BACKGROUND
pathogenic bacteria tightly interact with their host, often exploiting adhesin-mediated engagement of eukaryotic surface receptors to trigger intracellular signaling events  <cit> .
as bacteria-induced responses are of critical importance during the initiation and progression of the infection, signaling processes in the host cell are usually studied in molecular detail.
both biochemical and genetic approaches have shed light on protein-protein interactions and signaling connections that occur in infected eukaryotic cells.
however, widely used biochemical approaches to investigate protein-protein interactions such as glutathione s-transferase -pull-down assays or coimmunoprecipitation from cell lysates have two major drawbacks: firstly, it is always possible that the two associated proteins are not directly interacting, but rather are linked by a third protein; secondly, biochemical approaches disrupt the cellular context and therefore lack spatial resolution.
similarly, genetic methods such as yeast two-hybrid screens, although applicable even in a high-throughput format, do not provide any information on where these processes occur under physiological conditions at the subcellular level.
by contrast, the introduction of green fluorescent protein  from aequorea victoria, has greatly facilitated the microscopic investigation of proteins in living cells.
though the use of gfp and its spectral variants allows the observation of colocalization of multiple proteins in real time, the resolution of light microscopes  is too low to prove a direct interaction of two colocalized putative binding partners.
therefore, there is a need for methods that combine the power of biochemical studies to pinpoint molecular interactions with the ability to study the subcellular context as provided by fluorescence microscopy.
within the last few years, the phenomenon of fluorescence resonance energy transfer , first described by förster in  <dig>  has garnered increasing interest as a method to address protein-protein interactions in the context of the cell  <cit> .
during fret, energy is transferred from a donor fluorophore in its excited state in a non-radiative way by dipole-dipole interactions to an acceptor molecule  <cit> .
the efficiency of fluorescence resonance energy transfer is defined by: e = 1/.
apparently, the efficiency of energy transfer depends on the sixth power of the distance 'r' separating the donor and the acceptor molecule.
therefore, fret only takes place to a significant extent if molecules are spaced within a few nm   <cit> .
the additional parameter 'r0', called the förster radius, is defined as the distance where efficiency of energy transfer from donor to acceptor is 50%.
r <dig> is fret-pair specific and is influenced by the spectral overlap of donor emission and acceptor excitation, the quantum yield of the donor, the absorption coefficient of the acceptor and the relative orientation of donor and acceptor.
as a consequence, fret is only likely to occur if two proteins labeled with a donor and an appropriate acceptor molecule are in direct contact.
to exploit this methodology in the study of pathogen-induced host cell signaling and to provide a general framework on how to approach fret analysis in the context of receptor-initiated signaling cascades, we have used the example of carcinoembryonic antigen-related cell adhesion molecule -mediated contact with the gram-negative pathogen neisseria gonorrhoeae.
over the last few years, our group and others have demonstrated that ceacam <dig>  a granulocyte-expressed member of this receptor family, functions as an opsonin-independent phagocytic receptor  <cit> .
ceacam <dig> recognizes colony opacity associated  proteins of n. gonorrhoeae  as well as additional outer membrane adhesins of other gram-negative bacteria and, upon binding of bacteria, initiates an intracellular signaling cascade  <cit> .
efficient uptake of ceacam3-bound bacteria depends on an immunoreceptor tyrosine-based activation motif -like sequence in the cytoplasmic part of the receptor, which is phosphorylated within minutes of receptor engagement  <cit> .
biochemical analyses have demonstrated that src homology  <dig>  domains of several signaling molecules, including the protein tyrosine kinases  c-src and hck, are able to bind to the tyrosine-phosphorylated cytoplasmic domain of ceacam <dig>  <cit> .
as well as the sh <dig> domain of src ptks, the sh <dig> domains of phosphatidylinositol- <dig> kinase, phospholipase cγ, and syk have also been found to colocalize with the receptor upon bacterial binding  <cit> .
however, it is unclear if this colocalization is indeed due to direct interaction of the respective signaling molecule with the phosphorylated receptor, or if there are additional molecules involved.
in the present investigation, we took advantage of a novel fret pair of fluorophores, enhanced cyan fluorescent protein  and enhanced yellow fluorescent protein , which have been recently developed by random mutagenesis and which display enhanced fret efficiency compared to the often used cyan fluorescent protein -yellow fluorescent protein  pair  <cit> .
tagging the cytoplasmic domain of ceacam <dig> with cypet and coexpressing a ypet-sh <dig> domain fusion protein , we observed fret in cell lysates and by flow cytometry in intact cells expressing phosphorylated ceacam3-cypet together with ypet-sh <dig> hck, but not with ypet-sh <dig> sh <dig> domain-containing leukocyte protein of  <dig> kda .
importantly, fret measurements in infected cells using acceptor bleaching revealed the direct interaction between ceacam <dig> and hck exclusively at sites of bacterial contact with host cell.
together, these data highlight the use of fret approaches to visualize cellular signaling in response to bacterial host cell contact and provide conclusive evidence for a direct interaction between ceacam <dig> and hck upon n. gonorrhoeae infection.
