BACKGROUND
dna and protein microarrays have enabled the measurement of gene expression and biomarkers on an unprecedented scale, and have greatly affected fields ranging from oncology to medical diagnostics.
however, assaying biological events occurring on cell membranes, including signal transduction and intercellular communication, has proven to be considerably more difficult to industrialize in microarray format.
basic methods for reconstituting membrane proteins into phospholipid vesicles were developed quite a number of years ago  <cit> , facilitating fundamental discoveries in such diverse cellular processes as metabolism, solute transport, protein trafficking, and signal transduction.
in subsequent years, it was demonstrated that planar phospholipid bilayers could be generated simply by fusing these vesicles to various solid substrates , generating "supported membranes" which have lateral fluidity.
the characteristic fluidity, uniformity of lipid/protein orientation, and planarity make these supported membranes ideal for studying most membrane components, including receptors, transporters, channels, and adhesive factors.
alternatively, planar lipid bilayers can be deposited on surfaces derivatized with various polymeric films  <cit> .
regardless of how the planar membrane interacts with the solid support, display of these lipid bilayers represents a powerful way of developing technologies ranging from biosensors  <cit>  to platforms for the study of ion channel electrophysiology  <cit> .
given the increasing complexity of current basic research experiments and industrial drug discovery schemes, the ability to pattern arrays of lipid bilayers has become almost essential for performing multiplexed, high information-content assays.
to address this need, there have been a number of methods developed over the past few years to pattern arrays of lipid bilayers on solid supports for purposes of studying the biology of membranes and their component lipids and proteins  <cit> .
while these unique approaches all generate patterns of lipid bilayers on solid supports, they vary greatly in a number of important ways, including the manner of lipid bilayer display  and the means of generating the membrane pattern .
we have recently developed a technology for industrializing physiologically fluid supported membrane microarrays relying upon the discovery that fused silica, normally a compatible substrate for supported lipid bilayers, can readily be made membrane-incompatible through various lithographic procedures  <cit> .
this development led to the realization that fluid cell membranes could be isolated in independently-addressable corrals on a silicon chip through lithographic patterning.
in essence, microarrays consisting of spatially-indexed corrals of supported lipid bilayers can be generated at very high density   <cit> .
membrane corrals are regions of a solid substrate, in this case fused silica, which are compatible with lipid bilayer deposition and are isolated from each other by membrane-barriers composed of chrome.
in this manner, cell membranes are supported on a silicon chip with an aqueous interface between the membrane and solid support, as well as a bulk aqueous phase above the membrane  <cit> .
the isolated membranes in each corral of the array are physiologically fluid as confirmed by fluorescence recovery after photobleaching .
in this technique, excitation light shadows a small region of the corral, photobleaching the fluorescent molecules in that area.
following this excitation step, the photobleached region of the corral is examined for a recovery of fluorescence which is dependent upon the ability of fluorescent lipids in the surrounding membrane to diffuse into the photobleached area.
the extent of membrane fluidity determines the rate at which these fluorescent molecules diffuse into the photobleached area and diminish the shadow formed by the excitation step.
another technique used to measure the fluidity of supported lipid bilayers is the lateral migration of charged lipids in the presence of an electric field  <cit> .
given their aqueous surroundings, both upper and lower bilayers of the membrane exhibit a physiological level of fluidity.
the fluidity of our supported lipid bilayer arrays permits interactions among membrane components  on a physiological time scale, a phenomenon thought to underlie numerous signal transduction pathways.
this report demonstrates that the individually-corralled lipid bilayers can faithfully mimic the structural and functional characteristics of in vivo cell membranes, making them valuable for the study of biological processes ranging from simple ligand/receptor interactions to complex cell-cell signaling.
as mentioned previously, fluidity is a critical property of biological membranes in vivo.
according to biophysical models of membrane fluidity, there is a logarithmic dependence of the diffusion coefficient of a given molecule  on its radius, a phenomenon enabling large proteins embedded in the membrane to diffuse nearly as rapidly as lipid molecules  <cit> .
the fluidity dynamics are completely different for membranes interacting directly with solid substrates, where friction greatly attenuates the diffusion of larger molecules  in comparison to lipids  <cit> .
the characteristic fluidity of supported membranes depends entirely upon the manner in which the lipid bilayer is prepared and the type of solid substrate supporting the membrane.
in order to retain such a high level of fluidity, these membranes cannot be exposed to an air-water interface.
integrated membrane technologies, in which lipid bilayers are deposited on a self-assembled chemical monolayer coating the solid substrate instead of directly on the substrate itself, have sought to eliminate this drawback.
