mutant phenotypes ( stephan et  al . , 2011 ) .
in contrast to abiena , the expression of abienash3 surprisingly rescued the abi - dependent r - cell targeting defects ( figures 7a and 7b ) .
overexpression of the abi variants in a wild - type background , however , did not affect r - cell targeting excluding any dominant effects ( figure  s3 ) .
next , we analyzed the cellular requirement for the abi : ena interaction during oogenesis because , in contrast to the visual system , wrc and ena control cell - autonomous actin - based structures that are essential for normal egg development ( gates et  al .
loss of wave and arp2/3 in the  germline results in small and abnormally shaped eggs ( hudson and cooley , 2002 ; zallen et  al . , 2002 ) .
a similar dumpless mutant phenotype is also observed in flies lacking abi in the germline ( zobel and bogdan , 2013 ) .
rescue experiments using a puast - abi transgene that is only expressed in somatic follicle cells and not the germline rescues the lethality of abi mutants ( zobel and bogdan , 2013 ) . however , the integrity of cortical actin  in nurse cells within the egg chamber is disrupted and the rescued female flies are completely sterile ( figures 7c and 7d ) .
to overcome this problem , we generated puasp transgenes for efficient germline expression ( rrth , 1998 ) and performed rescue experiments with the different abi variants .
reexpression of wild - type abi rescues the egg morphology defects and female sterility of abi mutant flies ( figures 7c and 7d ) .
in contrast , abi mutant flies expressing abiena are completely sterile , containing smaller and abnormally round eggs ( figures 7c7e ) .
this round - egg phenotype resembles those seen in rac1 , rac2 mutant follicle cell clones defective for egg chamber elongation rather than flies lacking abi in the germline ( conder et  al .
, we observed a similar round - egg phenotype when we suppress wrc function in follicle cells using an sra-1 rnai transgene ( figure  7f ) .
late - stage abiena mutant egg chambers also show additional defects in nurse cell cortical actin integrity , resulting in detached cytoplasmic actin bundles and ring canals ( figure  7c ) .
these defects closely resemble phenotypes found in abi mutants lacking germline abi ( puast - abi rescue ; zobel and bogdan , 2013 ) and wave germline mosaics ( zallen et  al . , 2002 ) . because overexpression of abiena in a wild - type background did not affect fertility , dominant effects
can again be excluded ( figure  7d ) . finally , we examined the impact of an abi mutant lacking its ena binding sites and the c - terminal sh3 domain ( abienash3 ) .
consistent with our observations in the larval visual system , abienash3 rescued the egg morphology defects and female sterility of abi mutants ( figures 7c7f ) . in summary , our data demonstrate that the abi : ena interaction plays an important role in  vivo in regulating diverse actin - based structures and morphogenetic processes that require a functional wrc .
the wrc binds and activates the arp2/3 complex to drive actin polymerization at the plasma membrane in response to rac signaling during cell migration ( bisi et  al . , 2013 ) .
in contrast , ena / vasp proteins stimulate cell migration by antagonizing actin filament capping and acting as processive actin polymerases ( barzik et  al . , 2005 ; bear and gertler , 2009 ; bear et  al . , 2002 ; breitsprecher et  al . , 2008 , 2011 ; hansen and mullins , 2010 ; pasic et  al . ,
we have now demonstrated that ena / vasp proteins can be linked to the function of wrc by virtue of a direct interaction between their evh1 domains and abi , an integral component of the wrc .
our results have confirmed and extended previous yeast two - hybrid data and pull - downs from cell lysates demonstrating that the evh1 domains of mena and vasp can interact with human and mouse abi1 ( dittrich et  al . , 2010 ;
hirao et  al . , 2006 ; maruoka et  al . , 2012 ; tani et  al . ,
the structure of several evh1:fpppp complexes reveals that the  fpppp  motif adopts a type ii polyproline helix that is coordinated by three aromatic residues present in all ena / vasp family members ( peterson and volkman , 2009 ) .
in contrast , the evh1 domain interacts with an extended proline - rich binding site in human abi1 .
