BACKGROUND
the rho gtpases proteins are members of a large superfamily of regulatory proteins whose activities are controlled by regulated gdp/gtp cycling.
to date, a total of  <dig> rho family members have been suggested by the data available from the human genome sequence project.
the rho gtpases can be divided into six groups: rho , rac , cdc <dig> , rnd , rhobtb  and miro .
additional members, rhod, rif and ttf/rhoh, do not fall into any of these subfamilies  <cit> .
rho gtpases control a wide variety of signal transduction pathways regulating many fundamental processes of cell biology, such as organization of the actin cytoskeleton  <cit> , gene expression, cell proliferation and survival  <cit> .
most rho proteins cycle between an active gtp-bound state and an inactive gdp-bound state.
binding to gtp is promoted by rho guanine nucleotide exchange factors , and gtp hydrolysis is catalysed by rho gtpase-activating proteins .
rho-gdp dissociation inhibitors  stabilize the gdp-bound form of rho proteins.
rho proteins are also implicated in participating in several steps of tumor progression and development of metastasis  <cit> .
activated rho proteins cooperate strongly with oncogenes ras and raf in focus-formation assays, but either fail to independently induce transformation or else exhibit weak transforming activity  <cit> .
they function in cell cycle regulation by the modulation of cyclin d <dig>  <cit>  and by their involvement in endocytic traffic  <cit> , such as in regulation of epidermal growth factor receptor  <cit> .
furthermore, lacal et al have shown that rho gtpases are directly involved in signalling pathways that trigger either proliferation or cell death  <cit> .
moreover, expression of activated rac protects against ras-induced apoptosis  <cit> .
these studies linking rho proteins to many aspects of cellular proliferation are further extended by the study by gomez del pulgar, which revealed that several human tumors contained aberrant expression and activation of rho gtpases  <cit> .
elevated expression of rhoa and rhoc was found in breast, lung, ovarian, gastric, and bladder cancers.
the involvement of rhoa in testicular human tumors was demonstrated by increased rhoa mrna levels in relation to tumour grade  <cit> .
overexpression of the rhoc gene in adenocarcinoma of pancreas correlated with poorer prognosis of patients  <cit> , whereas rhob expression is lost in several tumors  <cit> .
moreover, unlike ras, no mutated, constitutively active forms of rho proteins in tumors have thus far been identified  <cit> , apart from one report linking hyperactive rac <dig> with highly proliferative human breast cancer cells and tumor tissues  <cit> .
whatever the level of gene expressions of rho gtpases and assuming that high level of protein could be associate with higher concentration of activated rho gtpase, the knowledge of accurate variations of the rho activation under treatment would be a significant progress in the understanding of the biological role of rho in oncogenesis.
to further develop understanding of rho activation, we identified a conformation-specific scfv against the active form of rhoa, rhob and rhoc gtpases.
we employed a phage display approach, which has previously demonstrated successful results in generating precise sensors of variations in molecular conformation  <cit> .
to this end, we utilized the q63l mutant  <cit>  of rhob , and expressed this mutant as a gst fusion protein in e. coli and used the purified recombinant protein as a target ligand for selection of recombinant scfv.
using the griffin.
<dig> library  <cit> , one clone  was selected for its ability to discriminate between the activated versus the unactivated form of rhob, as well as rhoa and rhoc, but not other rho family members.
the selected c <dig> scfv was fused to a fragment of phage coat protein piii gene to enhance its production, expressed in e. coli, and its specificity confirmed.
finally, its ability to detect endogenous activated rho in vivo in immunofluorescence studies is shown.
RESULTS
identification of rhobq63l specific interacting phages
peptide and protein libraries on filamentous phages, in combination with effective selection strategies, have proven to be a rapid and successful method to identify proteins with specific binding properties.
a major advantage of the in vitro selection process by phage display is the extensive control of the selection conditions, which, for example, allowed the preservation of the three-dimensional structure of gtp-bound form of rho.
using the griffin.
