Michael Caplan - US grants

The plasma membranes of polarized epithelial cells are divided into two distinct domains which manifest markedly different protein compositions. Among the proteins which are differentially distributed between these domains are members of the E1-E2 class of ion transporting ATPases. These ion pumps mediate numerous processes necessary for normal cell and tissue function, including the maintenance of osmotic balance, the generation of neural potentials, the renal and intestinal handling of solutes and the gastric secretion of acid. Alterations in the regulation of their activity have been implicated in the pathogenesis of numerous disease states. Two members of this family, the Na,K-ATPase and the H,K-ATPase, share a great deal of structural and mechanistic homology, yet differ dramatically in several important attributes. Thus, while the Na,K-ATPase is generally sorted to the epithelial basolateral membrane, the H,K-ATPase is directed to the apical surface. The two ion pumps also manifest distinct ion affinities, transport stoichiometries and inhibitor sensitivities. The research described in this proposal is designed to elucidate the molecular correlates of these differences. Our goal is to establish the structure-function relationships which determine the individual cell biologic and physiologic properties of these extremely important proteins. We will, therefore, undertake to 1) identify and characterize the sorting signals which are embedded in these proteins and are responsible for determining their subcellular distribution; 2) identify and characterize the cellular components which interpret and act upon these signals; and 3) map the protein determinants which bestow upon these ion transport ATPases their individual catalytic characteristic. These problems will be addressed using the cDNA clones encoding these pumps and the tools of genetic recombination and transfection. We will examine MDCK cells expressing the Na,K-ATPase, the H,K-ATPase or chimeric ion pumps created by genetically recombining portions of the two transport ATPases. The remarkable similarity relating the two parent proteins will greatly facilitate the construction of the relevant chimeras as well as the analysis of their properties. Observation of the subcellular distribution and ion transport reactions associated with the parent and chimeric pumps will allow us to assign sorting and transport functions to specific portions of the H,K-ATPase and Na,K-ATPase polypeptide chains. This system will be further exploited to identify protein components of MDCK cell which interact with these ion pumps in order to mediate their sorting. Use of the chimeras will allow us to establish the specificity of these associations and to analyze their role in membrane protein targeting.

This is a National Science Foundation Young Investigator Award. The goal of Dr. Caplan's laboratory is to identify the cellular machinery involved in the generation and maintenance of epithelial polarity. This will be done using the powerful tools of Drosophila genetics. A screening procedure already developed in the laboratory, together with P-element mediated mutagenesis, will be used to identify mutants carrying genetic alterations relevant to epithelial polarity. Genes identified through this process will be cloned and their protein products will be characterized. %%% This is a National Science Foundation Young Investigator Award. Dr. Caplan is interested in the fundamental and important question of how epithelial cells "know" how to maintain a distinct "up and down" polarity. This is a critical question, since the proper function of epithelial cells is dependent on this polarity (e.g., pumping of ions from one side of an epithelial cell layer to the other; secretion of hormones, enzymes, or mucous lubricants to the appropriate place; uptake of nutrients from the alimentary canal; etc.). Until now, cell biologists have addressed the question by direct observation and biochemical manipulation of epithelial cells. Dr. Caplan is developing a clever and novel approach to the problem by exploiting the powerful techniques of Drosophila genetics, which have already proven enormously useful in studies of development.

The plasma membranes of polarized epithelial cells are divided into two domains. The protein compositions of these domains are different, reflecting their physiologic functions. This compositional heterogeneity underlies the epithelial capacity to mediate vectorial solute transport. In order to catalyze transcellular fluxes against steep concentration gradients, epithelia must populate their apical and basolateral surfaces with distinct classes of transport proteins. The ability to spatially segregate transport activities is required for an enormous variety of homeostatic functions, an its impairment is implicated in numerous disease processes. The research described in this proposal will examine the mechanisms through which transport proteins are sorted to their appropriate domains. The studies outlined in this proposal are designed to identify epithelial sorting signals and to investigate their interaction with components of the sorting apparatus. Our strategy will take advantage of a family of ion transport proteins whose closely related members are localized in distinct sub-cellular compartments. The Na,K-ATPase and the H,K-ATPase belong to the E1-E2 class of ion pumps. The protein subunits which comprise these enzymes are highly homologous, yet the pump complexes are concentrated on different surfaces of epithelial plasma membranes and manifest different cell biologic properties. The two ATPases also differ in a number of interesting catalytic parameters. Both of these pumps subserve vital cellular and organismic functions, and both are involved in a number of clinically relevant conditions. Previous work in our laboratory has demonstrated that sorting mechanisms and structure-function relationships can be elucidated by studying the behavior of novel chimeric transport proteins. We will continue with this approach in order to: 1) identify narrowly defined domains of the H,K and Na,K-ATPase alpha- subunits which determine their parent pumps' subcellular distributions and enzymatic functions; and 2) identify narrowly defined sequence domains of the H,K and Na,K-Atpase beta-subunits which are important for the maturation, sorting and recycling of their parent pumps.Transporter chimeras will be expressed in polarized LLC-PK1 cells and their steady state distributions, dynamic properties and functional characteristics will be established. In this manner, it will be possible to identify sequence domains which determine the parent transport proteins' cell biologic and physiologic traits. Identification of these domains will provide insight into the mechanisms through which these proteins carry out their transport functions and will provide tools with which to probe the cellular machinery that governs their distribution and regulation.