RESULTS
bacterial engagement of ceacam <dig> is accompanied by hck-sh <dig> recruitment
receptor engagement by bacterial pathogens is known to trigger intracellular signaling cascades in the infected eukaryotic cell.
our group and others have shown previously that src kinases are critical for ceacam3-mediated uptake of n. gonorrhoeae  <cit> .
biochemically, gst-pull-down assays have demonstrated the ability of the src kinase hck to interact with the phosphorylated cytoplasmic domain  of ceacam <dig>  to demonstrate the recruitment of hck to ceacam <dig> upon bacterial binding and to investigate the kinetics of this process, 293t cells were cotransfected with the cdna of hck-sh2-far-red fluorescent protein  together with ceacam <dig> wt-gfp or ceacam <dig> Δct-gfp, respectively .
at  <dig> days later, the cells were infected with opacea-expressing n. gonorrhoeae and imaged once per min for  <dig> h using confocal microscopy .
representative images of live cells during the infection process are shown in figure 1b.
whereas hck-sh <dig> strongly colocalizes with ceacam <dig> wt at sites of bacterial contact, the unligated receptor does not recruit the kinase sh <dig> domain.
in cells expressing ceacam <dig> Δct, a mutant form of the receptor that lacks the complete cytoplasmic domain and that is not phosphorylated upon bacterial infection, hck-sh <dig> is distributed evenly in the cytoplasm, even though the bacteria bind to the extracellular domain .
clearly, hck-sh <dig> recruitment to the bacteria-bound ceacam <dig> wt is transient .
within  <dig> to  <dig> min, the sh <dig> domain disappears from cell-associated bacteria suggesting that the ceacam3-initiated signaling complex is changing its composition during bacterial internalization.
it is also interesting to note that cells expressing ceacam <dig> wt seem to polarize with regard to bacterial uptake: hck-sh <dig> is recruited to one side of the cell, where efficient receptor clustering and internalization takes place.
if this represents direct binding of the hck-sh <dig> domain to the phosphorylated cytoplasmic domain of ceacam <dig> or if the recruitment of hck-sh <dig> is due to some other phosphoprotein, which is found in the vicinity of the receptor, remains unresolved.
however, the results clearly demonstrate that the hck sh <dig> domain is effectively recruited to ceacam3-enriched parts of the cell membrane upon receptor engagement by bacteria.
hck-sh <dig> binds to phosphorylated cytoplasmic domain of ceacam3
fret is a powerful tool to investigate protein-protein interactions.
however, the ability to observe fret depends on multiple parameters, including the proper spatial orientation of the fluorescent molecules, which is hard to predict even for known interaction partners.
in our case, the cypet tag was added to the c-terminus of ceacam <dig> that is in close proximity to the tyrosine residues of the itam-like sequence  <cit> , whereas the ypet tag was added to the n-terminus of the sh <dig> domain of hck .
to investigate, if the association of the hck sh <dig> domain with the phosphorylated cytoplasmic domain of ceacam <dig> can be monitored in the chosen configuration, we first used a simple in vitro fret assay.
therefore, 293t cells were cotransfected with ceacam <dig> wt-cypet and ypet-hck-sh <dig> together with or without v-src.
indeed, coexpression of v-src leads to constitutive tyrosine phosphorylation of ceacam <dig> in the absence of bacterial infection .
as a further control, we coexpressed ceacam <dig> Δct-cypet together with ypet-hck-sh <dig> in the presence of v-src.
cells were lysed  <dig> days after transfection, when equivalent amounts of the receptor or the hck sh <dig> domain were expressed in the different samples .
the resulting cell lysates were analyzed for fret using a spectrofluorometer.
importantly, fret signals were only detected in samples coexpressing ceacam <dig> wt together with ypet-hck-sh <dig> and v-src .
by contrast, no fret was detected in the absence of v-src under conditions where ceacam <dig> wt is not tyrosine phosphorylated.
for ceacam <dig> Δct, marginal fret was detected both in the presence or absence of v-src.
these results suggest that efficient fret between cypet and ypet occurs only upon interaction of tyrosine-phosphorylated ceacam <dig> wt with hck-sh <dig>  demonstrating that these two molecules are closely associated.
the method of measuring fret between two potential interaction partners in cell lysates is less labor intensive than other biochemical approaches such as gst-pull-down assays or coimmunoprecipitations.
nevertheless, the sensitized emission generated by fret in these samples cannot be observed directly.