consistent with their ability to bind , abi2 has an almost identical sequence whereas abi3 has two  lpppp  motifs in this region . in many respects ,
the extended nature of the abi1 interaction resembles that of the n - wasp wh1 binding site in wip , which also  involves three regions of contact ( peterson et  al . , 2007 ) .
in classical evh1 interactions , the acidic residues flanking the  fpppp  motif play an important role in determining the affinity , orientation and specificity of evh1 binding ( ball et  al . , 2000 ; peterson and volkman , 2009 ) .
in contrast , the evh1 binding site in human abi1 contains two pairs of aspartic acid residues flanking the central phenylalanine in the middle of the motif as well as a downstream acidic patch ( dyedee ; figure  4a ) .
the molecular basis of the evh1 human abi1 interaction , including the extended peptide orientation and role of acidic residues , must await structural determination of the complex .
nevertheless , our data clearly demonstrate that the evh1 domain can bind additional proline rich ligands beyond  fpppp  motifs .
interestingly , the meander region of wave1 contains an  lpppp  motif that is capable of interacting with mena ( okada et  al . , 2012 ) .
the ability of mena to bind abi in the wrc presumably explains why it still associates with wave lacking its proline rich region ( okada et  al . , 2012 ) .
consistent with the presence of  lpppp  motifs pull - downs with recombinant proteins demonstrate that the evh1 domain of mena can interact with wave 1 and 2 , but not wave 3 ( figure  s2b ) .
our observations , however , suggest that the interaction with abi is more important for mena interactions with the wrc than wave ( figure  4d ) .
moreover , our in  vitro assays clearly demonstrate that the ability of rac to activate wrc - mediated actin polymerization via the arp2/3 complex is significantly enhanced by vasp binding to abi .
in contrast to the full - length protein , monomeric vasp or its isolated evh1 domain is unable to activate the wrc to stimulate arp2/3-mediated actin polymerization even at high concentrations .
this difference may reflect the ability of the vasp tetramer to induce oligomerization of the wrc , an effect that would enhance wrc potency toward the arp2/3 complex ( padrick et  al . , 2008 ; padrick and rosen , 2010 ) .
it is possible that the simultaneous engagement of a vasp tetramer with abi and the  lpppp  motif in wave increases the activity of the wrc .
however , oligomerization alone can not account for our data because mutating the actin binding elements of vasp , which should have no effect on tetramerization , abrogates activity .
furthermore , the vasp effect does not appear to be simple allosteric activation of the wrc ( i.e. , release of the vca ) , because this should produce activity equal to that of the vca alone .
while not definitive , our collective data are most consistent with a model in which vasp binds the rac - activated wrc with high affinity based on tetramerization - mediated avidity and also interacts with actin filaments , thus increasing the association of the wrc with filaments . because both the released wave vca and actin filaments activate the arp2/3 complex ( machesky et  al . , 1999 ) , assembling these two elements should enhance their cooperative actions and increase actin assembly .
in contrast to the situation in humans , the interaction between the evh1 domain of ena and abi in drosophila is mediated by two  lpppp 
the loss of these two  lpppp  motifs increases the dynamics of the wrc at the plasma membrane but did not affect lamellipodium formation in s2 cells in culture .
in contrast , the consequences of disrupting the interaction of ena with abi in  vivo are  more dramatic , as primary macrophages expressing abiena have reduced lamellipodial membrane protrusions and defects in cell morphology .
unlike the situation in s2 cells , which have been treated with dsrna and transiently transfected with gfp - tagged expression constructs , the abi transgenes ( abi and abiena ) are expressed from the same genomic locus .
these in  vivo rescue experiments therefore allow for a more precise analysis of the requirement of the interaction between ena and abi rather than in the hypomorphic situation in s2 cells .
the ability of abiena to rescue lamellipodium formation in s2 cells might reflect an incomplete abi knockdown or a difference in its expression level compared to endogenous abi in untreated cells .
consistent with this , in macrophages , we found that strong expression of abi in earlier larval stages using the da - gal4 driver results in a more robust rescue of lamellipodia protrusion and cell morphology defects as compared to macrophage - specific expression ( hml-gal4 ) at late larval stages .