DESCRIPTION: This proposal is focused on determining the mechanisms and interactions required for the differential localization of homologous members of the P-type ATPase family. Work outlined in this proposal stems from the observation that Na/K-ATPase is restricted to the basolateral surface of most epithelial cell types while the H/K-ATPase is present on apical surface of these cells. In addition, the H/K-ATPase also appears to be uniquely associated with a subapical population storage vesicles. The presence of this molecule in a storage compartment appears to be responsible for the stimulated secretion of gastric acid following the fusion of these vesicles with apical cell membranes while the sensation of acid secretion is associated with retrieval of the molecule from the cell surface. Results generated in the previous funding period have demonstrated that differential localization of each of the a subunits of these proteins appears to be determined by sequences found in each of the fourth transmembrane spanning sequences (TM4). In addition, studies in transgenic mice appear to have defined a tyrosine-based sorting signal in the cytoplasmic tail of the H/K-ATPase which is required for reinternalization of this pump following stimulation of gastric parietal cells. These studies establish the fact that the sorting signals embedded within both subunits play critical roles in regulating their trafficking. In the next period of funding, the sorting signals embedded in the H/K-ATPase and Na/K-ATPase a subunit will be further characterized following expression cells of molecules containing alterations in TM4. Initially, chimeras will be generated in which specific portions of TM4 of each protein are interchanged and, following expression in cultured polarized epithelial cells, the fate of chimeric molecules determined by immunoflorescence and surface biotinylation. Individual residues in each subdomain of the TM4 necessary for a proper sorting of each molecule will then be assessed by site-directed mutagenesis. Biochemical characterization of chimeras in which TM4 from the H/K-ATPase has been incorporated into the Na/K-ATPase show that the resulting protein is apparently able to transport both protons and sodium. It seems likely then that some or all of the residues in TM4 account for the proton transport specificity of the H/K-ATPase. To further define this property within the H/K-ATPase TM4 domain, all molecules developed for targeting studies will also be assessed for their role in determining the number and nature of actions transported by each pump. Tracer flux assays, intracellular pH measurements, ATPase assays, and electrophysiological assays of pump driven current will be used to assess the biochemical characteristics of each molecule. Experiments will also test whether the TM4 is sufficient to target molecules to either apical or basolateral surfaces when incorporated within the context of foreign molecules. Finally, similar experimental paradigms will be used to define sequences within the b subunit which are involved in the vectoral targeting and endocytosis of the H/K-ATPase. The second aim will investigate the molecular interactions defined by the TM4 domain of the a subunit and the tyrosine-based motif in the b subunit which are required for the proper molecular sorting of these molecules. In the case of TM4, experiments will focus on determining whether the sorting signal embedded in this domain interacts with specific lipids or proteins and subsequently determining whether these interactions are required for appropriate targeting. Parietal cell proteins which are responsible for mediating regulated internalization of the H/K-ATPase b subunit through interaction with tyrosine-based signals in the cytoplasmic tail will be identified through standard yeast two hybrid screens. Finally, the last aim will use a transgenic mouse model to determine whether domains in the H/K-ATPase a and b subunits responsible for appropriate targeting and recycling of this molecules are also acted upon in the context of the complex cell type in which they serve their critical physiological functions. Here, chimeric constructs created in relation to the goals of earlier specific aims will be expressed in transgenic mice lacking in endogenous H/K-ATPase and the localization, function, and pathology-associated mislocalization or misregulation of the chimeric proteins assessed in gastric parietal cells.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common cause of renal failure that can be attributed to a genetic mutation at a single locus. Two genes responsible for ADPKD have been identified and cloned. The PKD2 gene encodes polycystin-2, an integral membrane protein that has a molecular eight of 110 kDa and is predicted to span the membrane 6 times. In vitro experiments suggest that polycystin-2 forms homo- oligomers and can interact with polycystin-1, the product of the PKD1 gene. Polycystin-2 exhibits significant structural homology to a family of calcium channels, but nothing is yet known of its function nor of the structural mechanisms through which its mutation causes the dramatic pathology characteristic of ADPKD. The subcellular localization of polycystin-2 has not been established. The molecular signals and cellular mechanisms responsible for targeting polycystin-2 to its site of ultimate functional residence also remain to be determined. The dynamic regulation of the polycystin-2 protein in renal epithelial cells has been investigated, nor is it clear which domains of the protein participate in its proper functioning. We will define the cell biologic properties of polycystin-2 in situ and in heterologous expression systems. Toward this end we will: 1) examine the subcellular distribution of polycystin-2 in its native renal tubular epithelial cells by immunoelectron microscopy and determine the biochemical properties of he compartments in which it accumulates by subcellular fractionation; 2) identify and characterize renal epithelial cell proteins that interact with functionally important domains of the polycystin-2 protein; and 3) elucidate the molecular signals responsible for the sub-cellular distribution of polycystin-2 and identify the mechanisms through which these signals exert their effects. These studies will help to define the range of cellular processes in which polycystin-2 may participate. Investigating the structures with which polycystin-2 associates and elaborating the mechanisms which govern its cell biologic behavior should further our understanding of the pathways through which absent or aberrant polycystin- 2 protein induces disease.