<dig> phage display library, perez et al identified scfvs that act as molecular conformation sensors by showing selectivity towards the gtp-bound conformation of the rab <dig> gtpase  <cit> .
the present work used the same phagemid library containing more than  <dig> independent clones.
each clone expresses human recombinant antibodies as a single-chain fv , where both myc, 6xhis tags, and the piii protein of m <dig> are fused to the c-terminus of immunoglobulin vh-vl fusion .
due to its ability to decrease the kinetics of gtp hydrolysis, rhobq63l is considered as a "gtp-locked" mutant of rhob.
we expressed and purified the gst-tagged rhobq63l mutant in escherichia coli, and used the recombinant protein as target for phage selection.
for the first round of selection, we collected phage bound to gst-rhobq63l-bound beads.
for the subsequent four rounds, selection was a two-part process: we first preblocked phage on gst-beads, and the phage mixture was then loaded on gst-rhobq63l beads.
during the last round, soluble recombinant wild-type rhob was added into the phage mixture to specifically select phage that interact only with the active gst-rhobq63l form on the beads.
rounds iv and v contained polyclonal phage specific to gst-rhobq63l.
to increase isolation of positive clones, we initiated two independent screenings from round v: v- <dig> and v- <dig>  in total,  <dig>  clones were randomly picked and checked by elisa, yielding  <dig> rhobq63l selective phages.
characterization of the selected single chain antibodies
to characterize the  <dig> rhobq63l selective phages, their specificity was first examined by analyzing their interaction with gst, gst-rhob and gst-rhobq63l .
six of the phages showed preferential binding to the gtp-locked rhobq63l mutant, while two clones  failed to interact and were discarded .
to assess the specificity of the selected scfv against the gtp-bound form of the three rho proteins of interest, c <dig> and control phages were tested by elisa with the wild type form of rhoa, b and c  and their corresponding active forms .
as shown in fig 2b, c <dig> phage showed significant reactivity against the q63l mutant of all three rho proteins.
in contrast, the reactivity against the wt form was very low in most cases, and within the range of the background response.
as predicted, the c <dig> phage does not discriminate between the gtp-bound form of rhoa, b, c, suggesting that c <dig> is directed against the gtp binding domain or against the effector-binding domain shared at almost 100% identity among the three proteins.
this also suggests that the carboxy terminal hyper-variable domain of these proteins, which functions to target membrane localization, has little importance in the conformation of the active form.
dna sequencing of the  <dig> preselected clones revealed three unique scfv sequences for phages c <dig>  d <dig> and a <dig> , although scfv d <dig> and a <dig> only contained sequence for the variable heavy chain, vh.
the major differences between the amino-acid sequences of the three clones were concentrated in the hypervariable complementary-determining regions, cdr <dig> and cdr <dig>  of the vh.
the a <dig> and d <dig> phages proved to be unstable.
they quickly lost their recognition properties, and were discarded.
thus, only the c <dig> phage was kept for further analysis.
biochemical characterization of the specific binding of scfv c1
in order to further determine whether c <dig> specifically detected rho proteins in their gtp-bound conformation, we next assayed c <dig> binding to rho protein preloaded with gtp.
first, gst-rhoa expressed and purified onto glutathione beads was loaded with gdp or the slowly hydrolyzable analogue of gtp, gtpγs, to ensure maximum loading of rhoa with the nucleotide  <cit> .
c <dig> phage was next added and incubated for  <dig> hour, and bound c <dig> phages were measured with anti-m <dig>  as seen in fig 4a, the c <dig> binding increased up to  <dig> min after loading with gtpγs until a steady state was reached.
under similar incubation conditions, in the presence of gdp, the c <dig> phage recognition of rhoa did not change.
to further confirm the rho gtp-bound conformational specificity of c <dig>  nucleotide-binding assays with rhob and rhoc were tested in comparison with rhoa.
c <dig> phages bound specifically to gtpγs but not gdp preloaded rhoa, rhob and rhoc .
interestingly, when we tested the binding of c <dig> phage against the gtp-bound form of rac <dig> and cdc <dig>  two well-characterized members of two separate subfamilies of rho gtpases, c <dig> phages bound selectively on gtpγs-rhob but not gtpγs-rac <dig> or gtpγs-cdc <dig> , despite their ability to successfully bind gtp .
together this demonstrated that the scfv c1-piii fusion protein specifically targeted the gtp-bound form of rhoa, rhob and rhoc, and was unable to recognize gtpγs or gdp preloaded rac <dig> and cdc <dig>  two other rho subfamily members.
expression and activity analysis of recombinant scfvc1
to further characterize scfv c <dig> binding, we constructed and expressed recombinant scfv c <dig> protein.
the phen <dig> phagemid vector carries an amber codon  at the c-terminal of the scfv sequence and prior to the m <dig> piii sequence .