however, the increased convenience of coating the substrate with a chemical monolayer is offset by the observation that integrated membranes have considerably reduced fluidity  in comparison to supported lipid bilayers deposited directly on bare glass  <cit> .
consequently, the supported membranes described in this study display a level of fluidity more characteristic of the in vivo environment, a property likely to be critical for numerous cell biological events.
fluid membrane protein arrays are thus a powerful technology for displaying lipid bilayers which facilitates the study of cell surface processes.
planar synthetic membranes and their molecular components are readily displayed in a fluid and functional state characteristic of the in vivo environment.
while the basic principles of fluid bilayer arrays were invented in academia  <cit> , we have industrialized the technology to a level suitable for high throughput drug screening.
by combining lipid biochemistry with semiconductor microfabrication and robotic handling, membranes are arrayed at high density in discrete corrals on a silicon chip.
in order to complement the membrane array, we have also developed methods for efficiently expressing, purifying, and displaying the vast majority of membrane proteins in the genome.
taken together, the membrane array and membrane protein display technologies enable the study of biological processes occurring on cell membranes.
in this article, we report the validation of our membrane array technology for studies pertaining to ligand/receptor binding and live cell-based assays.
we initially characterized our supported lipid bilayers by displaying glycolipid molecules found in mammalian  and bacterial  cells.
we have chosen to study the interaction between ctb and the membrane ganglioside gm <dig>  cholera toxin, naturally secreted by the bacterium vibrio cholerae, is thought to exist as a hexamer consisting of two subunits in an ab <dig> configuration.
the pentameric ctb subunit is thought to bind up to five gm1gangliosides in a cooperative fashion  <cit> .
this multivalent interaction likely requires membrane fluidity for proper stoichiometric assembly of the ligand/receptor complex.
additionally, we demonstrate the successful display of various other membrane components, including lps from gram-negative bacteria and the mammalian proteins icam- <dig> and i-ek, molecules which are important drug targets for treating diseases ranging from septic shock in the case of the former to autoimmunity in the case of the latter.
gram-negative bacteria display lps in the outer membrane of their cell wall.
also referred to as endotoxin, lps consists of two distinct regions: a hydrophilic polysaccharide moiety and the highly conserved hydrophobic toxin lipid a  <cit> .
as lps is present only in bacterial membranes, it has been a target of continual interest to drug discovery programs aimed at developing more efficacious antibiotics.
our studies examining t cell adhesion and activation on membrane protein arrays displaying immunological proteins demonstrates the utility of our platform for biological assays.
in these t cell experiments, we have displayed proteins present on antigen presenting cells which are critical to generating an immunological response: the adhesion protein icam- <dig> and the class ii dimeric antigen-presentation molecule i-ek.
the ability to study complex multivalent ligand/receptor interactions and the engagement of live cells with our supported lipid bilayers could aid not only industrial drug screening against membrane targets, but also basic research studies in academia.
RESULTS
arraying high quality supported membranes
we sought to characterize the quality and precision of our fluid membrane arraying capabilities for industrial production.
the ctb/gm <dig> interaction afforded a system to study our supported lipid bilayers, as its pentavalent structure is likely to require a high degree of membrane fluidity for efficient complex formation.
as depicted in figure 2a, membranes containing unique lipid compositions  were arrayed at very high density in a manner which prevents mixing of contents from corral to corral.
moreover, the arrayed membranes were of high quality in terms of their physiological level of fluidity, as determined by frap measurements .
in order to demonstrate the precision of arraying membranes of varying compositions into specific corrals of an array, we used a pre-written program  to deposit  <dig> different concentrations of ganglioside gm <dig> on a chip in a manner so as to generate an arrow-shaped pattern .
gm <dig> was detected using fluorescence microscopy following an incubation with fluorescently-labeled ctb , a method we have previously used to investigate this ligand/receptor pair  <cit> .
high information-content binding of ctb to gm <dig> arrayed on supported membranes
given the ctb/gm <dig> binding illustrated in figure 3a, we next wished to quantify this ligand/receptor interaction.
figure 3b depicts a binding experiment performed using eight  <dig> ×  <dig>  supported membrane arrays displaying varying concentrations of gm <dig>  we used  <dig> different concentrations of gm <dig>  and  <dig> different concentrations of ctb .
hence, we performed a ligand binding experiment using  <dig> replicates for each concentration of ctb applied so as to obtain highly quantitative and information-rich binding data with very little reagent material required.