this is mostly due to the fact that the signal in the fret channel is contaminated by spectral bleed through of donor emission and direct excitation of the acceptor at the excitation wavelength of the donor.
therefore, the signal has to be adjusted with specific correction factors derived from equivalent lysates containing either the donor or the acceptor only  <cit> .
the sensitized emission calculated by linear unmixing of the signal in the fret channel is further normalized to acceptor intensity to obtain fret efficiency that can be compared between different samples  <cit> .
to further assure that the calculated fret efficiency between ceacam <dig> wt-cypet and ypet-hck sh <dig> is due to an sh <dig> domain-mediated molecular interaction, we introduced an additional internal control.
in this regard, we took advantage of a recombinant glutathione s-transferase fusion protein of the c-src-sh <dig>  that has shown strong binding to phosphorylated ceacam <dig> wt in in vitro gst-pull-down assays  <cit> .
we reasoned that an excess of non-fluorescent gst-src sh <dig> added to the lysates should act as a specific competitive inhibitor displacing ypet-hck-sh <dig> from the phosphorylated ceacam <dig> wt.
in line with this assumption, fret between ceacam <dig> wt-cypet and ypet-hck sh <dig> was almost completely abolished upon addition of the c-src sh <dig> domain .
as a further control, similar amounts of gst or gst-src sh <dig> were added to lysates of cells expressing ceacam <dig> wt-cypet, ypet-hck sh <dig> and v-src.
only in the case of gst-src sh <dig> was a dramatic decrease of fret efficiency observed, whereas gst alone had no effect on fret .
these results demonstrate that fret observed in whole cell lysates is due to a specific interaction between the ypet-tagged sh <dig> domain of hck and the tyrosine-phosphorylated cytoplasmic domain of ceacam <dig> wt-cypet.
accordingly, we confirmed a tight binding between the sh <dig> domain of the src family protein tyrosine kinase  hck and ceacam <dig>
fret between ceacam <dig> and hck-sh <dig> occurs in intact cells
in cell lysates, the cellular context of membrane receptors and cytoplasmic signaling molecules is disrupted, potentially enabling interactions of protein partners that are not found in the same subcellular location.
to investigate if the intimate association between ceacam <dig> and hck also occurs in intact cells, 293t cells were cotransfected as above and the intact cells were analyzed by flow cytometry .
similar to the fret determination in cell lysates, the intensity in the fret channel is corrected for spectral bleedthrough and cross-excitation by using control cells expressing either fluorescent protein alone.
cells expressing both constructs were identified on the basis of cypet and ypet fluorescence intensity .
in the presence or absence of v-src, similar amounts of cypet-positive and ypet-positive cells were observed and the double-positive cell populations in the samples did not differ with regard to the cypet or ypet intensities measured .
therefore, double-positive cells  were gated and their fluorescence intensity recorded in the fret channel detecting ypet emission  during excitation of cypet  .
whereas only low fluorescence intensity was observed for cells transfected with ypet-hck-sh <dig> and ceacam <dig> wt-cypet in the absence of v-src, a strong increase in the fret signal was obtained for cells coexpressing ceacam <dig> wt-cypet and ypet-hck-sh <dig> together with v-src .
furthermore, cells expressing ceacam <dig> Δct together with ypet-hck-sh <dig> and v-src showed no increase in fluorescence intensity in the fret channel .
though a fourfold increase in the mean fluorescence intensity in the fret channel was observed in samples where ceacam <dig> wt-cypet, ypet hck sh <dig>  and v-src are coexpressed, the extent of fret might still be underestimated.
this could be due to the fact that not every gated cell positive for the donor and acceptor fluorophore also expresses v-src.
nevertheless, the flow cytometric analysis demonstrates intimate binding of hck-sh <dig> to the phosphorylated cytoplasmic domain of ceacam <dig> in intact cells.
this suggests that recruitment of the hck-sh <dig> domain to the sites of ceacam <dig> engagement by bacteria as observed by live cell microscopy might be linked to the direct binding of these two proteins.
fret acceptor bleaching measurements reveal direct association between hck-sh <dig> and ceacam <dig> at sites of bacterial contact
the preceding experiments convincingly showed that fret occurs between ceacam <dig> wt-cypet and ypet-hck-sh <dig> upon constitutive receptor phosphorylation by v-src.
however, it was still unclear if and where this tight association takes place upon bacterial infection in intact cells.
therefore, nih 3t <dig> cells were cotransfected with expression vectors for ceacam <dig> wt-cypet and ypet-hck-sh <dig>  at  <dig> days later, cells were infected with alexafluor647-nhs-labeled opacea-expressing n. gonorrhoeae for  <dig> min and fixed.
images were recorded in all three fluorescence channels.
in the region surrounding ceacam3-bound bacteria, the acceptor fluorophore  was photochemically destroyed with a short high-intensity laser pulse .