non-suppressive strains, such as bl <dig>  interpret the amber codon as a translation stop signal, thus the scfv c <dig> is expressed in soluble form and can be purified via the histidine tags.
crude bacterial extract from cells expressing scfv c <dig>  as confirmed by western blot analysis with anti-c-myc antibodies, lost its ability to bind rhoaq63l .
the multiple supplementary bands observed in the blot would be explained by the renowned propensity of scfv to form dimeric and higher-order aggregates.
this loss of function is a common observation when phage-display derived scfvs are expressed as soluble scfvs.
jensen et al.
reported that an inactive antibody fragment can be functionally rescued by fusion to the n-terminal domain of the original phage display fusion partner, filamentous phage protein iii  <cit> .
we employed a similar approach and mutated the amber codon to allow translation and fusion to the n terminal of piii, as well as removed the c terminal of piii .
as shown in fig 5b, scfv c1-n1n <dig> exhibited the predicted molecular mass of  <dig> kda.
furthermore, the bacterial lysate expressing scfv c1-n1n <dig> was now able to selectively recognize rhoaq63l versus rhoa as determined by elisa.
competitive experiments between scfvc1-n1n <dig> and the rho binding domain of rhotekin  have been analyzed and are summarized in table  <dig>  rbd alone is not stable enough to be used in this study, however the chimere gst-rbd is able to compete with scfvc1-n1n <dig> on rhoaq63l and shows an affinity more than  <dig> fold higher than the scfv c1-n1n <dig>  affinities for gdp-rho, gtp-cdc <dig> and gtp-rac <dig> are too weak to be determined.
the new recombinant c1-n1n <dig> fusion protein was expressed more easily in a soluble form, and showed higher binding to rhoaq63l.
we then purified scfv c1-n1n <dig> from crude extract by incubation with ni-nta agarose beads and elution with imidazole, and aliquots from each step were analyzed by sds page .
the binding activity towards rhoaq63l of scfv c1-n1n <dig> crude extract and the four elutions fractions were tested and confirmed by elisa .
as seen on the sds page various proteins with lower molecular mass than scfvc1-n1n <dig> are co-purified with the desired probe.
as reported by others these proteins would be the result of translational arrest in the n1n <dig> sequence of the phage piii.
to measure the affinity of this scfv, surface plasmon resonance  experiments were performed on a biacore  <dig> instrument  <cit> .
using the previously described methodology by horn et al, we bound gst-rhoa loaded with gdp on one flowcell , and gst-rhoa loaded with gtpγs on a second flowcell   <cit> .
c1-n1n <dig> "antibody" was injected at a concentration of  <dig>  and  <dig> μm using a flow rate of  <dig> μl/min.
in order to test the specificity of scfv c1-n1n <dig> for the gtp-bound form of rhoa, differential responses  were recorded and analyzed using the biaevaluation  <dig>  software .
the results supported the gtp-bound conformational specificity, with an apparent dissociation constant  in the range of  <dig> ±  <dig> μm .
similarly, affinity of a scfv molecule for gtp-bound ras was previously reported in the range of  <dig>  μm  <cit> .
same results were found with gst-cdc  <dig> and gst-rac  <dig> loaded with gtpγs are coated on fc <dig> in front of gst-rhoa loaded with gtpγs on fc <dig>
recombinant scfv c1-n1n <dig> used as sensor of gtp-bound rho in vitro and in vivo
we determined the ability of the antibody to immunoprecipitate rhoa and rhob in their gtp-bound conformations.
crude bacterial extracts expressing scfv c1-n1n <dig> was incubated with ni-nta beads to immobilize the antibody, and bacterial lysate containing recombinant rhoa or rhob preloaded with gdp or gtp were mixed with these beads.
rhoa and rhob specifically bound to the scfv c1-n1n <dig> beads were analysed by western blot with commercial anti-rhoa and anti-rhob antibodies .
the scfv c1-n1n <dig> on ni-nta beads selectively associated with the gtp-bound form of both rhoa and rhob, confirming the specificity of this new antibody.
the ability of scfv c1-n1n <dig> to identify activated rho was next tested in vivo on activated hela cells by immunofluorescence.
after serum deprivation , hela cells were activated  <dig> hour with egf  and serum  and immunostained with scfvc1-n1n <dig>  irrelevant scfv  and anti-c-myc antibodies.
we observed that staining with scfv c1-n1n <dig> , but not the irrelevant scfv , produced a positive immunofluoresence signal specifically in activated cells .