the fluid membranes were arrayed using a pre-written program consisting of an  <dig> ×  <dig> standard loop with  <dig> samples.
as in the case of the membranes in figure  <dig>  fluidity was confirmed using the frap technique.
figure 3b is a contour plot of the quantitation of the ctb/gm <dig> data, indicating the expected specific and saturable binding of the ligand to its receptor.
incorporation of diverse membrane components into supported fluid lipid bilayers and live cell assays
in order to further validate our system for industrial drug discovery and basic research applications, we have purified and displayed important molecules mediating microbial pathogenesis  and various immunological disorders .
lipid a, the principal endotoxic moiety of lps, was displayed in lipid bilayers and detected specifically by the anti-lipid a antibody , yielding an approximately 4-fold increase in immunoreactivity compared with membranes devoid of the endotoxin .
we next sought to extend our technology for assaying membrane biology important to the immune system.
the ectodomains of icam- <dig>  and i-ek  were over-expressed in mammalian cells, purified, and displayed in supported lipid bilayers through glycosylphosphatidylinositol  tethering .
given that these two proteins are particularly important in mediating the adhesion and activation of t cells on antigen-presenting cells, we examined these proteins for their ability to capture a transgenic murine t cell line  <cit>  specific for the pigeon cytochrome c  antigen.
figure 4a illustrates that surfaces displaying either icam- <dig> or i-ek-complexed to pcc as gpi-linked proteins mediate specific adhesion of t cells when displayed either alone or in combination, while membranes devoid of these proteins confer almost no adhesion.
moreover, in order to mimic a potential therapeutic intervention, we show that monoclonal antibodies specific for either icam- <dig> or i-ek nearly abolish t cell adhesion, while having little effect on the low level of non-specific adhesion to membranes devoid of protein .
in addition to measuring adhesion, the physiological level of fluidity of our supported membranes makes them ideal for assaying the full t cell activation process.
following adhesion of t cells to antigen-presenting cells, there is a dramatic relocalization of signaling molecules at the cell-cell interface known as the "immunological synapse," which is thought to be the most proximal marker of antigen-dependent t cell activation  <cit> .
upon initial encounter of the pcc-specific murine t cells with a membrane displaying fluorescently-labeled icam- <dig> and i-ek complexed to pcc peptide, t cells adhere and form an immature "synapse" characterized by icam- <dig> localized centrally and i-ek at the periphery .
during the next  <dig> minutes of t cell engagement, however, this "synaptic" pattern changes dynamically, as the peptide-loaded i-ek migrates centrally and icam- <dig> adopts a more peripheral localization .
in addition to measuring "immunological synapse" formation qualitatively as has been previously demonstrated  <cit> , we can also obtain quantitative information about this marker of t cell activation by measuring the clustering of cell surface molecules.
as presented in figure 4b, we have quantified the intensity of clustered i-ek to measure the strength of "synapse" formation and, hence, t cell activation.
as a control for quantitative measurement, we have compared the strength of "synapse" formation in the presence of i-ek complexed to a wild-type pcc peptide to that complexed to either no peptide or a mutated pcc* peptide which cannot elicit t cell expansion.
figure 4b demonstrates not only the capability of obtaining robust quantitative data for t cell activation using our assay system, but also the high degree of antigen-specificity recorded in the experiment.
discussion
we report here the industrialization of membrane protein microarrays suitable for basic research and drug discovery applications in numerous aspects of membrane biology, from multivalent ligand/receptor interactions to live cell assays.
through robotic handling approaches, it is possible to construct high density arrays of supported lipid bilayers containing unlimited unique combinations of membrane components.
the experiments illustrated in figure  <dig> and figure  <dig> underscore the physiological integrity of our membranes as determined by their in vivo-like fluidity, as well as the ability to array these membranes at high density without causing mixing of contents among corrals.
the high quality of the arrayed membranes is extremely reproducible between corrals and among different chips.
further, the precision of the arraying equipment facilitates deposition of unique membrane compositions in specific corrals of a membrane array.
the physiological fluidity of each discretely-arrayed bilayer is truly what enables the study of membrane processes, which often require the dynamic lateral motion of proteins and lipids.
in order to validate our system for studying membrane biology, we first examined the interaction between ctb and gm <dig>  a multivalent binding event likely to require fluidity of the bilayer for proper complex formation.
as illustrated in figure  <dig>  a vast amount of high content data can be obtained rapidly  for the ctb/gm <dig> system, or nearly any other ligand/receptor system, given the ease with which concentrations of ligand and receptor can be varied and analyzed.