given that our in  vitro actin polymerization assays indicate that vasp ( ena ) is not an essential activator but rather acts cooperatively with rac1 to promote wrc activation , it is likely that in  vivo the requirement for this interaction depends on the level of abi .
this explanation may also partially account for the more dramatic phenotypes observed in the multicellular context .
remarkably , we found that the loss of the ability of abi to interact with ena resulted in a similar defect in r - cell targeting as the absence of the complete protein .
this suggests that ena has a nonautonomous role in the larval brain , as we have previously shown for wrc function in targeting of early retinal axons ( stephan et  al . , 2011 ) .
mosaic mutant analysis further supports a nonautonomous function for ena in retinal axon targeting ( data not shown ) .
thus , we propose that the interaction between ena and the wrc is required to regulate actin dynamics in the target area neurons .
however , since the precise projection pattern of early retinal axons depends on complex interactions between different populations of glia cells and neurons in the target field , it remains unclear how ena and the wrc function together in this developmental context .
in contrast , drosophila oogenesis provides an excellent model to study the cell autonomous function of the interaction between ena and the wrc .
previous phenotypic analyses of mutant egg chambers suggest ena and wrc have both distinct and overlapping functions during oogenesis ( gates et  al . , 2009 ; zallen et  al . , 2002 )
both are required for the integrity of the cortical actin in nurse cells and mutant egg chambers become multinucleated as the plasma membrane breaks down due to a loss of cortical actin integrity .
in contrast , to wave mutant egg chambers , disruption of ena function does not affect ring canal morphology but rather leads to a reduced and delayed formation of cytoplasmic actin filament bundles ( gates et  al . , 2009 ; zallen et  al . , 2002 ) .
similar to wave germline clones , the loss of abi in the germline results in a dumpless mutant phenotype and female flies are sterile ( zobel and bogdan , 2013 ) .
we have now found that these defects in egg morphology and female fertility can not be rescued by reexpression of a full - length abi deficient in ena binding .
abiena mutant egg chambers have defects in the integrity of the nurse cell cortical actin resulting in detached cytoplasmic actin bundles and ring canals .
the rupture of nurse cell membranes is even more obvious at later stages when the fast transport of nurse cell contents starts , as recently observed for ena , wave , and abi mutants ( gates et  al .
, 2009 ; zallen et  al . , 2002 ; zobel and bogdan , 2013 ) .
in addition to nurse cell dumping defects , we also observed a striking egg chamber elongation defect .
mutant eggs lacking the  interaction between abi and ena fail to elongate and remain spherical as similarly found in rac or pak mutants ( conder et  al . , 2007 ) .
the round egg phenotype observed in flies expressing abiena suggests that there might be a defect in the basal actin  cytoskeleton of the follicle cells that drives egg chamber elongation ( bilder and haigo , 2012 ; gates , 2012 ) .
consistently , reexpression of abiena in somatic follicle cells ( abi , da > uast - abiena ) also results in a round - egg phenotype .
these data suggest a requirement of wrc function in follicle cells during egg elongation . supporting this notion
, we found that a follicle cell - specific knockdown of sra-1 function results in a strong round - egg phenotype .
our rescue experiments additionally imply a more complex interaction network among ena , abi , and sh3 interacting proteins . whereas a minimal abi fragment lacking the ena - binding or proline - rich region and the c - terminal sh3 domain is able to rescue substantially abi mutant traits in drosophila and dictyostelium ( davidson et  al . , 2013 ; stephan et  al . , 2011 )
thus , we propose a scenario in which the influence of ena on wrc activity depends on additional proteins interacting with the abi - sh3 domain .
the most prominent candidate is the nonreceptor tyrosine kinase abelson ( abl ) that binds abi and ena proteins ( dai and pendergast , 1995 ; gertler et  al . , 1989 ) .
based on the antagonistic genetic interaction between ena and abl , it has been hypothesized that a precise balance between abl and ena activity is required for fly viability .
however , it is still unclear how abl affects the function of ena , because mutation of all known abl phosphorylation sites only has a modest effect on ena function in  vivo ( comer et  al . , 1998 ;
similarly , abl and abi have opposing roles in drosophila ( lin et  al . , 2009 ) .