to confirm the signal specificity, prior to staining, scfv c1-n1n <dig> was first incubated with gtpγs- preloaded rhoa to exhauste the selective scfv  or gdp-preloaded rhoa which would have any effect on scfv activity .
on serum starved cells, little signal was detected with either the scfv c1-n1n <dig> antibody preincubated with gdp-bound rhoa  or gtpγs-bound rhoa , although rho a is detected by a commercial antibody .
upon activation, a signal was visualized with the scfv c1-n1n <dig> preincubated with gdp-bound rhoa , showing labelling of the plasma membrane and punctate cytoplasmic structures consistent with previous reports for rho localization  <cit> .
similar labelling is obtained with commercial anti-rhoa .
moreover, this signal was suppressed by preincubation of the scfv c1-n1n <dig> antibody with gtpγs-preloaded rhoa .
this data strongly suggests that scfv c1-n1n <dig> is able to recognize endogenous activated and gtp-bound rho at the cellular level.
CONCLUSIONS
binding domains of effector proteins have proven to be useful as conformational sensors in analyzing the spatiotemporal activation of gtpases  <cit> .
in this study, we have shown that a phage display approach identified a specific phage for the gtp-bound form of rhoa, b and c. the expressed and purified scfvc1-n1n <dig> protein proceeded from this phage is able to recognize the three rho proteins  loaded with gtpγs with great specificity, and detect activation of endogenous rho protein in fixed cells.
this finding implicates the potential for scfv c1-n1n <dig> as a useful tool to compare the level of activated rho in cancer tissues versus normal tissues.

 the rho gtpases proteins are members of a large superfamily of regulatory proteins whose activities are controlled by regulated gdp/gtp cycling. to date, a total of rho family members have been suggested by the data available from the human genome sequence project. the rho gtpases can be divided into six groups: rho, rac, cdc, rnd, rhobtb and miro. additional members, rhod, rif and ttf/rhoh, do not fall into any of these subfamilies. rho gtpases control a wide variety of signal transduction pathways regulating many fundamental processes of cell biology, such as organization of the actin cytoskeleton, gene expression, cell proliferation and survival. most rho proteins cycle between an active gtp-bound state and an inactive gdp-bound state. binding to gtp is promoted by rho guanine nucleotide exchange factors, and gtp hydrolysis is catalysed by rho gtpase-activating proteins. rho-gdp dissociation inhibitors stabilize the gdp-bound form of rho proteins. rho proteins are also implicated in participating in several steps of tumor progression and development of metastasis. activated rho proteins cooperate strongly with oncogenes ras and raf in focus-formation assays, but either fail to independently induce transformation or else exhibit weak transforming activity. they function in cell cycle regulation by the modulation of cyclin d and by their involvement in endocytic traffic, such as in regulation of epidermal growth factor receptor. furthermore, lacal et al have shown that rho gtpases are directly involved in signalling pathways that trigger either proliferation or cell death. moreover, expression of activated rac protects against ras-induced apoptosis. these studies linking rho proteins to many aspects of cellular proliferation are further extended by the study by gomez del pulgar, which revealed that several human tumors contained aberrant expression and activation of rho gtpases. elevated expression of rhoa and rhoc was found in breast, lung, ovarian, gastric, and bladder cancers. the involvement of rhoa in testicular human tumors was demonstrated by increased rhoa mrna levels in relation to tumour grade. overexpression of the rhoc gene in adenocarcinoma of pancreas correlated with poorer prognosis of patients, whereas rhob expression is lost in several tumors. moreover, unlike ras, no mutated, constitutively active forms of rho proteins in tumors have thus far been identified, apart from one report linking hyperactive rac with highly proliferative human breast cancer cells and tumor tissues. whatever the level of gene expressions of rho gtpases and assuming that high level of protein could be associate with higher concentration of activated rho gtpase, the knowledge of accurate variations of the rho activation under treatment would be a significant progress in the understanding of the biological role of rho in oncogenesis. to further develop understanding of rho activation, we identified a conformation-specific scfv against the active form of rhoa, rhob and rhoc gtpases. we employed a phage display approach, which has previously demonstrated successful results in generating precise sensors of variations in molecular conformation. to this end, we utilized the q63l mutant of rhob, and expressed this mutant as a gst fusion protein in e. coli and used the purified recombinant protein as a target ligand for selection of recombinant scfv. using the griffin. library, one clone was selected for its ability to discriminate between the activated versus the unactivated form of rhob, as well as rhoa and rhoc, but not other rho family members. the selected c scfv was fused to a fragment of phage coat protein piii gene to enhance its production, expressed in e. coli, and its specificity confirmed. finally, its ability to detect endogenous activated rho in vivo in immunofluorescence studies is shown.  