if fret occurs between donor and acceptor fluorophore, the destruction of the acceptor  should induce a significant increase in donor  intensity.
importantly, such an increase in cypet intensity upon laser bleaching of ypet can be observed exactly at the point, where bacteria were in contact with ceacam <dig> .
these results not only verify an intimate binding of hck-sh <dig> to ceacam <dig> in response to receptor stimulation, but also localize this interaction precisely to the sites of bacteria-host cell contact.
fret measurements based on sensitized emission have been recently used to detect phosphatidylinositol-3' kinase activation in response to bacterial infection  <cit> .
the approach was based on the simultaneous recruitment of differentially labeled pleckstrin homology  domains to the cell membrane upon generation of 3'-phosphoinositides.
however, it was not designed to reveal protein-protein interactions.
in our case, we employed a different methodology to microscopically detect fret by acceptor photobleaching.
in contrast to fret determination based on sensitized emission, which requires extensive controls to exclude artifacts arising from variable concentrations and stoichiometry of acceptor or donor fluorophores in transiently transfected cells, acceptor bleaching displays fret in a straightforward way.
this is due to the fact that acceptor bleaching causes a positive signal due to an increase of donor fluorescence intensity following photochemical destruction of the acceptor.
one reported caveat of acceptor photobleaching is the photoconversion of different yfp variants into a cfp-like species  <cit> .
however, the photoconverted yfp with cfp-like properties appears to get excited primarily at  <dig> nm and to a lesser extent at  <dig> nm as used in the current investigation.
in fact, we did not observe an increase of cypet fluorescence in bleached regions without ceacam3-bound bacteria .
furthermore, we bleached cells expressing only the ypet acceptor construct and did not observe an increase of the signal in the cypet channel upon excitation at  <dig> nm .
one clear advantage of acceptor bleaching is that it generates additional internal controls: first, donor intensity in regions with unbleached acceptor fluorophore should be unaffected; second, in bleached areas, where no protein-protein interactions take place, no alteration of donor fluorescence should be observed.
indeed, upon comparison of prebleaching and postbleaching pictures of the same region, an increase of donor intensity can only be observed where bacteria contact the host cell .
these results demonstrate that early host signaling in this situation is confined to the sites of tight pathogen binding to the receptor ceacam <dig>  it can be envisioned that such an approach would be also ideal to visualize and localize signaling events in response to other pathogenic bacteria.
in particular receptor-dependent protein-protein interactions in the infected host cell that are induced upon binding of pathogens to host integrins  <cit> , cadherins  <cit> , tetraspanins  <cit> , or proteoglycans  <cit>  could yield valuable insight into the spatial organization of host responses.
furthermore, it would be highly desirable to study the subcellular distribution and place of action of bacterially secreted effector molecules  <cit> .
this would require tagging of the secreted bacterial protein  and would allow the validation of biochemically determined protein-protein interactions between bacterial effector molecules and host factors in the context of the intact infected cell.
as is often the case in signaling pathways, the recruitment of hck-sh <dig> to ceacam <dig> in living cells was transient .
therefore, it would be of interest to analyze the kinetics of the hck-sh2-ceacam <dig> association by fret in live infected cells.
however, acceptor photobleaching destroys the acceptor fluorophore, preventing the continuous analysis of protein-protein interactions.
therefore, additional methodological approaches such as fluorescence lifetime imaging microscopy  would be required.
measuring fret according to the lifetime of the donor is independent of fluorophore concentrations and is non-destructive.
however, the hardware requirements restrict the use of flim to dedicated microscope facilities that might be cautious of introducing live bacterial pathogens.
this might limit the widespread use of flim in the realm of infection biology and makes the described approach of acceptor bleaching a valuable and feasible method to validate and subcellularly localize pathogen triggered signaling events in infected host cells.
CONCLUSIONS
elucidation of protein-protein interactions is of intense interest to understand signaling pathways in cells.
numerous biochemical and genetic approaches such as gst-pull-down assays, coimmunoprecipitation, yeast two-hybrid screens and protein microarrays are well established techniques for analyzing protein-protein interactions.
however, utilization of fret techniques not only allows the determination of intimate binding of two proteins, but also enables the verification of protein-protein interactions in the physiological context of the cell.
measuring fret by acceptor bleaching allowed us to localize the biochemically established interaction between phosphorylated ceacam <dig> and the src ptk hck to the sites of pathogen binding in infected host cells.
our results demonstrate that receptor-initiated signaling events are not only transient, as observed by the recruitment of the hck sh <dig> domain in living cells, but the pathogen-induced protein-protein interaction is also spatially confined within the infected cell.
therefore, this study refines our understanding of bacteria-induced signaling in eukaryotic cells and provides a rational framework to harness the potential of fret in other close encounters between specialized microbes or their translocated effectors and the host organism.