Several members of the H,K-ATPase family of ion pumps participate in renal K transport. This class of P-type ATPases includes the gastric H,K-ATPase as well as a number of "non-gastric" H,K-ATPase isoforms. Physiologic studies suggest that these enzymes operate predominantly at the apical surfaces of tubule epithelial cells. While much has been learned about the patterns of K,K-ATPase isoform expression and its response to stress, the functional and cell biologic attributes of these pumps remain largely unelucidated. We have studied the properties which is responsible for the apical sorting of the gastric H,K-ATPase alpha-subunits, indicating that different mechanisms determine these molecules' polarized distributions. Our analysis of ion fluxes driven by a "non-gastric" H,K-ATPase isoform suggests that it exchanges Na (rather than H) for K under normal circumstances. Thus, the individual H, K-ATPase isoforms in situ are regulated by endocytosis, which is mediated by an endocytosis signal in the cytoplasmic tail of the gastric H,K-ATPase beta-subunit. Transgenic mice expressing a version of the protein in which the signal has been disabled exhibit constitutively active renal K resorption. The identities of the K,K-ATPase isoforms which are normally subject to endocytic regulation and the nature of the participating epithelial cell machinery have yet to be established. To further understand the processes which govern H,K-ATPase function in the kidney, we will: 1) identify the sorting signals which target these pumps to the appropriate surface domains of diverse renal epithelial cell types; 2) complete the functional characterization of a "non-gastric" H,K-ATPase in vitro and identify the individual transport processes driven by each pump isoform in situ and 3) examine the role of endocytosis in regulating renal H,K-ATPase activity in situ and establish the molecular interactions and cell biologic mechanisms through which a complex collection of ion pumps participate in the maintenance of systemic K balance.

The major goal of the imaging core is to facilitate the use of quantitative and qualitative imaging techniques in the analysis of transport physiology at the cellular level. New technologies are now available for greater resolution of localization of specific target proteins as well as permitting vital microscopy where dynamic changes can be identified. Paraffin section light microscopy has been expanded to include immunolocalization of specific proteins as well as the utilization of in situ hybridization and in situ reverse transcription for localization of specific mRNAs. The capability and resolution of immunofluorescence microscopy has been advanced by the introduction of confocal microscopy. In addition, confocal microscopy can be applied to vital tissues, and analyses of changes in intracellular ion concentration can be assessed at the tissue level using ion sensitive fluorochromes. Video imaging and analysis of intracellular ion concentrations using fluorochromes is now well established, and gives accurate quantitative information by using dual excitation/emission ratio analysis. The utilization of electron microscopic immunocytochemical techniques has also been improved for greater accuracy in localization using immunogold and in situ hybridization techniques. The value of all of these techniques has also been enhanced by the availability of computer driven image analysis systems. The imaging core will provide expertise and assistance in the utilization of light, immunofluorescence, confocal, atomic force and electron microscopy with particular emphasis on immunocytochemistry, vital microscopy and image analysis. The facility will give access to these specialized techniques and the equipment necessary to apply them and will form a focal point for collaboration between members of the program project.

The Na,K and gastric H,K pumps are members of the P-type family of ion transporting ATPases. While these two pumps share a great deal of structural and mechanistic homologies, they differ in their subcellular localizations, regulation, and substrate specificities. During the previous four year funding period we have identified domains in each of these molecules that account in part for these differences. We have found that motifs widely separated in these proteins' linear sequences collaborate to form conformationally-defined determinants that specify pump distribution and cation selectivity. Recently, the structure of the P-type sarcoplasmic reticulum Ca-ATPase has been solved at 2.6 Angstrom units resolution. The Na,K and H,K-ATPases are closely related to the Ca pump, and their structures are almost certain to reflect this homology. We will use the structure of the Ca-ATPase as a guide in generating novel chimeric polypeptides composed of complementary portions of the Na,K and H,K pumps. By assessing the sorting and catalytic properties of these chimeras, we will be able to identify the residues that comprise these determinants and to define the mechanism through which they are brought together in the tertiary structures of the pump proteins. We will also conduct two hybrid screens for proteins that interact with the Na,K and H,K pump polypeptides, using as baits portions of these proteins that correspond to autonomous units in the Ca-ATPase structure. Finally, we will utilize cell culture systems as well as newly generated knockout mouse models to assess the roles of pump domains and protein- protein interactions in regulating the function and distributions of ion pumps in situ. These studies will allow us to determine whether and how specific molecular signals and associations might be related to such clinically significant pathologies as gastric ulcer disease and hypertension.

DESCRIPTION (provided by applicant): This proposal takes a biomimetic approach to understanding how biomaterial composition and structure relate to the mechanical and integrin binding site environment experienced by contacted cells and then varies these parameters to elicit desired cell behaviors. Since basement membranes have wide relevance in the body (e.g., as a substrate for gingival epithelial cells), biomimetic basement membranes will be developed. The hypothesis that the elastic modulus of two-component collagen IV/laminin materials can be predicted from their molecular composition using the cellular solids model and visco-elastic theory and that their cell adhesion strength can be predicted from molecular composition through calculating the number of integrin bonds will be tested. A second hypothesis that it is possible to vary the membrane elastic modulus while holding the integrin bond number constant and vice versa, by variation of the laminin/collagen IV composition of the membrane, will also be tested. The Specific Aims are to assemble and characterize single-component membranes of collagen IV or laminin, assemble and characterize two-component membranes by co-assembling collagen IV and laminin, and finally systematically vary the mechanical modulus and integrin binding site density to achieve differences in cell migration behavior. Potential applications range from coatings for dental implants to artificial vascular grafts. In particular for dental applications, a bioactive material would be able to strengthen binding of the epithelial lining of the gingiva to the implant in order to exclude bacteria from the subepithelial gingival tissue. The candidate, having completed a PhD in the laboratories of Dr. Douglas Lauffenburger and Dr. Roger Kamm at MIT, has developed expertise in the field of self-assembling biomaterials that have potential use in medical implants. Current post-doctoral research in the laboratory of Dr. Harold Erickson at Duke University extends the candidate's knowledge into the self-assembly of naturally occurring extracellular matrix proteins. Having accepted a position as Assistant Professor at Arizona State University, the candidate's immediate goal is to lay a strong foundation for the learning environment and productivity of his laboratory. In the long term, this research will synergistically combine the candidate's expertise in the rational design of biomaterials and extracellular matrix self-assembly to produce bioactive materials.