identification of rhobq63l specific interacting phages peptide and protein libraries on filamentous phages, in combination with effective selection strategies, have proven to be a rapid and successful method to identify proteins with specific binding properties. a major advantage of the in vitro selection process by phage display is the extensive control of the selection conditions, which, for example, allowed the preservation of the three-dimensional structure of gtp-bound form of rho. using the griffin. phage display library, perez et al identified scfvs that act as molecular conformation sensors by showing selectivity towards the gtp-bound conformation of the rab gtpase. the present work used the same phagemid library containing more than independent clones. each clone expresses human recombinant antibodies as a single-chain fv, where both myc, 6xhis tags, and the piii protein of m are fused to the c-terminus of immunoglobulin vh-vl fusion. due to its ability to decrease the kinetics of gtp hydrolysis, rhobq63l is considered as a "gtp-locked" mutant of rhob. we expressed and purified the gst-tagged rhobq63l mutant in escherichia coli, and used the recombinant protein as target for phage selection. for the first round of selection, we collected phage bound to gst-rhobq63l-bound beads. for the subsequent four rounds, selection was a two-part process: we first preblocked phage on gst-beads, and the phage mixture was then loaded on gst-rhobq63l beads. during the last round, soluble recombinant wild-type rhob was added into the phage mixture to specifically select phage that interact only with the active gst-rhobq63l form on the beads. rounds iv and v contained polyclonal phage specific to gst-rhobq63l. to increase isolation of positive clones, we initiated two independent screenings from round v: v- and v- in total, clones were randomly picked and checked by elisa, yielding rhobq63l selective phages. characterization of the selected single chain antibodies to characterize the rhobq63l selective phages, their specificity was first examined by analyzing their interaction with gst, gst-rhob and gst-rhobq63l. six of the phages showed preferential binding to the gtp-locked rhobq63l mutant, while two clones failed to interact and were discarded. to assess the specificity of the selected scfv against the gtp-bound form of the three rho proteins of interest, c and control phages were tested by elisa with the wild type form of rhoa, b and c and their corresponding active forms. as shown in fig 2b, c phage showed significant reactivity against the q63l mutant of all three rho proteins. in contrast, the reactivity against the wt form was very low in most cases, and within the range of the background response. as predicted, the c phage does not discriminate between the gtp-bound form of rhoa, b, c, suggesting that c is directed against the gtp binding domain or against the effector-binding domain shared at almost 100% identity among the three proteins. this also suggests that the carboxy terminal hyper-variable domain of these proteins, which functions to target membrane localization, has little importance in the conformation of the active form. dna sequencing of the preselected clones revealed three unique scfv sequences for phages c d and a, although scfv d and a only contained sequence for the variable heavy chain, vh. the major differences between the amino-acid sequences of the three clones were concentrated in the hypervariable complementary-determining regions, cdr and cdr of the vh. the a and d phages proved to be unstable. they quickly lost their recognition properties, and were discarded. thus, only the c phage was kept for further analysis. biochemical characterization of the specific binding of scfv c1 in order to further determine whether c specifically detected rho proteins in their gtp-bound conformation, we next assayed c binding to rho protein preloaded with gtp. first, gst-rhoa expressed and purified onto glutathione beads was loaded with gdp or the slowly hydrolyzable analogue of gtp, gtpγs, to ensure maximum loading of rhoa with the nucleotide. c phage was next added and incubated for hour, and bound c phages were measured with anti-m as seen in fig 4a, the c binding increased up to min after loading with gtpγs until a steady state was reached. under similar incubation conditions, in the presence of gdp, the c phage recognition of rhoa did not change. to further confirm the rho gtp-bound conformational specificity of c nucleotide-binding assays with rhob and rhoc were tested in comparison with rhoa. c phages bound specifically to gtpγs but not gdp preloaded rhoa, rhob and rhoc. interestingly, when we tested the binding of c phage