this advantage would be extremely beneficial for investigating the pharmacodynamics of essentially any ligand against membrane components.
indeed, the experiment depicted in figure  <dig> was performed rapidly and only required  <dig> wells of a 96-well microtitre plate.
given the physiological level of fluidity in our supported membrane arrays, it is possible to study ligand/receptor interactions in a manner beyond capturing mere binding data.
actual signaling can be examined using our technology, given that the fluid and dynamic motion of membrane components is preserved.
we chose the ctb/gm <dig> interaction precisely for its multivalency, a property which may require fluidity of the surrounding lipid membrane environment for proper complex formation.
indeed, pentavalent ctb is thought to recruit five gm1molecules in a process that demonstrates positive cooperativity among the components  <cit> .
hence, the characteristic fluidity of our lipid bilayers is what permits the study of ctb/gm <dig> and likely numerous other membrane receptors on an almost in vivo-like level.
while the ctb/gm <dig> system is instructive for studying multivalent ligand/receptor interactions in the context of fluid membranes, we wanted to validate our technology for studying various other types of membrane components.
our ability to display bacterial lps could facilitate academic studies in the area of host/pathogen interaction, as well as lead to the identification of novel therapeutics to treat life-threatening septic shock.
while the polymyxin family of amphiphilic antibiotics target lps, there is a continuing push to develop more efficacious anti-endotoxin molecules to treat sepsis  <cit> .
for example, candidate antibody or small molecule therapeutics for septic shock could be identified from studying the interaction of immune system cells with membrane surfaces displaying lps.
in order to validate our technology for live cell-based assays, we have examined the activation of intact t cells on membrane arrays displaying molecules critical for adhesion and antigen-presentation.
as in the case of the ctb/gm <dig> interaction, we chose a cell-cell association  which would highlight the power of our fluid lipid bilayer technology to faithfully mimic in vivo membrane environments.
the adhesion of t cells to the supported membranes  models the first mechanistically-distinct stage of t cell activation, which happens to be an important point for therapeutic intervention in a number of immunological disorders.
indeed, raptiva, the recently-approved antibody therapeutic for psoriasis, directly targets the icam-1/lfa-1-mediated t cell adhesion process  <cit> .
in addition to cell adhesion, our ability to measure full t cell activation in both qualitative and quantitative formats underscores our ability to capture actual signal transduction events occurring on our fluid lipid bilayers, which enable the dynamic reorganization of surface receptors so crucial for this process.
while t cell adhesion and partial activation has been demonstrated on arrays consisting solely of mhc deposited on a glass surface without membranes or other molecules present, it is possible that this technology fails to capture important nuances of the activation pathway which may be critical in identifying novel membrane components capable of modulating these processes  <cit> .
in addition, drugs targeting the t cell activation process may have subtle effects which can be distinguished only through qualitative observation of the dynamic "immunological synapse."
studying t cell activation in array format could be quite useful in identifying the roles of various t cell or antigen-presenting cell membrane proteins in this process.
specifically, these proteins could be arrayed in the presence of antigen-presentation machinery in order to assess their precise roles in the distinct stages of t cell activation, including t cell adhesion, t cell stopping, and synapse formation.
in essence, activation of live t cells on our supported membranes greatly facilitates high information-content immunological assays.
CONCLUSIONS
our membrane protein microarray system extends the capabilities of dna and protein arrays.
while the latter have successfully industrialized the analysis of gene and protein expression, fluid cell membrane arrays represent the industrialization of membrane biology.
given that the vast majority of pharmaceuticals, including most of the recent protein- and antibody-based therapeutics, target membrane components, this system has the capability to advance the drug discovery process.
moreover, the technology is also useful for academic research applications, which will undoubtedly form the basis of many future therapeutic opportunities.
the combination of physiological in vivo-like qualities of our membranes with robotic arraying has created a powerful system for studying membrane biology in a high throughput fashion.