There is growing evidence that the cell biologic and physiologic properties of renal ion transport systems are determined in part through their interactions with a variety of regulatory and structural proteins. We have demonstrated that renal ion transport polypeptides interact with tetraspan proteins, both in vitro and in situ. As their name implies, members of the tetraspan family are transmembrane proteins that are predicted to possess four membrane spanning segments. Genomic sequencing data suggest that mammals express about 30 tetraspan family members. All of these are characterized by short N and C terminal tails facing the cytoplasm and two relatively large extracellular loops in which are found a number of conserved residues that constitute a molecular signature present in most tetraspan family members. While much has been learned about the expression patterns of tetraspan proteins, less is known of their functions. It has been demonstrated that tetraspans form macromolecular complexes with a number of transmembrane proteins. These associations may be involved in regulating membrane protein distribution, stability and access to regulatory molecules. We find that interactions with tetraspans can exert tramatic effects on the subcellular localizations of renal transport proteins. Thus, interaction with the tetraspan protein CD63 induces the rapid endocytosis of the H,K-ATPase. In contrast, association with the tetraspan-like protein VIP17/MAL prevents the internalization of the aquaporin 2 water channel and increases its functional presence at the plasma membrane. We will examine the role that tetraspan proteins play in regulating the distribution and function of a number of important renal transport systems. Towards this end we will define the profile of tetraspan partners that associate with each of the individual members of a selected subset of renal ion transport proteins. We will assess the impact of these associations on the physiologic properties of these transport systems and we will define molecular domains of the tetraspan and transport proteins that participate in specific interactions. Finally, we will take advantage of knockout mouse models to measure the impact of tetraspan interactions on renal transport processes in situ.

Abstract not provided.

ADPKD is one of the most common autosomal genetic disorders, affecting approximately 1/1000 individuals. It is characterized by progressive renal cyst development, typically leading to end stage renal disease in late middle age. ADPKD is caused by mutations in the PKD1 or PKD2 genes, which encode the polycystin-1 (PC1) and polycystin-2 (PC2) proteins, respectively. PC1 is a membrane protein that may be involved in signaling from sites of cell-cell contact, while PC2 is a transmembrane protein that shares homology with calcium channels. These two proteins appear to participate in the same signaling pathway; however, their functions are largely unknown. A new signaling paradigm known as regulated intramembrane proteolysis (RIP) has been recently described. In this model, the cytoplasmic portion of a transmembrane receptor is released after ligand interaction and enters the nucleus, where it directly acts as a modulator of gene expression. During the previous funding period of this award we have found that PC1 undergoes a RIP-like proteolytic cleavage that releases its C-terminal tail (CTT), which enters the nucleus and initiates signaling processes. The cleavage occurs in vivo in association with alterations in mechanical stimuli. PC2 modulates the signaling properties of the PC1 CTT, and appears to serve as a cytoplasmic buffer that modulates the quantity of CTT available to enter the nucleus. In order to explore further the role that this cleavage plays in the normal functioning of the polycystin proteins and in the pathogenesis of ADPKD we will 1) identify the enzyme responsible for the release of the PC1 CTT; 2) identify the stimuli and signaling pathways that induce or prevent the cleavage and 3) identify the protein partners with which the CTT fragment interacts and define the collection of genes whose expression is altered through nuclear translocation of the CTT fragment. These studies will begin to unravel the relationship between the cleavage of the PC1 CTT and the pathogenesis of ADPKD, and may perhaps suggest new targets for therapeutic intervention.

DESCRIPTION (provided by applicant): Recent advances in the fields of polymer science, bioengineering, and genomics present an exciting opportunity for a synergistic and strategic approach to developing specific cancer therapies. The multi-disciplinary team of investigators assembled here brings the important contributions from each field together to create a novel and potentially powerful general strategy for targeting therapeutic molecules to cells overexpressing two cell surface receptors. The hypothesis tested is that multivalent ligands (many ligands bound to one polymer) can increase specificity of that construct for a target cell type by at least one order-of magnitude, while decreasing the required concentration of unbound ligand by at least one order-of magnitude. Further, it is hypothesized that, if one of the ligand types binds to an integrin, it will decrease cell adhesion strength thus preventing tumor cell migration. Combined with genomic testing to determine which cell surface receptors are highly expressed in specific pathological cell types, this two-ligand type strategy has potential to target almost any cellular pathology in which the expression of at least two cell surface receptors is up-regulated; and the specific aims in this study are intended to demonstrate proof-of principle for a model system based on this approach. Ligands for a receptor target determined by global gene expression data will be synthesized or isolated from recombinant production. These will be bound in combination to a co-polymer with side chains each containing one of two chemically reactive groups. A mathematical model of these multivalent ligands binding to cells will be developed, and the accuracy of the model will be tested by comparing to in vitro affinity and specificity experiments. The model's predictions will be implemented by systematically varying the number of each ligand type on the polymer construct to optimize specificity for cancer cells versus normal brain cells. Finally, in vitro cell culture assays for cell adhesion, cell migration, and apoptosis will determine the therapeutic potential of these constructs.