 pathogenic bacteria tightly interact with their host, often exploiting adhesin-mediated engagement of eukaryotic surface receptors to trigger intracellular signaling events. as bacteria-induced responses are of critical importance during the initiation and progression of the infection, signaling processes in the host cell are usually studied in molecular detail. both biochemical and genetic approaches have shed light on protein-protein interactions and signaling connections that occur in infected eukaryotic cells. however, widely used biochemical approaches to investigate protein-protein interactions such as glutathione s-transferase -pull-down assays or coimmunoprecipitation from cell lysates have two major drawbacks: firstly, it is always possible that the two associated proteins are not directly interacting, but rather are linked by a third protein; secondly, biochemical approaches disrupt the cellular context and therefore lack spatial resolution. similarly, genetic methods such as yeast two-hybrid screens, although applicable even in a high-throughput format, do not provide any information on where these processes occur under physiological conditions at the subcellular level. by contrast, the introduction of green fluorescent protein from aequorea victoria, has greatly facilitated the microscopic investigation of proteins in living cells. though the use of gfp and its spectral variants allows the observation of colocalization of multiple proteins in real time, the resolution of light microscopes is too low to prove a direct interaction of two colocalized putative binding partners. therefore, there is a need for methods that combine the power of biochemical studies to pinpoint molecular interactions with the ability to study the subcellular context as provided by fluorescence microscopy. within the last few years, the phenomenon of fluorescence resonance energy transfer, first described by förster in has garnered increasing interest as a method to address protein-protein interactions in the context of the cell. during fret, energy is transferred from a donor fluorophore in its excited state in a non-radiative way by dipole-dipole interactions to an acceptor molecule. the efficiency of fluorescence resonance energy transfer is defined by: e = 1/. apparently, the efficiency of energy transfer depends on the sixth power of the distance'r' separating the donor and the acceptor molecule. therefore, fret only takes place to a significant extent if molecules are spaced within a few nm. the additional parameter'r0', called the förster radius, is defined as the distance where efficiency of energy transfer from donor to acceptor is 50%. r is fret-pair specific and is influenced by the spectral overlap of donor emission and acceptor excitation, the quantum yield of the donor, the absorption coefficient of the acceptor and the relative orientation of donor and acceptor. as a consequence, fret is only likely to occur if two proteins labeled with a donor and an appropriate acceptor molecule are in direct contact. to exploit this methodology in the study of pathogen-induced host cell signaling and to provide a general framework on how to approach fret analysis in the context of receptor-initiated signaling cascades, we have used the example of carcinoembryonic antigen-related cell adhesion molecule -mediated contact with the gram-negative pathogen neisseria gonorrhoeae. over the last few years, our group and others have demonstrated that ceacam a granulocyte-expressed member of this receptor family, functions as an opsonin-independent phagocytic receptor. ceacam recognizes colony opacity associated proteins of n. gonorrhoeae as well as additional outer membrane adhesins of other gram-negative bacteria and, upon binding of bacteria, initiates an intracellular signaling cascade. efficient uptake of ceacam3-bound bacteria depends on an immunoreceptor tyrosine-based activation motif -like sequence in the cytoplasmic part of the receptor, which is phosphorylated within minutes of receptor engagement. biochemical analyses have demonstrated that src homology domains of several signaling molecules, including the protein tyrosine kinases c-src and hck, are able to bind to the tyrosine-phosphorylated cytoplasmic domain of ceacam. as well as the sh domain of src ptks, the sh domains of phosphatidylinositol- kinase, phospholipase cγ, and syk have also been found to colocalize with the receptor upon bacterial binding. however, it is unclear if this colocalization is indeed due to direct interaction of the respective signaling molecule with the phosphorylated receptor, or if there are additional molecules involved. in the present investigation, we took advantage of a novel fret pair of fluorophores, enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, which have been recently developed by random mutagenesis and which display enhanced fret efficiency compared to the often used cyan fluorescent protein -yellow fluorescent protein pair. tagging the cytoplasmic domain of ceacam with cypet and coexpressing a ypet-sh domain fusion protein, we observed fret in cell lysates and by flow cytometry in intact cells expressing phosphorylated ceacam3-cypet together with ypet-sh hck, but not with ypet-sh sh domain-containing leukocyte protein of kda. importantly, fret measurements in infected cells using acceptor bleaching revealed the direct interaction between ceacam and hck exclusively at sites of bacterial contact with host cell. together, these data highlight the use of fret approaches to visualize cellular signaling in response to bacterial host cell contact and provide conclusive evidence for a direct interaction between ceacam and hck upon n. gonorrhoeae infection.  