 dna and protein microarrays have enabled the measurement of gene expression and biomarkers on an unprecedented scale, and have greatly affected fields ranging from oncology to medical diagnostics. however, assaying biological events occurring on cell membranes, including signal transduction and intercellular communication, has proven to be considerably more difficult to industrialize in microarray format. basic methods for reconstituting membrane proteins into phospholipid vesicles were developed quite a number of years ago, facilitating fundamental discoveries in such diverse cellular processes as metabolism, solute transport, protein trafficking, and signal transduction. in subsequent years, it was demonstrated that planar phospholipid bilayers could be generated simply by fusing these vesicles to various solid substrates, generating "supported membranes" which have lateral fluidity. the characteristic fluidity, uniformity of lipid/protein orientation, and planarity make these supported membranes ideal for studying most membrane components, including receptors, transporters, channels, and adhesive factors. alternatively, planar lipid bilayers can be deposited on surfaces derivatized with various polymeric films. regardless of how the planar membrane interacts with the solid support, display of these lipid bilayers represents a powerful way of developing technologies ranging from biosensors to platforms for the study of ion channel electrophysiology. given the increasing complexity of current basic research experiments and industrial drug discovery schemes, the ability to pattern arrays of lipid bilayers has become almost essential for performing multiplexed, high information-content assays. to address this need, there have been a number of methods developed over the past few years to pattern arrays of lipid bilayers on solid supports for purposes of studying the biology of membranes and their component lipids and proteins. while these unique approaches all generate patterns of lipid bilayers on solid supports, they vary greatly in a number of important ways, including the manner of lipid bilayer display and the means of generating the membrane pattern. we have recently developed a technology for industrializing physiologically fluid supported membrane microarrays relying upon the discovery that fused silica, normally a compatible substrate for supported lipid bilayers, can readily be made membrane-incompatible through various lithographic procedures. this development led to the realization that fluid cell membranes could be isolated in independently-addressable corrals on a silicon chip through lithographic patterning. in essence, microarrays consisting of spatially-indexed corrals of supported lipid bilayers can be generated at very high density. membrane corrals are regions of a solid substrate, in this case fused silica, which are compatible with lipid bilayer deposition and are isolated from each other by membrane-barriers composed of chrome. in this manner, cell membranes are supported on a silicon chip with an aqueous interface between the membrane and solid support, as well as a bulk aqueous phase above the membrane. the isolated membranes in each corral of the array are physiologically fluid as confirmed by fluorescence recovery after photobleaching. in this technique, excitation light shadows a small region of the corral, photobleaching the fluorescent molecules in that area. following this excitation step, the photobleached region of the corral is examined for a recovery of fluorescence which is dependent upon the ability of fluorescent lipids in the surrounding membrane to diffuse into the photobleached area. the extent of membrane fluidity determines the rate at which these fluorescent molecules diffuse into the photobleached area and diminish the shadow formed by the excitation step. another technique used to measure the fluidity of supported lipid bilayers is the lateral migration of charged lipids in the presence of an electric field. given their aqueous surroundings, both upper and lower bilayers of the membrane exhibit a physiological level of fluidity. the fluidity of our supported lipid bilayer arrays permits interactions among membrane components on a physiological time scale, a phenomenon thought to underlie numerous signal transduction pathways. this report demonstrates that the individually-corralled lipid bilayers can faithfully mimic the structural and functional characteristics of in vivo cell membranes, making them valuable for the study of biological processes ranging from simple ligand/receptor interactions to complex cell-cell signaling. as mentioned previously, fluidity is a critical property of biological membranes in vivo. according to biophysical models of membrane fluidity, there is a logarithmic dependence of the diffusion coefficient of a given molecule on its radius, a phenomenon enabling large proteins embedded in the membrane to diffuse nearly as rapidly as lipid molecules. the fluidity dynamics are completely different for membranes interacting directly with solid substrates, where friction greatly attenuates the diffusion of larger molecules in comparison to lipids. the characteristic fluidity of supported membranes depends entirely upon the manner in which the lipid bilayer is prepared and the type of solid substrate supporting the membrane. in order to retain such a high level of fluidity, these membranes cannot be exposed to an air-water interface. integrated membrane technologies, in which lipid bilayers are deposited on a self-assembled chemical monolayer coating the solid substrate instead of directly on the substrate itself, have sought to eliminate this drawback. however, the increased convenience of coating the substrate with a chemical monolayer is offset by the observation that integrated membranes have considerably reduced fluidity in comparison to supported lipid bilayers deposited directly on bare glass. consequently, the supported membranes described in this study display a level of fluidity more characteristic of the in vivo environment, a property likely to be critical for numerous