against the gtp-bound form of rac and cdc two well-characterized members of two separate subfamilies of rho gtpases, c phages bound selectively on gtpγs-rhob but not gtpγs-rac or gtpγs-cdc, despite their ability to successfully bind gtp. together this demonstrated that the scfv c1-piii fusion protein specifically targeted the gtp-bound form of rhoa, rhob and rhoc, and was unable to recognize gtpγs or gdp preloaded rac and cdc two other rho subfamily members. expression and activity analysis of recombinant scfvc1 to further characterize scfv c binding, we constructed and expressed recombinant scfv c protein. the phen phagemid vector carries an amber codon at the c-terminal of the scfv sequence and prior to the m piii sequence. non-suppressive strains, such as bl interpret the amber codon as a translation stop signal, thus the scfv c is expressed in soluble form and can be purified via the histidine tags. crude bacterial extract from cells expressing scfv c as confirmed by western blot analysis with anti-c-myc antibodies, lost its ability to bind rhoaq63l. the multiple supplementary bands observed in the blot would be explained by the renowned propensity of scfv to form dimeric and higher-order aggregates. this loss of function is a common observation when phage-display derived scfvs are expressed as soluble scfvs. jensen et al. reported that an inactive antibody fragment can be functionally rescued by fusion to the n-terminal domain of the original phage display fusion partner, filamentous phage protein iii. we employed a similar approach and mutated the amber codon to allow translation and fusion to the n terminal of piii, as well as removed the c terminal of piii. as shown in fig 5b, scfv c1-n1n exhibited the predicted molecular mass of kda. furthermore, the bacterial lysate expressing scfv c1-n1n was now able to selectively recognize rhoaq63l versus rhoa as determined by elisa. competitive experiments between scfvc1-n1n and the rho binding domain of rhotekin have been analyzed and are summarized in table rbd alone is not stable enough to be used in this study, however the chimere gst-rbd is able to compete with scfvc1-n1n on rhoaq63l and shows an affinity more than fold higher than the scfv c1-n1n affinities for gdp-rho, gtp-cdc and gtp-rac are too weak to be determined. the new recombinant c1-n1n fusion protein was expressed more easily in a soluble form, and showed higher binding to rhoaq63l. we then purified scfv c1-n1n from crude extract by incubation with ni-nta agarose beads and elution with imidazole, and aliquots from each step were analyzed by sds page. the binding activity towards rhoaq63l of scfv c1-n1n crude extract and the four elutions fractions were tested and confirmed by elisa. as seen on the sds page various proteins with lower molecular mass than scfvc1-n1n are co-purified with the desired probe. as reported by others these proteins would be the result of translational arrest in the n1n sequence of the phage piii. to measure the affinity of this scfv, surface plasmon resonance experiments were performed on a biacore instrument. using the previously described methodology by horn et al, we bound gst-rhoa loaded with gdp on one flowcell, and gst-rhoa loaded with gtpγs on a second flowcell. c1-n1n "antibody" was injected at a concentration of and μm using a flow rate of μl/min. in order to test the specificity of scfv c1-n1n for the gtp-bound form of rhoa, differential responses were recorded and analyzed using the biaevaluation software. the results supported the gtp-bound conformational specificity, with an apparent dissociation constant in the range of ± μm. similarly, affinity of a scfv molecule for gtp-bound ras was previously reported in the range of μm. same results were found with gst-cdc and gst-rac loaded with gtpγs are coated on fc in front of gst-rhoa loaded with gtpγs on fc recombinant scfv c1-n1n used as sensor of gtp-bound rho in vitro and in vivo we determined the ability of the antibody to immunoprecipitate rhoa and rhob in their gtp-bound conformations. crude bacterial extracts expressing scfv c1-n1n was incubated with ni-nta beads to immobilize the antibody, and bacterial lysate containing recombinant rhoa or rhob preloaded with gdp or gtp were mixed with these beads. rhoa and rhob specifically bound to the scfv c1-n1n beads were analysed by western blot with commercial anti-rhoa and anti-rhob antibodies. the scfv c1-n1n on ni-nta beads selectively associated with the gtp-bound form of both rhoa and rhob, confirming the specificity of this new antibody. the ability of scfv c1-n1n to identify activated rho was next tested in vivo on activated hela cells by immunofluorescence. after serum deprivation, hela cells were activated hour with egf and serum and immunostained with scfvc1-n1n irrelevant scfv and anti-c-myc antibodies. we observed that staining with scfv c1-n1n, but not the irrelevant scfv, produced a positive immunofluoresence signal specifically in activated cells. to confirm the signal specificity, prior to staining, scfv c1-n1n was first incubated with gtpγs- preloaded rhoa to exhauste