bioimaging /biomedical imaging; technology /technique development

DESCRIPTION (provided by applicant): The Na, K and H, K-ATPases are members of the P-type family of ion pumps and play critical roles in maintaining cellular homeostasis and transepithelial transport. The Na, K-ATPase creates ion gradients that maintain cellular osmotic balance and membrane potential. These gradients are exploited by transporting epithelia to drive the import and export of a wide range of solutes against steep concentration gradients. The H, K-ATPase is responsible for gastric acid secretion and also appears to play a role in renal potassium reabsorption. To carry out these functions, these pumps must be restricted to specific domains of the plasma membranes of polarized epithelial cells. The Na, K-ATPase resides at the basolateral surfaces of most polarized epithelial cell types. In the acid secreting parietal cells of the stomach the H, K-ATPase is stored in an intracellular vesicular compartment that fuses with apical plasma membrane in response to secretagogue stimulation. The activities of these ion pumps are tightly controlled through a variety of processes that regulate both their subcellular trafficking and their catalytic properties. In order to attain their appropriate subcellular distributions and to participate in their respective regulatory pathways, both pumps are likely to interact with a large number of accessory proteins. During the previous funding period we have identified a several novel partner polypeptides that appear to interact with these pumps to modulate their cell biologic and functional properties. We find that the Na,K-ATPase associates with both neurabin II/spinophilin and arrestin, suggesting that the sodium pump may be susceptible to regulatory mechanisms similar to those that govern G protein coupled receptor signaling. In addition, the Na, K and H, K-ATPase each form complexes with a member of the tetraspan family of transmembrane interacting proteins, and these associations profoundly influence pump trafficking. In the present proposal we will carry out studies designed to 1) determine the molecular and cell biologic mechanisms through which neurabin II/spinophilin and arrestin modulate the function of the Na, KATPase, 2) characterize the physiologic effects of interactions between ion pumps and tetraspans, and 3) examine the role of interacting proteins in governing ion pump function in vivo. These studies will allow us to define the physiological significance of these novel interactions, and to understand their involvement in governing pump function both under normal circumstances and in the context of pathological conditions.

Proposal Title: NIRT: Molecular Nanodevices for Creation of Smart, Adaptable Vaccine Delivery Vehicles
CTS-0609326
Principal Investigator: Tarek Fahmy
Institution: Yale University

This proposal was received in response to Nanoscale Science and Engineering Initiative,
NSF 05-610, category NIRT.

The proposed work, which will be conducted by an interdisciplinary team of established investigators (engineers, cell biologists, and immunologists), will lead to improvements in the state of the art in the preparation of a new generation of vaccine systems, and will provide the first systematic study of the interactions of polymer nanoparticles with both immune system cells such as dendritic cells and tissues in live animals. Our team has considerable expertise with each individual element of the proposed smart nanoparticle system; a dedicated effort in combining these elements will be the major goal of the proposal. The proposed research program will have broad impact because it will explore new methods for synthesis of modular nanoparticles, which may be useful in a wide variety of settings. By focusing on use of these new materials as vaccine vectors, the work will provide an improved understanding of the mechanisms of nanoparticle interaction with dendritic cells, and will therefore contribute understanding that can be applied to significant problems in world health. In addition, this work will lead to a better understanding and better design of materials needed to address important challenges in biology. Unlike approaches relying on the use of engineered recombinant antibodies, our strategy should produce a stable, easy to fabricate vaccine product that might be useful as an agent useful for the challenges posed by global health problems. This research team will approach an important technological problem in nanoscale science within the context of an educational and outreach program that seeks to train young scientists and engineers in a new area of interdisciplinary science that is of immediate societal relevance and public interest: vaccination against viral and biothreat agents. In addition, this novel nanoscale science project, which addresses a problem of biomedical significance, provides an opportunity to attract the attention of young people; the investigators will work with high school students and educators in the great New Haven area through well-established and successful outreach and education programs. This proposal addresses the following research and education theme listed in the program announcement: Biosystems at the Nanoscale.