cell biological events. fluid membrane protein arrays are thus a powerful technology for displaying lipid bilayers which facilitates the study of cell surface processes. planar synthetic membranes and their molecular components are readily displayed in a fluid and functional state characteristic of the in vivo environment. while the basic principles of fluid bilayer arrays were invented in academia, we have industrialized the technology to a level suitable for high throughput drug screening. by combining lipid biochemistry with semiconductor microfabrication and robotic handling, membranes are arrayed at high density in discrete corrals on a silicon chip. in order to complement the membrane array, we have also developed methods for efficiently expressing, purifying, and displaying the vast majority of membrane proteins in the genome. taken together, the membrane array and membrane protein display technologies enable the study of biological processes occurring on cell membranes. in this article, we report the validation of our membrane array technology for studies pertaining to ligand/receptor binding and live cell-based assays. we initially characterized our supported lipid bilayers by displaying glycolipid molecules found in mammalian and bacterial cells. we have chosen to study the interaction between ctb and the membrane ganglioside gm cholera toxin, naturally secreted by the bacterium vibrio cholerae, is thought to exist as a hexamer consisting of two subunits in an ab configuration. the pentameric ctb subunit is thought to bind up to five gm1gangliosides in a cooperative fashion. this multivalent interaction likely requires membrane fluidity for proper stoichiometric assembly of the ligand/receptor complex. additionally, we demonstrate the successful display of various other membrane components, including lps from gram-negative bacteria and the mammalian proteins icam- and i-ek, molecules which are important drug targets for treating diseases ranging from septic shock in the case of the former to autoimmunity in the case of the latter. gram-negative bacteria display lps in the outer membrane of their cell wall. also referred to as endotoxin, lps consists of two distinct regions: a hydrophilic polysaccharide moiety and the highly conserved hydrophobic toxin lipid a. as lps is present only in bacterial membranes, it has been a target of continual interest to drug discovery programs aimed at developing more efficacious antibiotics. our studies examining t cell adhesion and activation on membrane protein arrays displaying immunological proteins demonstrates the utility of our platform for biological assays. in these t cell experiments, we have displayed proteins present on antigen presenting cells which are critical to generating an immunological response: the adhesion protein icam- and the class ii dimeric antigen-presentation molecule i-ek. the ability to study complex multivalent ligand/receptor interactions and the engagement of live cells with our supported lipid bilayers could aid not only industrial drug screening against membrane targets, but also basic research studies in academia.  arraying high quality supported membranes we sought to characterize the quality and precision of our fluid membrane arraying capabilities for industrial production. the ctb/gm interaction afforded a system to study our supported lipid bilayers, as its pentavalent structure is likely to require a high degree of membrane fluidity for efficient complex formation. as depicted in figure 2a, membranes containing unique lipid compositions were arrayed at very high density in a manner which prevents mixing of contents from corral to corral. moreover, the arrayed membranes were of high quality in terms of their physiological level of fluidity, as determined by frap measurements. in order to demonstrate the precision of arraying membranes of varying compositions into specific corrals of an array, we used a pre-written program to deposit different concentrations of ganglioside gm on a chip in a manner so as to generate an arrow-shaped pattern. gm was detected using fluorescence microscopy following an incubation with fluorescently-labeled ctb, a method we have previously used to investigate this ligand/receptor pair. high information-content binding of ctb to gm arrayed on supported membranes given the ctb/gm binding illustrated in figure 3a, we next wished to quantify this ligand/receptor interaction. figure 3b depicts a binding experiment performed using eight × supported membrane arrays displaying varying concentrations of gm we used different concentrations of gm and different concentrations of ctb. hence, we performed a ligand binding experiment using replicates for each concentration of ctb applied so as to obtain highly quantitative and information-rich binding data with very little reagent material required. the fluid membranes were arrayed using a pre-written program consisting of an × standard loop with samples. as in the case of the membranes in figure fluidity was confirmed using the frap technique. figure 3b is a contour plot of the quantitation of the ctb/gm data, indicating the expected specific and saturable binding of the ligand to its receptor. incorporation of diverse membrane components into supported fluid lipid bilayers and live cell assays in order to further validate our system for industrial drug discovery and basic research applications, we have purified and displayed important molecules mediating microbial pathogenesis and various immunological disorders. lipid a, the principal endotoxic moiety of lps, was displayed in lipid bilayers and detected specifically by the anti-lipid a antibody, yielding an approximately 4-fold increase in immunoreactivity compared with membranes devoid of the endotoxin. we next sought to extend our technology for assaying membrane biology important to the immune system. the ectodomains of icam- and i-ek were over-expressed in mammalian cells, purified, and displayed in supported lipid bilayers through glycosylphosphatidylinositol tethering. given that these two proteins are particularly important in mediating