the selective scfv or gdp-preloaded rhoa which would have any effect on scfv activity. on serum starved cells, little signal was detected with either the scfv c1-n1n antibody preincubated with gdp-bound rhoa or gtpγs-bound rhoa, although rho a is detected by a commercial antibody. upon activation, a signal was visualized with the scfv c1-n1n preincubated with gdp-bound rhoa, showing labelling of the plasma membrane and punctate cytoplasmic structures consistent with previous reports for rho localization. similar labelling is obtained with commercial anti-rhoa. moreover, this signal was suppressed by preincubation of the scfv c1-n1n antibody with gtpγs-preloaded rhoa. this data strongly suggests that scfv c1-n1n is able to recognize endogenous activated and gtp-bound rho at the cellular level.  binding domains of effector proteins have proven to be useful as conformational sensors in analyzing the spatiotemporal activation of gtpases. in this study, we have shown that a phage display approach identified a specific phage for the gtp-bound form of rhoa, b and c. the expressed and purified scfvc1-n1n protein proceeded from this phage is able to recognize the three rho proteins loaded with gtpγs with great specificity, and detect activation of endogenous rho protein in fixed cells. this finding implicates the potential for scfv c1-n1n as a useful tool to compare the level of activated rho in cancer tissues versus normal tissues. 
after five rounds of phage selection using a constitutively activated mutant of rhob, three scfvs were selected for subsequent analysis. further biochemical characterization was pursued for the single clone, c exhibiting an scfv structure. although overexpression of rho gtpases has been correlated with tumorigenesis, only their gtp-bound forms are able to activate the signalling pathways implicated in tumorigenesis. c was selective for the gtp-bound form of rhoa, rhob, as well as rhoc, and failed to recognize gtp-loaded rac or cdc two other members of the rho family. we identified an antibody, c1-n1n specific for the gtp-bound form of rhob from a phage library, and confirmed its specificity towards gtp-bound rhoa and rhoc, as well as rhob. to enhance its production, soluble c was expressed in fusion with the n-terminal domain of phage protein piii, it appeared specifically associated with gtp-loaded recombinant rhoa and rhob via immunoprecipitation, and endogenous activated rho in hela cells as determined by immunofluorescence. the rho gtpases a, b and c proteins, members of the rho family whose activity is regulated by gdp/gtp cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. the aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable that recognizes the active, gtp-bound, form of rho gtpases and is able to discriminate it from the inactive, gdp-bound, rho in endogenous settings. however useful, these tools used to study the mechanism of rho activation still have limitations. the success of c1-n1n in discriminating activated rho in immunofluorescence studies implies that this new tool, in collaboration with currently used rhoa and b antibodies, has the potential to analyze rho activation in cell function and tumor development.
 the rho gtpases a, b and c proteins, members of the rho family whose activity is regulated by gdp/gtp cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. although overexpression of rho gtpases has been correlated with tumorigenesis, only their gtp-bound forms are able to activate the signalling pathways implicated in tumorigenesis. thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular gtp-bound rho, or determining the subcellular location of activation. however useful, these tools used to study the mechanism of rho activation still have limitations. the aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable that recognizes the active, gtp-bound, form of rho gtpases and is able to discriminate it from the inactive, gdp-bound, rho in endogenous settings.  after five rounds of phage selection using a constitutively activated mutant of rhob, three scfvs were selected for subsequent analysis. further biochemical characterization was pursued for the single clone, c exhibiting an scfv structure. c was selective for the gtp-bound form of rhoa, rhob, as well as rhoc, and failed to recognize gtp-loaded rac or cdc two other members of the rho family. to enhance its production, soluble c was expressed in fusion with the n-terminal domain of phage protein piii, it appeared specifically associated with gtp-loaded recombinant rhoa and rhob via immunoprecipitation, and endogenous activated rho in hela cells as determined by immunofluorescence.  we identified an antibody, c1-n1n specific for the gtp-bound form of rhob from a phage library, and confirmed its specificity towards gtp-bound rhoa and rhoc, as well as rhob. the success of c1-n1n in discriminating activated rho in immunofluorescence studies implies that this new tool, in collaboration with currently used rhoa and b antibodies, has the potential to analyze rho activation in cell function and tumor development. 