bacterial engagement of ceacam is accompanied by hck-sh recruitment receptor engagement by bacterial pathogens is known to trigger intracellular signaling cascades in the infected eukaryotic cell. our group and others have shown previously that src kinases are critical for ceacam3-mediated uptake of n. gonorrhoeae. biochemically, gst-pull-down assays have demonstrated the ability of the src kinase hck to interact with the phosphorylated cytoplasmic domain of ceacam to demonstrate the recruitment of hck to ceacam upon bacterial binding and to investigate the kinetics of this process, 293t cells were cotransfected with the cdna of hck-sh2-far-red fluorescent protein together with ceacam wt-gfp or ceacam Δct-gfp, respectively. at days later, the cells were infected with opacea-expressing n. gonorrhoeae and imaged once per min for h using confocal microscopy. representative images of live cells during the infection process are shown in figure 1b. whereas hck-sh strongly colocalizes with ceacam wt at sites of bacterial contact, the unligated receptor does not recruit the kinase sh domain. in cells expressing ceacam Δct, a mutant form of the receptor that lacks the complete cytoplasmic domain and that is not phosphorylated upon bacterial infection, hck-sh is distributed evenly in the cytoplasm, even though the bacteria bind to the extracellular domain. clearly, hck-sh recruitment to the bacteria-bound ceacam wt is transient. within to min, the sh domain disappears from cell-associated bacteria suggesting that the ceacam3-initiated signaling complex is changing its composition during bacterial internalization. it is also interesting to note that cells expressing ceacam wt seem to polarize with regard to bacterial uptake: hck-sh is recruited to one side of the cell, where efficient receptor clustering and internalization takes place. if this represents direct binding of the hck-sh domain to the phosphorylated cytoplasmic domain of ceacam or if the recruitment of hck-sh is due to some other phosphoprotein, which is found in the vicinity of the receptor, remains unresolved. however, the results clearly demonstrate that the hck sh domain is effectively recruited to ceacam3-enriched parts of the cell membrane upon receptor engagement by bacteria. hck-sh binds to phosphorylated cytoplasmic domain of ceacam3 fret is a powerful tool to investigate protein-protein interactions. however, the ability to observe fret depends on multiple parameters, including the proper spatial orientation of the fluorescent molecules, which is hard to predict even for known interaction partners. in our case, the cypet tag was added to the c-terminus of ceacam that is in close proximity to the tyrosine residues of the itam-like sequence, whereas the ypet tag was added to the n-terminus of the sh domain of hck. to investigate, if the association of the hck sh domain with the phosphorylated cytoplasmic domain of ceacam can be monitored in the chosen configuration, we first used a simple in vitro fret assay. therefore, 293t cells were cotransfected with ceacam wt-cypet and ypet-hck-sh together with or without v-src. indeed, coexpression of v-src leads to constitutive tyrosine phosphorylation of ceacam in the absence of bacterial infection. as a further control, we coexpressed ceacam Δct-cypet together with ypet-hck-sh in the presence of v-src. cells were lysed days after transfection, when equivalent amounts of the receptor or the hck sh domain were expressed in the different samples. the resulting cell lysates were analyzed for fret using a spectrofluorometer. importantly, fret signals were only detected in samples coexpressing ceacam wt together with ypet-hck-sh and v-src. by contrast, no fret was detected in the absence of v-src under conditions where ceacam wt is not tyrosine phosphorylated. for ceacam Δct, marginal fret was detected both in the presence or absence of v-src. these results suggest that efficient fret between cypet and ypet occurs only upon interaction of tyrosine-phosphorylated ceacam wt with hck-sh demonstrating that these two molecules are closely associated. the method of measuring fret between two potential interaction partners in cell lysates is less labor intensive than other biochemical approaches such as gst-pull-down assays or coimmunoprecipitations. nevertheless, the sensitized emission generated by fret in these samples cannot be observed directly. this is mostly due to the fact that the signal in the fret channel is contaminated by spectral bleed through of donor emission and direct excitation of the acceptor at the excitation wavelength of the donor. therefore, the signal has to be adjusted with specific correction factors derived from equivalent lysates containing either the donor or the acceptor only. the sensitized emission calculated by linear unmixing of the signal in the fret channel is further normalized to acceptor intensity to obtain fret efficiency that can be compared between different samples. to further assure that the calculated fret efficiency between ceacam wt-cypet and ypet-hck sh is due to an sh domain-mediated molecular interaction, we introduced an additional internal control. in this regard, we took advantage of a recombinant glutathione s-transferase fusion protein of the c-src-sh that has shown strong binding to phosphorylated ceacam wt in in vitro gst-pull-down assays. we reasoned that an excess of non-fluorescent gst-src sh added to the lysates should act as a specific competitive inhibitor displacing ypet-hck-sh from the phosphorylated ceacam wt. in line with this assumption, fret between ceacam wt-cypet and ypet-hck sh was almost completely abolished upon addition of the c-src sh domain. as a further control, similar amounts of gst or gst-src sh were added to lysates of cells expressing ceacam wt-cypet, ypet-hck sh and v-src. only in the case of gst-src sh was a dramatic decrease of fret efficiency observed, whereas gst alone had no effect on fret. these results demonstrate that fret observed in whole cell lysates is due to a specific interaction between the ypet-tagged sh domain of hck and the