the adhesion and activation of t cells on antigen-presenting cells, we examined these proteins for their ability to capture a transgenic murine t cell line specific for the pigeon cytochrome c antigen. figure 4a illustrates that surfaces displaying either icam- or i-ek-complexed to pcc as gpi-linked proteins mediate specific adhesion of t cells when displayed either alone or in combination, while membranes devoid of these proteins confer almost no adhesion. moreover, in order to mimic a potential therapeutic intervention, we show that monoclonal antibodies specific for either icam- or i-ek nearly abolish t cell adhesion, while having little effect on the low level of non-specific adhesion to membranes devoid of protein. in addition to measuring adhesion, the physiological level of fluidity of our supported membranes makes them ideal for assaying the full t cell activation process. following adhesion of t cells to antigen-presenting cells, there is a dramatic relocalization of signaling molecules at the cell-cell interface known as the "immunological synapse," which is thought to be the most proximal marker of antigen-dependent t cell activation. upon initial encounter of the pcc-specific murine t cells with a membrane displaying fluorescently-labeled icam- and i-ek complexed to pcc peptide, t cells adhere and form an immature "synapse" characterized by icam- localized centrally and i-ek at the periphery. during the next minutes of t cell engagement, however, this "synaptic" pattern changes dynamically, as the peptide-loaded i-ek migrates centrally and icam- adopts a more peripheral localization. in addition to measuring "immunological synapse" formation qualitatively as has been previously demonstrated, we can also obtain quantitative information about this marker of t cell activation by measuring the clustering of cell surface molecules. as presented in figure 4b, we have quantified the intensity of clustered i-ek to measure the strength of "synapse" formation and, hence, t cell activation. as a control for quantitative measurement, we have compared the strength of "synapse" formation in the presence of i-ek complexed to a wild-type pcc peptide to that complexed to either no peptide or a mutated pcc* peptide which cannot elicit t cell expansion. figure 4b demonstrates not only the capability of obtaining robust quantitative data for t cell activation using our assay system, but also the high degree of antigen-specificity recorded in the experiment. discussion we report here the industrialization of membrane protein microarrays suitable for basic research and drug discovery applications in numerous aspects of membrane biology, from multivalent ligand/receptor interactions to live cell assays. through robotic handling approaches, it is possible to construct high density arrays of supported lipid bilayers containing unlimited unique combinations of membrane components. the experiments illustrated in figure and figure underscore the physiological integrity of our membranes as determined by their in vivo-like fluidity, as well as the ability to array these membranes at high density without causing mixing of contents among corrals. the high quality of the arrayed membranes is extremely reproducible between corrals and among different chips. further, the precision of the arraying equipment facilitates deposition of unique membrane compositions in specific corrals of a membrane array. the physiological fluidity of each discretely-arrayed bilayer is truly what enables the study of membrane processes, which often require the dynamic lateral motion of proteins and lipids. in order to validate our system for studying membrane biology, we first examined the interaction between ctb and gm a multivalent binding event likely to require fluidity of the bilayer for proper complex formation. as illustrated in figure a vast amount of high content data can be obtained rapidly for the ctb/gm system, or nearly any other ligand/receptor system, given the ease with which concentrations of ligand and receptor can be varied and analyzed. this advantage would be extremely beneficial for investigating the pharmacodynamics of essentially any ligand against membrane components. indeed, the experiment depicted in figure was performed rapidly and only required wells of a 96-well microtitre plate. given the physiological level of fluidity in our supported membrane arrays, it is possible to study ligand/receptor interactions in a manner beyond capturing mere binding data. actual signaling can be examined using our technology, given that the fluid and dynamic motion of membrane components is preserved. we chose the ctb/gm interaction precisely for its multivalency, a property which may require fluidity of the surrounding lipid membrane environment for proper complex formation. indeed, pentavalent ctb is thought to recruit five gm1molecules in a process that demonstrates positive cooperativity among the components. hence, the characteristic fluidity of our lipid bilayers is what permits the study of ctb/gm and likely numerous other membrane receptors on an almost in vivo-like level. while the ctb/gm system is instructive for studying multivalent ligand/receptor interactions in the context of fluid membranes, we wanted to validate our technology for studying various other types of membrane components. our ability to display bacterial lps could facilitate academic studies in the area of host/pathogen interaction, as well as lead to the identification of novel therapeutics to treat life-threatening septic shock. while the polymyxin family of amphiphilic antibiotics target lps, there is a continuing push to develop more efficacious anti-endotoxin molecules to treat sepsis. for example, candidate antibody or small molecule therapeutics for septic shock could be identified from studying the interaction of immune system cells with membrane surfaces displaying lps. in order to validate our technology for live cell-based assays, we have examined the activation of intact t cells on membrane arrays displaying molecules critical for adhesion and antigen-presentation. as in the case of the