[unreadable] DESCRIPTION (provided by applicant): This exploratory study will broaden the existing paradigm on which biomaterials science is based by demonstrating that the cell biology literature can be used to guide development of biomaterial surface modifications that can elicit desired cell behaviors. In this study we aim to identify the main factors important in regulation of endothelial cell recruitment of inflammatory cells. We hypothesize that these factors include the composition of the extracellular matrix (ECM) proteins to which the cells bind, the mechanical properties of the ECM substrate, and the shear stress imparted on the cells by fluid flow. In normal physiology, endothelial cells produce high levels of nitric oxide (NO) and low numbers of Intracellular Adhesion Molecule 1 (ICAM-1) and E-selectin molecules on their surfaces. ICAM-1, and E-selectin are important molecules in the initiation of inflammation because correponding molecules on neutrophils can bind to the endothelial lining of blood vessels through them, and low concentrations of NO lead to up-regulation of one of these. Reduction of inflammation is an important goal of biomaterials science as two of the most prevalent reasons for device failure are acute inflammation and chronic scarring. Our first aim will be accomplished by comparing the levels of NO, ICAM-1, and E-selectin produced by endothelial cells adhered to Matrigel, laminin-1, collagen IV, collagen III, collagen I, and fibronectin surfaces which have been created either by adsoption or gelation. Experiments will be performed under flow and static conditions. In the second aim, we will measure leukocyte adhesion to endothelial cells cultured on the same substrates also under flow and static conditions. The applications to which this study has direct applicability are vascular grafts and arteriovenous shunts which often lose patency by vascular wall thickening at the edges of the implant that occludes the vessel lumen and decreases blood flow. Eventually we will use the model system developed here to study the usual lack of correlation between in vitro results and in vivo efficacy of biomaterials. Here we use an experimental technique originally developed for intravital microscopy, and this will greatly facilitate such studies by allowing us to use identical experimental measurement techniques in vivo. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION, OVERALL (provided by applicant): [unreadable] The overall goal of this Program Project is to understand the mechanisms underlying renal fluid and [unreadable] electrolyte homeostasis and renal epithelial function in health and the processes that modulate these [unreadable] mechanisms in disease. A broad spectrum of techniques will be used to address a continuum of problems ranging from the molecular characterization of individual transport-related proteins to the contribution of these proteins to integrated renal function at the level of the intact tubule, the organ, and the whole animal. Our strategy to pursue these themes successfully will include close collaboration on interrelated research projects; sharing of expertise, concepts and techniques by Directors of the five individual Projects and the four research Cores; joint use of the research core facilities and single administrative core. The research projects comprise a broad range of experimental preparations including ion/solute transport proteins, transport regulatory proteins, transfected mammalian cells in tissue culture, isolated cell membrane vesicles, Xenopus oocyte expression system, isolated kidney cells and tubules, and whole kidney in vivo. We shall use a wide range of methods including molecular cloning and mutagenesis, functional cDNA expression, generation and use of transgenic mice, immune-cytochemistry, phosphopeptide enrichment coupled to mass spectral identification of phosphorylation sites, confocal microscopy, fluorometric assays of cell ion activities, whole cell clamp and patch- and giant patch-clamp techniques, in vivo and in vitro perfusion of defined tubule segments, and clearance studies. Each of the projects and cores is concerned with the central themes of the Program: to provide important new insights into individual transport proteins that play a role in renal electrolyte homeostasis, to elucidate the regulation of these transport-related proteins, to elucidate the cellular pathways involved in epithelial polarity that is an absolute prerequisite for vectorial solute and fluid transport, and to assess the relative contributions of these proteins to tubule and organ function under conditions of normal and deranged electrolyte and energy metabolism. Our strategy to pursue these themes successfully will include close collaboration on interrelated research projects; sharing of expertise, concepts and techniques by Program investigators; and joint use of core facilities. [unreadable] [unreadable] [unreadable]

Abstract The Na,K-ATPase, or sodium pump, generates the ion gradients responsible for most fluid and electrolyte transport processes in the kidney. In keeping with this central role in homeostasis, the function of the renal sodium pump is regulated by signals that contribute to the control of body fluid volume and blood pressure. To drive vectorial trans-epithelial transport, the Na,K-ATPase must be restricted to the basolateral surfaces of renal tubule epithelial cells. While a great deal has been learned about the structure and catalytic cycle of the sodium pump, much less is known about the partner proteins and trafficking pathways that determine its subcellular distribution and modulate its activity. During the previous 4 year funding period of this award we have identified new sodium pump interacting proteins and explored their roles in controlling its properties. We have also adapted a novel labeling methodology that allows us to investigate the attributes of temporally defined cohorts of Na,K-ATPase. The SNAP tag technique endows a protein of interest with the ability to become covalently coupled to fluorophores or biochemically useful ligands. We have generated a SNAP- tagged Na,K-ATPase [unreadable]-subunit fusion protein and expressed it in MDCK renal epithelial cells. The SNAP tagged [unreadable]-subunit assembles with Na,K-ATPase [unreadable]-subunit to form functionally competent pumps that are sorted appropriately. We have developed pulse-chase protocols with which we can observe directly the trafficking itinerary pursued by newly synthesized Na,K-ATPase and isolate newly synthesized Na,K-ATPase in association with its collections of partner proteins. These efforts have already yielded surprising new insights into the mechanisms through which the pump is assembled and transported through epithelial cells. We will expand our analysis to: 1) define the pathways and parameters that govern the trafficking of newly synthesized Na,K-ATPase in polarized renal epithelial cells;2) identify and characterize the protein partners that interact with a temporally-defined cohort of Na,K-ATPase at each stage of its post-synthetic life span;and 3) establish the pathways and partners that regulate Na,K-ATPase sorting and function in the epithelial cells of each of the segments of the nephron in situ. This approach permits us to ask entirely new and previously experimentally inaccessible questions about the biology of the sodium pump, and will yield valuable insights into the cell biological and biochemical factors that govern this physiologically critical ion transport system.

The principal goal of the Center for Polycystic Kidney Disease Research at Yale is to facilitate translational research that will advance the understanding and treatment of polycystic kidney diseases. The Center will provide investigators both at Yale and across the country with access to highly specialized services and research tools not otherwise routinely available to support their research. Three Center Cores will be established whose specific objectives are to 1) generate, maintain and provide small animal models of polycystic kidney disease, 2) provide detailed characterizations of the proteomic profiles of urine specimens derived from these mouse models during the course both of disease progression and of experimental therapies, and 3) offer polycystic kidney disease researchers access to advanced physiological tools, including characterization of ion channel activities and state of the art imaging facilities, with which to explore the cellular and molecular basis of polycystic kidney disease. We have identified a research user base of 31 investigators, including 11 at outside institutions, who have expressed specific interest in using core services of the Polycystic Kidney Disease Center at Yale. A Pilot and Feasibility Program will be established to provide initial project funding for young investigators, to attract new investigators into the field of polycystic kidney disease research, and to foster basic and translational studies directly related to polycystic kidney diseases. In addition, an Enrichment Program will be established to promote interdisciplinary interactions and collaborations among investigators participating in the Center;facilitate the application of new technologies to polycystic kidney disease research;and provide an online index of resources developed in the Center (e.g. mouse and zebrafish models and urine proteomes) so that they can be made available to polycystic kidney disease investigators both at Yale and at other institutions. The Center will also include a Research Training Program to enhance the training of graduate students, medical students, and postdoctoral fellows in polycystic kidney disease research in general, and in the specialized models and methodologies provided by the Center Cores in particular.