tyrosine-phosphorylated cytoplasmic domain of ceacam wt-cypet. accordingly, we confirmed a tight binding between the sh domain of the src family protein tyrosine kinase hck and ceacam fret between ceacam and hck-sh occurs in intact cells in cell lysates, the cellular context of membrane receptors and cytoplasmic signaling molecules is disrupted, potentially enabling interactions of protein partners that are not found in the same subcellular location. to investigate if the intimate association between ceacam and hck also occurs in intact cells, 293t cells were cotransfected as above and the intact cells were analyzed by flow cytometry. similar to the fret determination in cell lysates, the intensity in the fret channel is corrected for spectral bleedthrough and cross-excitation by using control cells expressing either fluorescent protein alone. cells expressing both constructs were identified on the basis of cypet and ypet fluorescence intensity. in the presence or absence of v-src, similar amounts of cypet-positive and ypet-positive cells were observed and the double-positive cell populations in the samples did not differ with regard to the cypet or ypet intensities measured. therefore, double-positive cells were gated and their fluorescence intensity recorded in the fret channel detecting ypet emission during excitation of cypet. whereas only low fluorescence intensity was observed for cells transfected with ypet-hck-sh and ceacam wt-cypet in the absence of v-src, a strong increase in the fret signal was obtained for cells coexpressing ceacam wt-cypet and ypet-hck-sh together with v-src. furthermore, cells expressing ceacam Δct together with ypet-hck-sh and v-src showed no increase in fluorescence intensity in the fret channel. though a fourfold increase in the mean fluorescence intensity in the fret channel was observed in samples where ceacam wt-cypet, ypet hck sh and v-src are coexpressed, the extent of fret might still be underestimated. this could be due to the fact that not every gated cell positive for the donor and acceptor fluorophore also expresses v-src. nevertheless, the flow cytometric analysis demonstrates intimate binding of hck-sh to the phosphorylated cytoplasmic domain of ceacam in intact cells. this suggests that recruitment of the hck-sh domain to the sites of ceacam engagement by bacteria as observed by live cell microscopy might be linked to the direct binding of these two proteins. fret acceptor bleaching measurements reveal direct association between hck-sh and ceacam at sites of bacterial contact the preceding experiments convincingly showed that fret occurs between ceacam wt-cypet and ypet-hck-sh upon constitutive receptor phosphorylation by v-src. however, it was still unclear if and where this tight association takes place upon bacterial infection in intact cells. therefore, nih 3t cells were cotransfected with expression vectors for ceacam wt-cypet and ypet-hck-sh at days later, cells were infected with alexafluor647-nhs-labeled opacea-expressing n. gonorrhoeae for min and fixed. images were recorded in all three fluorescence channels. in the region surrounding ceacam3-bound bacteria, the acceptor fluorophore was photochemically destroyed with a short high-intensity laser pulse. if fret occurs between donor and acceptor fluorophore, the destruction of the acceptor should induce a significant increase in donor intensity. importantly, such an increase in cypet intensity upon laser bleaching of ypet can be observed exactly at the point, where bacteria were in contact with ceacam. these results not only verify an intimate binding of hck-sh to ceacam in response to receptor stimulation, but also localize this interaction precisely to the sites of bacteria-host cell contact. fret measurements based on sensitized emission have been recently used to detect phosphatidylinositol-3' kinase activation in response to bacterial infection. the approach was based on the simultaneous recruitment of differentially labeled pleckstrin homology domains to the cell membrane upon generation of 3'-phosphoinositides. however, it was not designed to reveal protein-protein interactions. in our case, we employed a different methodology to microscopically detect fret by acceptor photobleaching. in contrast to fret determination based on sensitized emission, which requires extensive controls to exclude artifacts arising from variable concentrations and stoichiometry of acceptor or donor fluorophores in transiently transfected cells, acceptor bleaching displays fret in a straightforward way. this is due to the fact that acceptor bleaching causes a positive signal due to an increase of donor fluorescence intensity following photochemical destruction of the acceptor. one reported caveat of acceptor photobleaching is the photoconversion of different yfp variants into a cfp-like species. however, the photoconverted yfp with cfp-like properties appears to get excited primarily at nm and to a lesser extent at nm as used in the current investigation. in fact, we did not observe an increase of cypet fluorescence in bleached regions without ceacam3-bound bacteria. furthermore, we bleached cells expressing only the ypet acceptor construct and did not observe an increase of the signal in the cypet channel upon excitation at nm. one clear advantage of acceptor bleaching is that it generates additional internal controls: first, donor intensity in regions with unbleached acceptor fluorophore should be unaffected; second, in bleached areas, where no protein-protein interactions take place, no alteration of donor fluorescence should be observed. indeed, upon comparison of prebleaching and postbleaching pictures of the same region, an increase of donor intensity can only be observed where bacteria contact the host cell. these results demonstrate that early host signaling in this situation is confined to the sites of tight pathogen binding to the receptor