ctb/gm interaction, we chose a cell-cell association which would highlight the power of our fluid lipid bilayer technology to faithfully mimic in vivo membrane environments. the adhesion of t cells to the supported membranes models the first mechanistically-distinct stage of t cell activation, which happens to be an important point for therapeutic intervention in a number of immunological disorders. indeed, raptiva, the recently-approved antibody therapeutic for psoriasis, directly targets the icam-1/lfa-1-mediated t cell adhesion process. in addition to cell adhesion, our ability to measure full t cell activation in both qualitative and quantitative formats underscores our ability to capture actual signal transduction events occurring on our fluid lipid bilayers, which enable the dynamic reorganization of surface receptors so crucial for this process. while t cell adhesion and partial activation has been demonstrated on arrays consisting solely of mhc deposited on a glass surface without membranes or other molecules present, it is possible that this technology fails to capture important nuances of the activation pathway which may be critical in identifying novel membrane components capable of modulating these processes. in addition, drugs targeting the t cell activation process may have subtle effects which can be distinguished only through qualitative observation of the dynamic "immunological synapse." studying t cell activation in array format could be quite useful in identifying the roles of various t cell or antigen-presenting cell membrane proteins in this process. specifically, these proteins could be arrayed in the presence of antigen-presentation machinery in order to assess their precise roles in the distinct stages of t cell activation, including t cell adhesion, t cell stopping, and synapse formation. in essence, activation of live t cells on our supported membranes greatly facilitates high information-content immunological assays.  our membrane protein microarray system extends the capabilities of dna and protein arrays. while the latter have successfully industrialized the analysis of gene and protein expression, fluid cell membrane arrays represent the industrialization of membrane biology. given that the vast majority of pharmaceuticals, including most of the recent protein- and antibody-based therapeutics, target membrane components, this system has the capability to advance the drug discovery process. moreover, the technology is also useful for academic research applications, which will undoubtedly form the basis of many future therapeutic opportunities. the combination of physiological in vivo-like qualities of our membranes with robotic arraying has created a powerful system for studying membrane biology in a high throughput fashion. 
incorporation of transmembrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as interactions with live intact cells. in order to demonstrate the utility of our fluid membrane array technology to ligand/receptor studies, we investigated the multivalent binding of the cholera toxin b-subunit to the membrane ganglioside gm we have also displayed a number of bona fide drug targets, including bacterial endotoxin ) and membrane proteins important in t cell activation. we have demonstrated the applicability of our fluid cell membrane array technology to both academic research applications and industrial drug discovery. our technology facilitates the study of ligand/receptor interactions and cell-cell signaling, providing rich qualitative and quantitative information. by contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult. membrane array elements consist of a solid substrate, above which resides a fluid supported lipid bilayer containing biologically-active molecules of interest. the fluidity of these molecules in the planar lipid bilayer facilitates dimerization and other higher order interactions necessary for biological signaling events. microarray technology has been used extensively over the past years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. our robotically-arrayed supported membranes are physiologically fluid, a critical property which differentiates this technology from other previous membrane systems and makes it useful for studying cellular processes on an industrialized scale.
 microarray technology has been used extensively over the past years for assessing gene expression, and has facilitated precise genetic profiling of everything from tumors to small molecule drugs. by contrast, arraying cell membranes in a manner which preserves their ability to mediate biochemical processes has been considerably more difficult.  in this article, we describe a novel technology for generating cell membrane microarrays for performing high throughput biology. our robotically-arrayed supported membranes are physiologically fluid, a critical property which differentiates this technology from other previous membrane systems and makes it useful for studying cellular processes on an industrialized scale. membrane array elements consist of a solid substrate, above which resides a fluid supported lipid bilayer containing biologically-active molecules of interest. incorporation of transmembrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as interactions with live intact cells. the fluidity of these molecules in the planar lipid bilayer facilitates dimerization and other higher order interactions necessary for biological signaling events. in order to demonstrate the utility of our fluid membrane array technology to ligand/receptor studies, we investigated the multivalent binding of the cholera toxin b-subunit to the membrane ganglioside gm we have also displayed a number of bona fide drug targets, including bacterial endotoxin ) and membrane proteins important in t cell activation.  we have demonstrated the applicability of our fluid cell membrane array technology to both academic research applications and industrial drug discovery. our technology facilitates the study of ligand/receptor interactions and cell-cell signaling, providing rich qualitative and quantitative information. 