thus , we propose a model in which ena synergizes with rac to activate the wrc , but also inhibits abl function .
abl in turn inhibits wrc function as previously shown ( lin et  al . , 2009 )
thus , the disruption of ena binding to dabi would simultaneously decrease wrc stimulation by ena and increase its inhibition by abl .
such a scenario would explain why loss of ena binding to abi ( wrc ) phenocopies the abi mutants .
this also suggests that the interaction among wrc , abl , and ena function is of more general relevance for actin - based processes in multicellular contexts .
furthermore , recent data also suggest that lamellipodin , which cooperates with the wrc to promote cell migration in  vivo , is also likely to be part of this complex regulatory network , because it can bind both the evh1 domain of vasp and the sh3 domain of abi ( krause et  al .
our in  vitro data clearly demonstrate that ena / vasp proteins can directly affect the activity of the wave complex , whereas our observations in drosophila have revealed that , in  vivo , the function and activity of ena / vasp proteins and the wave complex are intimately linked .
bacterial expression vectors for gst - tagged evh1 domains of vasp , evl , and mena as well as the lim3 domain of tes and fpppp region of zyxin have been described ( boda et  al . , 2007 ) .
the evh1 domain of drosophila ena ( residues 1115 ) as well as human abi1 - 3 , wave1 - 3 , vasp , nap1 , and pir121 were amplified by pcr and cloned into the not1-ecor1 sites of pmw172-gst-3c or pmw172-his-3c to generate a gst or his - tagged e.  coli expression vectors .
the vasp 1335 and the gab ( l226a / i230a / l235a / r236e / k237e ) and fab ( r273e / r274e / r275e / k276e ) mutants ( breitsprecher et  al . , 2011
proteins were typically expressed in bl21(de3 ) rosetta cells and purified as previously described ( boda et  al .
all his - tagged vasp proteins were expressed in bl21 ( de3 ) t1 cells at 18c and purified by ni - nta agarose beads , a source sp15 column , and finally a superdex200 or superfex75 column . other proteins , including arp2/3 complex , actin , vca , rac1 q61l , and tev protease were purified as previously described ( ismail et  al . , 2009 ) .
pull - down assays with recombinant proteins on mv and s2 cell extracts were performed as previously described ( boda et  al . , 2007 , 2011 ) . stained protein bands ( imperial protein stain , thermo scientific )
were reduced , alkylated , and digested with trypsin , as previously described ( collazos et  al . , 2011 ) .
the digests were analyzed with liquid chromatography - tandem mass spectroscopy ( lc - ms / ms ) on an agilent 6510 mass spectrometer ( agilent ) .
lc - ms / ms data were searched against a protein database ( ncbinr 20080210 ) using the mascot search engine ( matrix science ) .
the human wrc was purified as previously described ( chen et  al . , 2014 ) .
gst pull - down was performed by mixing 380 pmol of bait proteins ( gst or gst - tagged evh1 or rac1 ) with equimolar prey proteins ( wrc or wrc - abi1[1159 ] ) and 20  l of glutathione sepharose beads ( ge healthcare ) in 1  ml of binding buffer ( 20  mm hepes , 100  mm nacl , 5% [ w / v ] glycerol , 2  mm mgcl2 , 1  mm egta , and 5  mm -mercaptoethanol , ph 7 ) at 4c for 30  min .
bound proteins were eluted with 30  mm reduced glutathione and examined with sds - page .
actin polymerization assays were performed at 22c using a pti fluorometer ( photon technology international ) as previously described with slight modifications ( ismail et  al . , 2009 ) .
reactions contained 4  m rabbit muscle actin with 5% labeled with pyrene , 10  nm bovine arp2/3 complex , 100  nm human wrc , and/or other proteins of interest in 10  mm imidazole , 100  mm kcl , 1  mm mgcl2 , 1  mm egta , 20% ( w / v ) glycerol , and 1  mm dithiothreitol , ph 7.0 .