DESCRIPTION (provided by applicant): In 2006 Yale started the innovative Medical Research Scholars Program (MRSP) with the mission of providing broad and deep Ph.D. training in the core basic science concepts of molecular biology, cell biology, biochemistry, genetics, physiology, pharmacology, and pathology, integrated with intensive exposure to medically oriented coursework and mentored clinical experiences designed specifically for Ph.D. students. The ultimate goal of this training is to prepare our students to be future interdisciplinary leader in the biomedical sciences, with a unique ability to pursue clinically relevant fundamental basic science inquiry aimed at elucidating the molecular mechanisms of disease. The MRSP has been supported by a combination of institutional funds and funds provided by the Howard Hughes Medical Institute (HHMI). The combination of substantial unmet demand from highly qualified students for admission (warranting expansion of the size of MRSP), the termination of HHMI support after the 2013-2014 academic year, and the desire to continue this highly successful ongoing program constitute the rationale for our Molecular Medicine (MM) Training Grant application. MRSP students participate in a variety of program activities designed to engage Ph.D. students in medically relevant training and experiences throughout their graduate studies. The classes focus first on normal human physiology, organ-based cell biology, and biostatistics, followed by human pathobiology and an introduction to drug discovery, validation, and clinical trials. MRSP students also have the option to participate with first-year Yale MD students in weekly small-group physiology case conference tutorials, in which they explore in depth the physiological underpinnings of particular disease states. The cornerstone of the MRSP is a two-year mentored clinical experience that provides students with in-depth exposure to the science behind human diseases and first-hand longitudinal encounters with patients to contextualize the mechanistic basis of disease in a way that is not possible through traditional classroom learning. In addition, MRSP students participate in a special research-in-progress series held jointly with the Yale Center for Clinical Investigation (YCCI)-an NIH-funded center that forms the nucleus for translational and mechanistic disease-focused research at Yale-and as the program grows we plan to incorporate an Annual Retreat (also in conjunction with the YCCI). In addition to these formal activities, the MRSP also provides ongoing mentoring to all MRSP trainees regarding selection of a thesis research laboratory and assembly of the dissertation committee to support the goal of the NIGMS Molecular Medicine program to provide training in the basic biomedical sciences with a focus on elucidating the mechanisms of human disease. Accordingly, we request funds for this MM Training Grant to support a total of five MRSP Ph.D. students per class year, with five second-year and five third-year students being supported at any given time (all first-year students are fully supported by institutional funds), with the expectation that MRSP students will complete the Ph.D. within six years.

Renal epithelial cell plasma membranes are divided into two domains whose distinct biochemical compositions reflect their individual roles. This polarity is required for vectorial solute and fluid transport in the kidney. To achieve this asymmetry, an epithelial cell must be able to establish distinct surface domains and to maintain these domains' distinct identities. The generation and maintenance of epithelial polarity is dependent upon the formation of contacts between neighboring cells, which initiates signals that activate the assembly of intercellular junctions and their associated polarization machinery. We and others have shown that adenosine monophosphate-stimulated protein kinase (AMPK) plays a key role in the formation of intercellular junctions and in the establishment of polarity. AMPK is a cellular energy sensor activated by intracellular ATP depletion that helps to protect tissues from the consequences of energy deprivation. AMPK governs numerous pathways that modulate cell growth and metabolism. Extracellular calcium plays a critical and complex role in inducing junction assembly, and we find that the calcium sensing receptor (CaSR) contributes to calcium- dependent junction assembly. We have also found that initiation of junction formation leads to inhibition of the GSK3? kinase, which regulates a number of cell differentiation and polarization signaling pathways. We wish to understand the roles that AMPK, GSK3?, the CaSR and other regulatory molecules play in the establishment and preservation of epithelial polarity under normal circumstances and in the face of pathological perturbations such as energy deprivation. We propose to determine 1) how modulation of the activities of AMPK, GSK3? and the CaSR initiate tight junction formation and influence tight junction protein expression; 2) how the AMPK, GSK3? and CaSR pathways influence established junctions in intact epithelia; and 3) how loss of AMPK activity affects renal function and the susceptibility of renal tubular epithelial cells to ischemic damage. Through these studies, we will identify new regulatory targets and control points in the complex process of epithelial polarization. We will also invesigate cellular pathways involved in acute kidney injury and possible therapeutic interventions that may mitigate its severity.

? DESCRIPTION (provided by applicant): Development of polarized plasma membrane domains is a prerequisite for renal epithelial function and its perturbation contributes to a variey of pathologies. Thus, elucidating the mechanisms through which polarity is generated and maintained is fundamental to developing a thorough understanding of renal physiology. Epithelial cells avail themselves of a complex sorting apparatus to create and maintain the compositional integrity of their plasma membrane domains. During the previous funding period of this award we have employed new approaches that have illuminated aspects of the sorting and signaling systems involved in generating and maintaining renal epithelial cell polarity. Our previous efforts focused in part upon the sorting and trafficking properties of the Na,K-ATPase, the basolateral ion pump that generates the driving force for the vast majority of renal fluid and electrolyte transport. A number of recent studies suggest that the Na,K-ATPase is not simply an occupant of the basolateral cell surface, but is in addition an active participant in the intercelllar communication and signaling pathways that establish the composition and structure of the basolateral plasma membrane domain. In the present application we propose to apply innovative tools to investigate further the pathways and partners involved in the sorting and delivery of apical and basolateral membrane proteins. We will carry out studies that explore novel hypotheses relating to both the cellular mechanisms responsible for epithelial polarity and to their physiological implications. Towards this end we will 1) Define pathways pursued by newly synthesized proteins as they travel to the apical and basolateral domains of the plasma membrane; 2) Determine how sorting and trafficking behaviors vary among renal epithelial cell types and during the course of renal development; and 3) Identify and characterize temporal and polarity-dependent interactomes. Through this work we will develop new insights into the mechanisms that mediate membrane protein trafficking in renal epithelial cells and that therefore determine the physiological properties of the renal tubule.

Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic diseases, affecting >1:1000 individuals worldwide. It is characterized by large fluid-filled renal cysts that remodel, compress and destroy surrounding normal tissue, and that progressively reduce kidney function, leading to end stage renal disease in about 50% of patients by the sixth decade of life. Most ADPKD results from mutations in two genes, PKD1, which encodes the polycystin-1 protein (PC1), and PKD2, which encodes the polycystin-2 protein (PC2). PC1 and PC2 interact with one another and are thought to play a role in cilia signaling. It is generally accepted that the cilium is a central component in the pathways that drive ADPKD pathogenesis. Although their mechanistic connection to the functional PC complex in cilia is unclear, numerous signaling pathways are perturbed in cysts. In the past few years the list of disease-related pathways has grown through new evidence that implicates metabolism as a novel pathway that is profoundly affected in ADPKD and that may both participate in disease pathogenesis and serve as a target for therapeutic development. While it remains to be established whether the newly-identified metabolic derangements that characterize ADPKD are direct drivers of cyst formation, it is clear that the nature and activities of a cell's many and varied intertwined metabolic circuits plays a central role in determining its capacity to invest the energy required in order to participate in the proliferation and active solute and fluid transport that are required for cyst growth. The main goal of this proposal is to provide the research community with novel tools and data sets that will substantially enhance efforts to explore and exploit the metabolic changes that characterize ADPKD. We will produce a uniquely designed and rigorously curated resource based upon novel in vivo models of the cell specific transcriptomic, mitochondrial proteomic and mitochondrial metabolic effects that result from the earliest stages after loss of the PC proteins and that are further informed by the effects of concomitant cilia loss and PC protein reactivation. This program will make use of adult inducible conditional PC knockout mouse models and will employ strategies that will permit conditional isolation of ribosomes (TRAP) and conditional isolation of mitochondria, thus enabling cell-type- specific transcriptomic and mitochondrial proteomic and metabolomic studies. State of the art in vivo metabolic flux studies will be applied to the kidney cortices of these novel genetic mouse models. The results of these analyses will be combined to produce robust biological data sets that will be assembled through application of the requisite informatics mechanisms in order to disseminate these data to the broader research community in near real time. Critically, our in vivo studies are designed to discover the earliest changes that occur after kidney tubules lose polycystin protein expression?at time points well before cysts form. The research team brings together extensive and complementary expertise in ADPKD animal models, PC signaling and biology and in vivo metabolic studies coupled with strong biostatistical and bioinformatics support to produce the proposed resource.

Each of the cell types of the nephron possesses segment-specific structural and biochemical specializations that both reflect and define its physiological properties. Nephron transport requires the asymmetric apportioning of ion transport proteins among the apical and basolateral plasma membrane domains of tubule epithelial cells. Polarized plasma membrane domains are a prerequisite for normal renal function, and their perturbation contributes to a variety of pathologies. To accommodate transport proteins, renal epithelial cells sculpt their plasma membranes into organized domains whose designs are exquisitely well suited to their physiological occupations. Furthermore, the organelles that populate renal epithelial cells participate in contacts and communication pathways through which they help to meet the metabolic, structural and biosynthetic demands imposed by renal transport. Elucidating the network of interactions that generate and maintain renal epithelial cell structure is central to understanding renal physiology. The past several years have seen the emergence of completely new understandings of the mechanisms through which the membranes of subcellular organelles communicate with one another and with the molecules that govern vesicular transport. A wide variety of membrane contact sites have been identified that not only tether subcellular structures to one another but also mediate inter-organelle communication and exchange. The inositol phospholipid compositions of organelle membranes and of sub-domains of the plasmalemma help to establish the identities of these membranes and define their interactions with the cellular sorting and trafficking machinery. While the roles of these contact sites and of the segregated distributions of inositol phospholipids has been established extensively in cultured cell systems, very little is known of their physiologic functions in epithelial cells that line renal tubules in situ. We hypothesize that the unique architecture of renal epithelial cells is predicated upon contact-mediated and inositol phospholipid-mediated communication among subcellular compartments. The studies outlined in the present proposal will explore the mechanisms through which renal tubule epithelial cell plasma membranes acquire and modulate some of their characteristic adaptations. The hypothesis that motivates these studies is that renal epithelial cell structure and function are predicated upon networks of protein-protein interactions and signaling pathways that collaborate with one another to communicate and respond to environmental cues. To explore this hypothesis and its implications in depth we will: 1) Define the nature, localization and ontogeny of membrane contact sites in renal epithelial cells; 2) Define the biochemical compositions of contact sites in the kidney and the roles of contact sites in renal development and function; and 3) Define the distributions of inositol phospholipids in renal epithelial cells in vivo and determine their roles in determining these cells' polarized structures. Thus, the studies outlined in this proposal will provide new cellular and molecular insights into the processes through which renal epithelial cells acquire the structures that their function demands.