ceacam it can be envisioned that such an approach would be also ideal to visualize and localize signaling events in response to other pathogenic bacteria. in particular receptor-dependent protein-protein interactions in the infected host cell that are induced upon binding of pathogens to host integrins, cadherins, tetraspanins, or proteoglycans could yield valuable insight into the spatial organization of host responses. furthermore, it would be highly desirable to study the subcellular distribution and place of action of bacterially secreted effector molecules. this would require tagging of the secreted bacterial protein and would allow the validation of biochemically determined protein-protein interactions between bacterial effector molecules and host factors in the context of the intact infected cell. as is often the case in signaling pathways, the recruitment of hck-sh to ceacam in living cells was transient. therefore, it would be of interest to analyze the kinetics of the hck-sh2-ceacam association by fret in live infected cells. however, acceptor photobleaching destroys the acceptor fluorophore, preventing the continuous analysis of protein-protein interactions. therefore, additional methodological approaches such as fluorescence lifetime imaging microscopy would be required. measuring fret according to the lifetime of the donor is independent of fluorophore concentrations and is non-destructive. however, the hardware requirements restrict the use of flim to dedicated microscope facilities that might be cautious of introducing live bacterial pathogens. this might limit the widespread use of flim in the realm of infection biology and makes the described approach of acceptor bleaching a valuable and feasible method to validate and subcellularly localize pathogen triggered signaling events in infected host cells.  elucidation of protein-protein interactions is of intense interest to understand signaling pathways in cells. numerous biochemical and genetic approaches such as gst-pull-down assays, coimmunoprecipitation, yeast two-hybrid screens and protein microarrays are well established techniques for analyzing protein-protein interactions. however, utilization of fret techniques not only allows the determination of intimate binding of two proteins, but also enables the verification of protein-protein interactions in the physiological context of the cell. measuring fret by acceptor bleaching allowed us to localize the biochemically established interaction between phosphorylated ceacam and the src ptk hck to the sites of pathogen binding in infected host cells. our results demonstrate that receptor-initiated signaling events are not only transient, as observed by the recruitment of the hck sh domain in living cells, but the pathogen-induced protein-protein interaction is also spatially confined within the infected cell. therefore, this study refines our understanding of bacteria-induced signaling in eukaryotic cells and provides a rational framework to harness the potential of fret in other close encounters between specialized microbes or their translocated effectors and the host organism. 
within the eukaryotic cell, signaling complexes are spatially organized. however, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. fret measurements on the basis of whole cell lysates revealed intimate binding between hck-sh -hck-sh2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type ceacam and a flow cytometry-based fret approach verified this association in intact cells. live-cell microscopy revealed the transient recruitment of the src family tyrosine kinase hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule. fret-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell. here, we provide a methodological approach to exploit fluorescence resonance energy transfer to visualize pathogen-initiated signaling events in human cells. using confocal microscopy and acceptor photobleaching, fret between hck-sh and ceacam was localized to the sites of bacteria-host cell contact. these data demonstrate not only the intimate binding of the sh domain of hck to the tyrosine-phosphorylated cytoplasmic domain of ceacam in intact cells, but furthermore, fret measurements allow the subcellular localization of this process during bacterial infection. bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. in cells expressing a ceacam variant lacking the cytoplasmic domain, the src homology domain of hck was not recruited, even though bacteria still bound to the receptor.
 bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. within the eukaryotic cell, signaling complexes are spatially organized. however, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. here, we provide a methodological approach to exploit fluorescence resonance energy transfer to visualize pathogen-initiated signaling events in human cells.  live-cell microscopy revealed the transient recruitment of the src family tyrosine kinase hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule. in cells expressing a ceacam variant lacking the cytoplasmic domain, the src homology domain of hck was not recruited, even though bacteria still bound to the receptor. fret measurements on the basis of whole cell lysates revealed intimate binding between hck-sh -hck-sh2) and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type ceacam and a flow cytometry-based fret approach verified this association in intact cells. using confocal microscopy and acceptor photobleaching, fret between hck-sh and ceacam was localized to the sites of bacteria-host cell contact.  these data demonstrate not only the intimate binding of the sh domain of hck to the tyrosine-phosphorylated cytoplasmic domain of ceacam in intact cells, but furthermore, fret measurements allow the subcellular localization of this process during bacterial infection. fret-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell. 