note that under these conditions , the wrc fully activated by rac1 ( or equivalently , 100  nm vca ) only exhibits modest activity toward the arp2/3 complex .
monoclonal antibodies gfp 3e1 ( cancer research uk ) , actin ac-74 ( sigma ) , and zyxin 164d4 ( sysy ) as well as rabbit antibodies against abi1 ( sigma a5106 ) and vasp ( cell signaling 3132s ) were used .
antibodies against human abi1 , nap1 , and pir121 ( steffen et  al . , 2004 ) were kindly provided by dr theresia stradal ( university of muenster , germany ) .
immunofluorescence and immunoblot analyses were performed as described ( boda et  al . , 2007 ) .
projection patterns of all photoreceptor axons of the indicated genotypes were depicted using mouse anti-24b10 ( -chaoptin , dshb ; stephan et  al . , 2011 ) .
human abi1 or abi1evh1 were cloned into the not1/ecori sites of a modified plvx - puro - gfp vector and used to generate lentiviruses .
ht1080 cell lines stably expressing gfp or the gfp - tagged proteins were selected with puromycin and facs sorting . for scratch assays ,
the cell lines were transfected with qiagen allstars negative control or sirna hs abi1 9 which targets the 3utr of abi1 . a day later
, 5   10 cells were seeded into each well of a 96-well imagelock microplates ( six replicate wells / condition ; essen bioscience ) and after 24  hr , a scratch was made in each well using the essen instruments woundmaker 96 .
the cells were washed three times with pbs and serum - free dulbecco 's modified eagle 's medium was added .
the plates were equilibrated at 37c for 30  min before scanning in the essen instruments incucyte flr .
images were captured every 30  min for 20  hr and percentage relative wound density was calculated using incucyte software . a representative graph and  images from one experiment are shown .
numbers are mean of six wells   sem and similar results were obtained in five other experiments .
all abi mutants were generated using the quikchange site - directed mutagenesis kit ( stratagene ) .
abi and evh1 binding mutants were cloned into cb6-n - gfp and pac - c - gfp ( ac5 promoter ) modified from pac5.1/v5-his ( invitrogen ) for expression in mammalian and drosophila cells , respectively .
293 t and s2 cells were transfected with abi expression vectors using calcium phosphate or effectene ( qiagen ) .
coimmunoprecipitations and pull - down assays using gst - evh1-ena were performed as described previously ( boda et  al . , 2007 , 2011 ) .
far western analysis of overlapping 20-mer human and drosophila abi peptide arrays were probed with gst or gst - tagged evh1 domains of ena , evl , mena , and vasp as described previously ( postigo et  al .
the 3utr and orf of dabi were amplified from a drosophila cdna library by pcr using primers containing a t7 rna polymerase binding site at their 5 ends .
purified pcr products served as templates for single - stranded rna ( ssrna ) synthesis using ambion megascript high yield transcription t7 kit .
the ssrnas were annealed and the resulting double - stranded rna ( dsrna ) was purified using the ambion megacleartm kit .
s2 cells were treated with dsrna as described previously ( kiger et  al . , 2003 ) .
after 3  days , dabi expression was assessed with qrt - pcr and immunoblot . to express dabi - gfp clones ,
for frap assays , drosophila s2 cells were seeded on to concanavalin a - coated mattek dishes for 30  min and imaged using a zeiss lsm 710 confocal with a 63/1.4na objective .
a region of 300   100 pixels was recorded using a scan speed of 1.27  s / pixel with a pinhole of 90  m .
a selected region within the imaging area of 90   25 pixels covering the protruding lamellipodia was imaged 5 times before bleaching with 30 iterations of the 488  nm laser at full power . immediately after bleaching , 260 images were acquired .
analysis of fluorescence recovery and curve fitting was performed as previously described ( weisswange et  al . , 2009 ) . all drosophila strains and crosses
the following strains were used : frt82b abi20 ( stephan et  al . , 2011 ) ,
elavc155gal4 , da - gal4 , hml-gal4 , and egfp ( bloomington stock center ) .
uast / p - abi , uast / p - abiena and uast / p - abienash3 transgenes were generated by  c31-integrase - mediated integration into the landing site m{3xp3-rfp.attp}zh-68e as previously described ( stephan et  al . , 2011 ) .
full - length abi , abiena , and abienash3 fragments were amplified by pcr and cloned into gateway entry vectors ( pentr dtopo , invitrogen ) .
the inserts were sequenced and subcloned into puast - attb - rfa , puasp - attb - rfa puast - attb - rfa - egfp ( drosophila genomics resource center ) by in  vitro lambda recombination ( invitrogen ) . to force early expression and to simultaneously depict cell dynamics , we combined the ubiquitous da - gal4 driver with the macrophage - specific expression of cytoplasmic egfp ( hml-gal4 , uas - egfp ) .
macrophages were isolated by dissecting larvae in a drop of m3 medium ( invitrogen ) .
glass slides were pretreated with 0.5  mg / ml concanvalina ( sigma ) for 30  min .
cells were fixed for 15  min in 4% paraformaldehyde , and shortly rinsed in pbs and 0.1% triton ( pbst ) .
after fixation , cells were incubated for 2  hr with primary antibody , rinsed twice with pbs , and incubated for 1  hr with secondary antibody and with phalloidin - alexa488 ( 1:100 ) and dapi ( 1:1000 , invitrogen ) .
wild - type ovaries ( w ) and ovaries from rescued abi20 mutant females ( abi20 , da - gal4/abi20 , uasp - abi ; abi20 , da - gal4/abi20 , uasp - abiena and abi20 , da - gal4/abi20 , uasp - abienash3 ) were dissected in cold pbs , fixed in 4% paraformaldehyde in pbs for 20  min , and stained with alexa - fluor-488 phalloidin and dapi ( invitrogen ) . to test fertility ,
sim images were taken with an elyra s.1 microscope ( zeiss ) with the software zen 2010 d ( zeiss ) . for image acquisition ,
, cells were plated on chambered cover glass ( lab - tek ) without concanvalina .
movies were taken with a spinning disc cell observer sd zeiss microscope and cropped so that only a single cell was present within the field of view . following kalman - filtering to suppress noise ,
cells were segmented in each frame using a region - growing algorithm implemented as a plug - in for imagej and velocity maps constructed as described previously ( dbereiner et  al . , 2006 ) .
the circularity of cells ( c  = 4[area]/[perimeter ] ) was estimated in each movie frame and an average taken for all frames .
the dynamic dimension is a measure we define as the slope of the line of best fit to the histogram of a velocity map , plotted logarithmically , for velocities greater than or equal to zero ( r2  > 0.9 ) .
the software necessary to perform these calculations was coded in java and implemented as a plug - in for imagej .
x.j.c . identified and mapped all ena / vasp evh1 interactions in human and drosophila abi .
x.j.c also generated all abi mutants and vasp 1 - 335 and performed all experiments in s2 cells , including the frap analysis .
b.c . purified the wrc and vasp proteins and performed gst - evh1/wrc pull downs , as well as all in  vitro actin polymerization assays , which were overseen by m.k.r .
generated ht1080 cell lines , analyzed their migration , generated vasp gab and fab mutants , and performed abi1 - 3 and wave1 - 3 in  vitro evh1 pull - down assays .
 summaryena / vasp proteins and the wave regulatory complex ( wrc ) regulate cell motility by virtue of their ability to independently promote actin polymerization . we demonstrate that ena / vasp and the wrc control actin polymerization in a cooperative manner through the interaction of the ena / vasp evh1 domain with an extended proline rich motif in abi 
 . this interaction increases cell migration and enables vasp to cooperatively enhance wrc stimulation of arp2/3 complex - mediated actin assembly in  vitro in  the presence of rac . 
 loss of this interaction in drosophila macrophages results in defects in lamellipodia formation , cell spreading , and redistribution of ena to the tips of filopodia - like extensions . 
 rescue experiments of abi mutants also reveals a physiological requirement for the abi : ena interaction in photoreceptor axon targeting and oogenesis . 
 our data demonstrate that the activities of ena / vasp and the wrc are intimately linked to ensure optimal control of actin polymerization during cell migration and development . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions