1978 — 1980 |
Burridge, Keith |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Purchase of Equipment For Computer Processing of Two-Dimensional Gels @ Cold Spring Harbor Laboratory |
0.915 |
1983 |
Burridge, Keith |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A Conference On Actin-Membrane Interaction At the Universityof North Carolina, Chapel Hill, Nc April 10-13, 1983 @ University of North Carolina At Chapel Hill |
1 |
1985 — 1992 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Actin-Membrane Interaction @ University of North Carolina Chapel Hill
Proteins involved in the attachment of actin to cell membranes, particularly plasma membranes, will be investigated. Two types of actin-membrane association will be analyzed: (1) that which may involve a class of proteins found in many cell types that we and others have shown to be closely related to erythrocyte spectrin, and (2) that which occurs in specialized regions of membranes, such as in fibroblast adhesion plaques, where bundles of actin filaments terminate. A strategy of sequential analysis will be used to identify and characterize the components that link actin to the membrane in these different locations. During the investigation of non-erythrocyte spectrins we will attempt to identify proteins involved in linking spectrin itself to membranes. A variety of biochemical, immunological and electron microscopic approaches will be used to define the relationship between spectrin and cell surface and membrane components in non-erythroid cells. The role of spectrin in regulating the lateral mobility and distribution of cell surface components will be studied using fluorescence recovery after photobleaching and other techniques. We will try to dissociate spectrin from the plasma membrane by microinjecting live cells with antibodies against spectrin or against proteins to which it binds. Regarding the proteins involved in attachment of actin microfilament bundles to the plasma membrane, we will concentrate on the properties of vinculin and 215K, a new protein that we have recently identified infibroblast adhesion plaques. New proteins interacting with vinculin and 215K will be identified using affinity chromatography and chemical cross-linking reagents. In an attempt to develop a new model system for examining "end-on" attachment of actin filaments to membranes, we will isolate thefascia adherens from cardiac muscle. This structrure is responsible for the membrane anchorage of actin in cardiac myofibrils. Again a strategy of sequential analysis will be used to define the components involved in attaching the actin filaments to this membrane.
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1 |
1991 — 1992 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrin-Cytoskeletal Interactions in Platelets @ University of North Carolina Chapel Hill
How the integrin family of receptors for the extracellular matrix associates with the cytoskeleton remains a largely unanswered question. This proposal is aimed at identifying and characterizing proteins that bind to platelet integrin cytoplasmic domains. These proteins may function as links between these receptors and the cytoskeleton. Synthetic peptides that correspond to platelet integrin cytoplasmic domains will be immobilized and used for affinity chromatography to isolate proteins from resting, activated or aggregated platelets that bind to these integrins. Initially we will use peptides that are identical to the cytoplasmic domains of glycoproteins IIb/IIIa, the most abundant platelet integrin. Later we will use peptides that correspond to other platelet integrin cytoplasmic sequences. In addition, we will covalently link the two cytoplasmic domain peptides of glycoproteins Ilb/IIIa at the site where they would emerge from the membrane. Using these joined peptides, we will look for proteins which interact with the complex rather than the individual sequences. Proteins binding to these peptide columns will be purified and characterized with special attention paid to their interactions with intact integrins and cytoskeletal proteins, such as actin, talin and vinculin. These studies will be modeled after our recent experiments in which we have demonstrated an association between alpha-actinin and the cytoplasmic domain of the integrin beta l subunit. The localization of integrin-binding proteins will be determined in normal resting, activated and aggregated platelets by immunoelectron microscopy, and will be compared with their distribution in thrombasthenic platelets, lacking glycoproteins IIb/IIIa. Many of the same integrins are found in endothelial cells, permitting us to use the same peptides to look for related endothelial cell proteins that bind these integrins. To probe the function of these proteins, antibodies will be raised and introduced into endothelial cells by microinjection. We will look for effects on the organization of the actin cytoskeleton and for altered cell adhesion. Finally, using the peptide affinity columns, we will assay for regulatory proteins, particularly protein kinases, that bind to platelet integrin cytoplasmic domains. If kinases are discovered, we will examine their substrates to determine whether phosphorylation modifies their interaction with integrins or cytoskeletal proteins.
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1 |
1993 — 1996 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure and Function of Focal Adhesions @ University of North Carolina Chapel Hill
Focal adhesions (FAs), the sites where cells in culture adhere most tightly to the underlying extracellular matrix (ECM), provide a model for analyzing the interaction of cells with the ECM. The goals of this proposal are to characterize the links between the ECM and the actin cytoskeleton in FAs, and to identify the signalling pathways at these sites of cell-ECM interaction. How integrin-mediated cell adhesion stimulates the tyrosine kinase (TK) p125FAK will be investigated. Binding assays will be used to determine whether p125FAK interacts with integrin cytoplasmic domains, and kinase assays will reveal whether integrin binding stimulates the activity of p125FAK. TK inhibitors that prevent FA formation will be examined for their action on p125FAK in vitro. Microinjection of antibodies against p125FAK will be employed as an alternative way to test the functions of this enzyme. The cloned SH2 domain of tensin will be used to determine whether tensin binds FA proteins that become tyrosine phosphorylated in response to adhesion. Is elevated tyrosine phosphorylation in FAs critical for anchorage- dependent growth? Is adhesion-induced expression of growth-associated genes blocked by inhibitors of p125FAK? Potential signalling proteins, such as PI-3 kinase and phospholipase Cgamma, will be assessed for adhesion-induced tyrosine phosphorylation. The src family of TKs will be studied for activation in response to ECM adhesion. FA assembly and tyrosine phosphorylation will be examined in cells that lack pp60c-src. The protein that binds the myristylated N-terminus of the src protein will be isolated, cloned and sequenced; its interaction with FA components will be studied. Proteins binding to the cytoplasmic domains of integrin alpha and beta subunits will continue to be investigated, using synthetic peptides to map binding sites. Proteins binding to the 47 kD talin domain will be identified, and the interactions and functions of the new FA protein, aciculin, will be explored. FA disassembly will be studied as cells enter mitosis and under conditions where motility is promoted. Does the mitotic phosphorylation of the beta1 integrin cytoplasmic domain inhibit its binding of talin, alpha-actinin or p125FAK? FA proteins that become phosphorylated as FAs disassemble will be studied and the effects of these phosphorylations on protein interactions determined. Does tyrosine dephosphorylation have a role in FA disassembly or merely accompany it? Phosphatase inhibitors will be used to inhibit dephosphorylation and to determine whether this prevents FA disassembly. These studies should provide an understanding of the structural organization of FAs, the regulation of their assembly and disassembly, and their role in signalling from the ECM.
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1 |
1997 — 2000 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure/Function of Focal Adhesions @ University of North Carolina Chapel Hill
Focal adhesions (FAs) are sites where cells in culture adhere tightly to the underlying extracellular matrix (ECM). They serve to anchor bundles of actin filaments to the plasma membrane and are sites of adhesion-mediated cell signaling, implicated in growth control. They are particularly rich in phosphotyrosine. This grant is aimed at understanding how FAs assemble, at determining the role of tyrosine phosphatases (PTPases) in FAs and at exploring the consequences of the interaction between the FA protein vinculin and the lipid PIP2. Activation of the GTP-binding protein Rho stimulates assembly of FAs. Determining the pathway from Rho to FA assembly is a major goal of this proposal. Following our discovery that Rho-stimulated contractility drives the formation of FAs, we will determine whether constitutively active myosin light chain (MLC) kinase or inhibitors of myosin phosphatase (MPP) induce contractility and FA assembly. We will test the hypothesis that Rho activates a kinase, such as p160ROCK or PKN, that phosphorylates and inhibits MPP and thereby elevates MLC phosphorylation. Dominant negative mutants of Rho- activated kinases will be used to explore this pathway. Mutant Rho constructs will be generated to define the Rho-activated kinase(s) that is responsible for stimulating contractility. The activation of Rho in response to integrin-mediated adhesion will be explored. Using dominant negative mutants of CDC42 and Rac, we will test the hypothesis that adhesion to ECM stimulates a cascade involving successively CDC42, Rac and Rho. The PTPases that regulate FA tyrosine phosphorylation will be examined. We will attempt to identify and characterize the PTPase that we have detected in association with integrins. We will explore the consequences of overexpressing or inhibiting this PTPase. Finally, we will determine whether changes in vinculin expression affect anchorage-dependent growth by modulating PIP2 availability. The effect of vinculin on PIP2 hydrolysis will be studied. We will determine which domain of vinculin restores anchorage-dependent growth when transfected into cells with reduced vinculin expression.
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1 |
1998 — 2002 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Integrin/Cytoskeleton Interactions in Platelets @ University of North Carolina Chapel Hill
Platelets play a critical role in the development of blood clots. This is an important physiological event in normal hemostasis, but the pathological development of blood clots can lead to heart attacks and strokes. During the formation of a blood clot, platelets become activated and rapidly assemble their actin cytoskeletons. These become linked to the major transmembrane integrin alphaIIb/beta3, which mediates adhesion to fibrin on the outside of the platelet. The coupling of alphaIIb/beta3 to the cytoskeleton allows platelets to contract clots. The first aim of this grant is to determine how the cytoplasmic domains of alphaIIb/beta3 interact with specific cytoskeletal components. Several strategies will be used to identify cytoskeletal proteins that associate with alphaIIb/beta3 in vivo. The interactions of alphaIIb/beta3 mutated in the cytoplasmic domains will be investigated, as will the interactions of integrin chimeras, in which the cytoplasmic domains are ligated onto the transmembrane and extracellular domains of other proteins. A scheme is proposed to permit analysis of the cytoskeletal links to individual cytoplasmic domains of other proteins. A scheme is proposed to permit analysis of the cytoskeletal links to individual cytoplasmic domain chimeras as well to chimeras to have been induced to dimerize. The role of specific proteins, such as talin, vinculin and alpha-actinin, will be explored in cells from which vinculin has been deleted and in which talin or alpha-actinin have been functionally disrupted. Many agents which prevent platelet activation raise cyclic nucleotide levels and stimulate protein kinase A or G. A prominent substrate for these kinases in platelets in the vasodilator-stimulated phosphoprotein (VASP). We will explore the function of VASP and the consequence of its phosphorylation in relation both to actin polymerization and the activation of alphaIIb/beta3. In response to platelet activation and aggregation, several tyrosine kinases become activated. We will investigate the regulation of platelet tyrosine phosphorylation by tyrosine phosphatases (PTPs). Specifically, we will look for PTPs that associate with and regulate tyrosine kinases, and PTPs that associate with alphaIIb/beta3.
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1 |
1998 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrin/Cytoskeletal Interactions in Platelets @ University of North Carolina Chapel Hill
DESCRIPTION: (Adapted from investigator's abstract) Platelets play a critical role in the development of blood clots. This is an important physiological event in normal hemostasis, but the pathological development of blood clots can lead to heart attacks and strokes. During the formation of a blood clot, platelets become activated and rapidly assemble their actin cytoskeletons. These become linked to the major transmembrane integrin alphaIIb/beta3, which mediates adhesion to fibrin on the outside of the platelet. The coupling of alphaIIb/beta3 to the cytoskeleton allows platelets to contract clots. The first aim of this proposal is to determine how the cytoplasmic domains of alphaIIb/beta3 interact with specific cytoskeletal components. Several strategies will be used to identify cytoskeletal proteins that associate with alphaIIb/beta3 in vivo. The interactions of alphaIIb/beta3 mutated in the cytoplasmic domains will be investigated, as will the interactions of integrin chimeras in which the cytoplasmic domains are ligated onto the transmembrane and extracellular domains of other proteins. A scheme is proposed to permit analysis of the cytoskeletal links to individual cytoplasmic domain chimeras as well as to chimeras that have been induced to dimerize. The roles of specific proteins, such as talin, vinculin, and alpha-actinin, will be explored in cells from which vinculin has been deleted and in which talin or alpha-actinin have been functionally disrupted. Many agents which prevent platelet activation raise cyclic nucleotide levels and stimulate protein kinase A or G. A prominent substrate for these kinases in platelets is the vasodilator-stimulated phosphoprotein (VASP). The applicant will explore the function of VASP and the consequences of its phosphorylation in relation both to actin polymerization and the activation of alphaIIb/beta3. In response to platelet activation and aggregation, several tyrosine kinases become activated. The applicant will investigate the regulation of platelet tyrosine phosphorylation by tyrosine phosphatases. Specifically, he will look for tyrosine phosphatases that associate with and regulate tyrosine kinases, and those that associate with alphaIIb/beta3.
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1 |
1999 — 2002 |
Burridge, Keith |
P60Activity Code Description: To support a multipurpose unit designed to bring together into a common focus divergent but related facilities within a given community. It may be based in a university or may involve other locally available resources, such as hospitals, computer facilities, regional centers, and primate colonies. It may include specialized centers, program projects and projects as integral components. Regardless of the facilities available to a program, it usually includes the following objectives: to foster biomedical research and development at both the fundamental and clinical levels; to initiate and expand community education, screening, and counseling programs; and to educate medical and allied health professionals concerning the problems of diagnosis and treatment of a specific disease. |
Regulation of Motility and Adhesion During Inflammation @ University of North Carolina Chapel Hill
Leukocyte extravasation (diapedesis) is a critical inflammatory event in which leukocytes exist from the circulation by migrating between endothelial cells. A goal of the project is to understand the cascade of adhesive, motile and signaling events in this complex, but poorly understood process. Our hypothesis is that monocyte adhesion to endothelial cells triggers sequential activation of Cdc42, Rac and Rho, and that these activated G proteins regulate successive steps in leukocyte extravasation. We will measure the activity of Rho, Rac and Cdc42 during monocyte adhesion and extravasation. We will perturb the functions of these proteins by introducing dominant negative and constitutively active forms of the proteins. A extravasating cell must dissociated endothelial adherens junctions made by VE-cadherin. We will investigate signaling cascades that affect the strength of VE-cadherin homophilic interactions. Beads coated with the VE-cadherin ectodomain will be adhered to endothelial cells in culture and the strength of adhesion measured using laser tweezers under a variety of conditions likely to affect adherens junction integrity. Because tyrosine phosphorylation has been implicated in regulating adherens junctions, we will examine the tyrosine phosphorylation of catenins during monocyte passage through endothelial monolayers. Using in vitro assays, the effect of phosphorylation on binding of catenins to cadherin cytoplasmic domains will be studied. During inflammation, cytokines stimulate migration of fibroblasts and epithelial cells. For migration to proceed, adhesions to extracellular matrix, such as focal adhesions, must be released. We will determined whether IL-8 induced focal adhesion disassembly occurs by inhibition of cell contractility or through phosphorylation of focal adhesion components. Upon completion of the inflammatory response, migration is inhibited. We will examine whether the clustering of cadherins inhibits cell migration, using cadherin constructs that can be aggregated by addition of chemical dimerizers. The juxtamembrane region of cadherins has been implicated in regulating migration. This region binds to p120cas, leading to our hypothesis that p120cas can bind either to cadherins or to a guanine nucleotide exchange factor that regulates Rac/Cdc42 activity, thereby controlling motility. We will look for guanine nucleotide exchange activity complexed with p120cas and determine whether this activity is lost when p120cas binds to aggregated cadherins.
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1 |
2001 — 2004 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure and Function of Fiscal Adhesions @ University of North Carolina Chapel Hill
DESCRIPTION (PROVIDED BY APPLICANT): Focal adhesions (FAs) are sites where cells in culture adhere tightly to the underlying extracellular matrix (ECM). They serve to anchor bundles of actin filaments to the plasma membrane and are sites of adhesion-mediated signaling, implicated in cell migration, growth and differentiation. The assembly of FAs is regulated by the GTPase RhoA. This grant is aimed at understanding how adhesion to ECM components, such as fibronectin (FN), activates RhoA, how FAs disassemble during mitosis and in response to the development of cell-cell junctions, and how specific tyrosine phosphatases (PTPases) regulate FA assembly and function. We will explore the hypothesis that adhesion to FN activates RhoA via engagement of two types of receptor, integrins and syndecan-4, which bind to different domains of FN. We will determine whether the increase in RhoA activity results from inhibition of GTPase activating proteins (GAPs), from stimulation of guanine nucleotide exchange factors (GEFs) or from both. We will use dominant negative constructs of RhoA, as well as trapping mutants of tyrosine phosphatases to isolate RhoA GEFs downstream from integrin and syndecan-4 engagement. To examine the mitotic disassembly of FAs, we have generated an antibody that binds to the phosphorylated integrin beta 1 cytoplasmic domain. This will be used to monitor changes in integrin phosphorylation that occur during mitosis. We will measure the affinity of integrins for FN and cytoskeletal proteins during mitosis and determine whether changes in affinity are regulated by cytoplasmic domain phosphorylation. Our preliminary data reveal that the development of cell-cell junctions is associated with a sharp decrease in active RhoA. We will explore the pathway that leads to this decrease and determine whether it is responsible for the parallel loss of FAs. With respect to PTPases, we will test the hypothesis that PTPa mediates the activation of c-Src in response to integrin engagement and look for an association between this phosphatase and integrins. The PTPase, FTP-PEST has been shown to regulate cell migration and the tyrosine phosphorylation of several FA proteins. We will test the hypothesis that PTP-PEST negatively regulates the activities of both RhoA and Rac1, either by acting on tyrosine phosphorylated GEFs, such as Vav2, or by acting on FA substrates, such as p13Ocas, to disrupt their interactions.
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1 |
2002 — 2004 |
Burridge, Keith |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Fogarty International Research Collaboration Award @ University of North Carolina Chapel Hill
DESCRIPTION (provided by applicant) The Foreign Investigator (Fl) of this project has recently discovered that alphavbeta3 integrin in astrocytes is a receptor for Thy-1 and shown that treatment of astrocytes with Thy- 1 induces signaling linked to focal adhesions (FA) and stress fiber formation. FAs anchor stress fibers to the plasma membrane, contain multiple structural, signaling and adaptor molecules and are implicated in cell migration, growth and differentiation. Astrocytes change from stellate to fibroblast-like morphology upon brain damage and the GTPase RhoA has been implicated in these changes. Our preliminary data reveal that RhoA is implicated in Thy-1-induced focal adhesion formation in astrocytes. This collaborative grant is aimed at understanding how cell-cell interaction mediated by Thy-1 binding to integrin alphavBeta3 activates RhoA, whether integrin signaling triggered by Thy-1 binding stimulates cell migration and what are the signaling events occurring downstream from FAs. We will explore the possibility that Thy-1-astocyte interaction is mediated by two different domains on Thy-1, one binding integrins and one binding syndecans. We will look for possible guanine nucleotide exchange factors (GEFs) that may activate RhoA using dominant negative constructs of RhoA, which have high affinity for Rho GEFs. To study the effect of Thy-1- alphavBeta3 integrin interaction on cell migration we will use different wound healing assays currently in use in the PI's lab. We will also test whether changes in motility correlate with modulation of Rac 1 or Cdc42 activity, measured with pulldown assays. We will test the hypothesis that Thy-1 activated integrins trigger signaling events in astrocytes that are similar to those described in fibroblasts, with particular interest on the involvement of PI 3-kinase and MAP kinase pathways, given their known participation in cell migration and proliferation, respectively. Thus, in this proposal we will study the molecular response of astrocytes to Thy-1- alphavBeta3 integrin interaction in vitro. Learning about such changes should lead to a better understanding of "astrogliosis," a migratory and proliferative reaction of astrocytes to wounding, that creates an environment antagonistic to neuronal regeneration following brain injury.
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1 |
2004 — 2005 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cytoskeletal Regulationm of Endothelial Cell Junctions @ University of North Carolina Chapel Hill |
1 |
2006 — 2018 |
Burridge, Keith |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Adhesion and the Regulation of Rho Gtpases @ Univ of North Carolina Chapel Hill
Abstract Adhesion of cells to extracellular matrices is a fundamental characteristic of all multicellular organisms. Adhesion provides not only structural links between the intracellular cytoskeleton and the extracellular environment, but also provides sites of signal transduction that affect many aspects of cell behavior. This grant is aimed at understanding how signals from cell-matrix adhesions regulate members of the Rho family of GTPases, which are themselves key regulators of the cytoskeleton and many intracellular processes. The goal of the first aim is to determine how adhesion to the extracellular matrix protein fibronectin stimulates RhoA. Having recently identified guanine nucleotide exchange factors, such as Lsc, that are involved in this process, we aim to determine the signaling pathways that lead to their activation. Our finding that adhesion stimulates Lsc tyrosine phosphorylation and promotes Lsc binding to vinculin suggests possible mechanisms of regulation and will be investigated further. We will also examine how mechanical tension and substrate rigidity/pliability regulate RhoA activity. Here we have found that some but not all of the same exchange factors are involved. We will investigate the signaling pathways downstream from mechanical tension that lead to exchange factor activation. In the second aim, we will follow up the discovery that reactive oxygen species can activate Rho proteins by transient oxidation of cysteine sulfhydryls without involvement of exchange factors. Using redox-resistant mutants of Rho GTPases, we will determine the role of Rho protein activation by cysteine oxidation in events such as adhesion and in response to growth factor stimulation. In the final aim, we will focus on the Rho GTPase sequestering protein RhoGDI, which we have recently found contributes to crosstalk between different Rho family members. Competitive interactions of Rho proteins for binding to RhoGDI will be explored. We will investigate how Rho proteins are released from RhoGDI in response to adhesion and determine the pathways that lead to their degradation following displacement from RhoGDI. Finally, we will explore the role of RhoGDI in mediating the trafficking of Rho proteins from their sites of postranslational modification to their sites of action at the plasma membrane.
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1 |
2006 — 2010 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Endothelial Responses to Leukocyte Engagement @ University of North Carolina Chapel Hill
Leukocyte migration across the endothelium is a critical event in inflammation. The goals of this project are[unreadable] to understand the signals initiated by leukocyte adhesion to endothelial cells that promote passage of[unreadable] leukocytes across the endothelium. We will focus on two aspects of this process: the generation of[unreadable] leukocyte-induced cup-like structures that form on the surfaces of endothelial cells, and on leukocyte[unreadable] passage through endothelial cell-cell junctions. In the first aim, we will test the hypothesis that leukocyte[unreadable] adhesion to endothelial cells activates specific Rho GTPases and that these contribute both to formation of[unreadable] cups and to the disassembly of endothelial cell-cell junctions. We will explore the pathways by which[unreadable] leukocyte adhesion regulates these GTPases, using techniques to identify relevant guanine nucleotide[unreadable] exchange factors (GEFs) and GTPase activating proteins (GAPs). Particular attention will be paid to SGEF,[unreadable] which co-localizes with ICAM-1 in cups. SGEF activates RhoG, a Rho protein which induces dorsal[unreadable] membrane ruffles. We will also investigate the pathways downstream from RhoA and Rac1 that promote[unreadable] junctional disassembly. In the second aim, the hypothesis that endothelial junctions are regulated by Rap1[unreadable] activity in response to leukocyte adhesion will be examined. We will use mouse models of inflammation to[unreadable] investigate the roles of Rap isoforms in endothelial cells in mice that are null for Rapla or Raplb. In[unreadable] preliminary work, we have shown that leukocyte adhesion stimulates the tyrosine phosphorylation of[unreadable] endothelial junctional components. In the third aim, we will investigate the pathway by which this occurs,[unreadable] whether it is in response to the activation of Rac1 and generation of reactive oxygen species. We will look[unreadable] for the tyrosine kinases and phosphatases involved and determine whether the tyrosine phosphorylation of[unreadable] VE-cadherin leads to its removal from junctions by endocytosis. Several receptor tyrosine phosphatases[unreadable] reside in endothelial junctions. We will test the hypothesis that these may interact with and be inhibited by[unreadable] extravasating leukocytes so as to elevate levels of phosphotyrosine in junctions. Because leukocyte[unreadable] migration across the endothelial barrier lining blood vessels is a critical step in inflammation, the elucidation[unreadable] of signaling pathways that regulate this process may reveal novel targets for the development of therapies to[unreadable] control inflammation and inflammatory diseases.[unreadable]
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1 |
2006 — 2010 |
Burridge, Keith |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Biosensor @ University of Virginia Charlottesville
The primary goals for the Biosensor Initiative are to continue to develop biosensors for key regulatory and effector nodes in cell migration and to use existing and proposed biosensors to acquire quantitative data that can be used to[unreadable] develop an experimentally verified theoretical model of cell migration by the Modeling Initiative and others[unreadable] in the modeling community. The Initiative builds on the sensor technologies, fluorescence assays, high[unreadable] throughput microscopy and collaborations from the first CMC funding period to move into Phase II,[unreadable] wherein we plan to visualize signaling events in living cells and to acquire and analyze large microscopic[unreadable] data sets. The modeling will be carried out as a close collaboration with the Modeling Initiative. A major[unreadable] focus of the Initiative will be Rho family regulators since they control key migration nodes. In addition[unreadable] the Initiative will focus on construction of biosensors and photoactivatable reagents, using both existing[unreadable] and new technologies, for key upstream and downstream members of these signaling pathways.[unreadable] In the end we produce a detailed experimental and mathematical analysis of a specific, critical signaling[unreadable] pathway in cell migration.
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0.961 |
2006 — 2007 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Cytoskeletal Regulation of Endothelial Cell Junctions @ University of North Carolina Chapel Hill |
1 |
2006 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Administrative Core @ University of North Carolina Chapel Hill |
1 |
2006 — 2010 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Adhesion and Migration in Inflammation @ University of North Carolina Chapel Hill
[unreadable] DESCRIPTION (provided by applicant): [unreadable] During inflammation leukocytes are recruited from the circulation into tissues. In order to leave the bloodstream leukocytes must adhere to and migrate across the endothelial lining of blood vessels. This proposal is aimed at understanding the complex series of adhesive and migratory events in this process. The first project will investigate the signals generated in endothelial cells as they respond to adhering leukocytes. We will investigate the roles of Rho GTPases in the assembly of leukocyte-induced endothelial cups. How leukocytes manipulate the integrity of endothelial cell-cell junctions to allow leukocyte passage will be studied. The roles of the Rapt, RhoA and Rac1 GTPases will be investigated in this process. Additionally, the role of tyrosine phosphorylation of endothelial junctional proteins will be determined during leukocyte transendothelial migration. The second project will focus on leukocyte-platelet interactions in the context of vascular injury where endothelial cells are lost and platelets provide the cellular layer lining damaged blood vessels. The roles of Rap1 isoforms and the tyrosine kinase Pyk2 will be studied in leukocytes interacting with platelets. The work will be extended to in vivo mouse models of arterial injury and inflammation. Filopodia are slender actin-based protrusions that mediate the initial contacts between leukocytes and endothelial cells during inflammation. The third project will investigate myosin X in filopodial function and endothelial cup assembly. All of the projects will take advantage of an imaging and biosensor core that will permit state of the art live cell imaging with special emphasis on the visualization of active signaling molecules by fluorescence resonance energy transfer (FRET). The long term goal of this program project proposal is to elucidate critical signaling events during inflammation that may lead to the development of novel therapies for cardiovascular disease and other inflammatory disorders.
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1 |
2010 — 2012 |
Burridge, Keith |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Cb2 Cannabinoid Receptor-Mediated Regulation of Prostate Cancer Growth @ Univ of North Carolina Chapel Hill
Abstract: Prostate cancer is a major health problem and a significant cause of mortality in men woridwide. Family history and race are the two major risk factors for this disease. The age-adjusted incidence rate and mortality rate of prostate cancer is significantly higher in African-Americans compared to Caucasian-Americans or other races in the US and worldwide. Thus, an understanding of the molecular mechanism responsible for the development and progression of prostate cancer is extremely important to the development of more effective therapeutic strategies. Cannabinoids (including endocannabinoids) regulate cell death or cell growth, depending on the cell type and concentration of the cannabinoid. Cannabinoids inhibit the growth of prostate cancer cells. We have found that activation of cannabinoid receptor-2 (CB2) inhibits androgen-sensitive prostate cancer (AS PC) cell proliferation and motility. Our preliminary data also suggest that cannabinoid compounds possess selective efficacy, producing less adverse effects on normal prostate epithelial cells compared to LNCaP prostate cancer cells. To date most of the anti-tumor effects of cannabinoids have been correlated with the CB1 receptors rather than CB2 receptor activation, although CB2 receptor expression is high in many tumor tissues including prostate tumor. However downstream mechanisms mediaflng anti-tumor effects of cannabinoids under/f7 wVo conditions are poorly understood. Further, CBI receptors are highly expressed in neuronal cells and brain tissue. Therefore, unlike activation of CB2 receptors, CBI receptor activation produces neurobehavioral and psychotropic side effects. Thus, CB2 receptor-mediated therapeutic intervenflon of prostate cancer has clinical advantages. Based on our preliminary data we hypothesize that activation of CB2 receptor inhibits androgen-sensitive prostate cancer (AS PC) growth. To test this hypothesis, we have developed the following 2 specific aims: (1) To determine the effects of CB2 receptor activation on cultured LNCaP and LAPC4 prostate cancer cell proliferation, viability and migratlon in relation to activatlon of RhoA and the focal adhesion kinase (FAK) signaling pathway;and (2) To determine the effects of exogenous activation of CB2 receptor and increase in endogenous cannabinoid activity on AS prostate cancer growrth in mice in relation to FAK activity.
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0.988 |
2012 — 2016 |
Burridge, Keith Doerschuk, Claire M Hahn, Klaus M. (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rho-Mediated Signaling in Lung Endothelial Cells Induced by Neutrophil Adhesion @ Univ of North Carolina Chapel Hill
DESCRIPTION (provided by applicant): The recruitment of neutrophils out of the blood and into surrounding lung tissues is a critical event in pulmonary inflammation. For this to occur, neutrophils must first adhere to cell adhesion molecules (particularly E-selectin and ICAM-1) expressed on the surface of endothelial cells lining blood vessels. These adhesive interactions provide attachment and allow neutrophils to generate traction on the endothelial surface, so that they can migrate over it as they probe for endothelial junctions or other sites where they can cross the endothelial barrier. Engagement of these adhesion molecules also triggers signaling pathways in the endothelial cells that promote transmigration. Of the many signaling pathways that have been identified downstream from E-selectin and ICAM-1, several Rho family GTPases have been implicated in mediating the changes in the cytoskeleton and cell junctions that allow neutrophil passage. Little is known about the co-ordination of the different Rho proteins and how they become activated in response to E-selectin and ICAM-1 ligation. Additionally, it is not known whether tractional forces exerted by neutrophils on these adhesion molecules affect their signaling pathways to promote neutrophil transit across the endothelium. However, these processes are highly co- ordinated and tightly regulated to maximize the benefits of host defense and minimize the injury resulting from endothelial cell damage, particularly in the lungs where edema interferes with gas exchange. To tackle these questions, we propose the following specific aims. Aim 1 will examine the dynamics of activation of key Rho proteins (RhoA, Rac1, RhoG and Cdc42) in response to engagement and crosslinking of E-selectin and ICAM- 1 on lung microvascular endothelial cells. FRET-based biosensors for each Rho GTPase will be used to follow the time and location of their activation. Novel photomanipulation techniques will be used to activate or inhibit specific GTPases at precise times and places to examine how interactions of the GTPases affect neutrophil transmigration. Aim 2 will identify and manipulate guanine nucleotide exchange factors (GEFs) that are downstream of E-selectin and ICAM-1 and that regulate Rho protein activity. Aim 3 will determine whether tension on E-selectin and ICAM-1 initiates activation of Rho proteins. Using 3D force microscopy, we will examine whether mimicking the tension applied by neutrophils on E-selectin and ICAM-1 initiates or modulates signaling to Rho GTPases. Aim 4 will determine how neutrophil migration over endothelial cell surfaces induces tension along and across endothelial cells through E-selectin and ICAM-1, and whether their ligation modulates disassembly of VE-cadherin complexes. Taken together, the proposed studies address important signaling pathways that regulate neutrophil passage across the endothelium during inflammation. They will contribute to an integrated model of endothelial adhesion molecule signaling, incorporating spatial and temporal control that is novel and important to a comprehensive understanding of inflammation. These studies may identify new targets for therapies in the treatment of inflammatory diseases.
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0.988 |
2013 — 2017 |
Burridge, Keith |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Rhoa Gef Regulation by Mechanotransduction @ Univ of North Carolina Chapel Hill
Cells respond to mechanical force in many ways. Recent work has shown that mechanical force applied to cell adhesion molecules can affect the activities of Rho family GTPases, thereby influencing the organization of the cytoskeleton, cell adhesion, migration and many other cellular activities. This project is aimed at elucidating the signaling pathways by which mechanical force affects Rho GTPases, particularly RhoA, under a number of situations. In the first part of this project, we will focus on migrating cells and use shRNA knockdown strategies to identify the GEFs responsible for RhoA activation at the leading edge. Using biosensors for RhoA and relevant GEFs developed by Hahin and Sondeic, combined with the multiplexing imaging strategies of Danuser and i-laiin, we will determine whether GEF activation occurs at the leading edge as a result of integrin engagement or by mechanical force. Our preliminary studies show that mechanical tension on cadherins also activates RhoA and in the second part of the project, working with Hall, we aim to identify the GEFs and signaling pathways involved. Using magnetic tweezers and beads coated with the extracellular domain of E-cadherin we will explore how force leads to the strengthening of cadherin-mediated adhesions. The final section of the project examines how mechanical tension applied to the cell surface affects RhoA signaling in the nucleus. Based on our preliminary work showing that stretching isolated nuclei activates RhoA, we will target the RhoA biosensor to the nucleus so that we can examine RhoA activity in this compartment as mechanical force is applied to the cell surface. We will screen for the GEFs responsible and then examine the consequences of RhoA activation within the nucleus, examining nuclear stiffening and effects on gene expression.
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0.988 |
2015 — 2020 |
Burridge, Keith |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Full Project 1: Lsr Alters Metabolic Signaling to Drive Aggressive Breast Cancer Behaviors @ Univ of North Carolina Chapel Hill
Project Summary/Abstract African American (AA) women have higher breast cancer mortality rates compared to other races. A greater prevalence of basal-like breast cancers in AA women explain some disparities, as these tumors are clinically the most aggressive, characterized by cancer stem No effective therapies exist and cell features. survival is poor. Escalating this disparity is the disease promoting effects of obesity, which is significantly higher in AA. The majority of studies on breast cancer disparities examine tumor characteristics, but the etiologic factors that lead to this disparity remain undefined. Our primary objective is to identify the mechanisms involved in promoting aggressive breast cancer in AAs, as a consequence of stromal effects at the site of the cancerous lesion. A key molecular pathway has been identified which integrates these clinical, microenvironmental and biological factors to affect tumor behavior. Aim 1 takes a novel approach, combining our published proteomic and gene expression datasets detailing race and tumor-subtype specific stromal interactions with our recently published data on the identified pathway promoting cancer stem cell- like, aggressive behaviors. Aims 2 and 3 extend these data to experimental studies to elucidate mechanistic details. To accomplish these tasks, our team established research partnerships that provides access to resources including the Normal Breast Study: a unique epidemiologic study from ethnically diverse patients at UNC Hospitals. There are several important biological implications of this work. The observation that AA breast tissue is enriched with distinct proteins, the prevalence of obesity in AA, and the predisposition to develop basal-like tumors strongly suggests a biological link between race, metabolism and cancer subtype. The role of differentially regulated metabolic-sensitive proteins in the tumor microenvironment will provide novel insights into the biological basis of racial disparities. The long-term goal of our collaboration is to understand tumor-microenvironment interactions and their influence on breast cancer disparities. Our distinctive approach will expose unique characteristics of the breast tissue microenvironment and will integrate advances in the field of tumor microenvironment with health disparities research. Relevance This proposal addresses breast cancer disparities by studying a key pathway that drive the aggressiveness of breast cancers. By combining observational studies on differences in this pathway by race, BMI, and breast cancer subtype with experimental studies to understand the role of this pathway in cellular phenotypes, this project will provide important and novel insight into the biological basis of racial disparities.
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0.988 |
2015 — 2016 |
Burridge, Keith Cerami, Carla Gomez, Shawn Michael |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Endothelial Cell Uptake of Infected Erythrocytes in Cerebral Malaria @ Univ of North Carolina Chapel Hill
? DESCRIPTION (provided by applicant): Malaria due to Plasmodium falciparum remains one of the largest global infectious disease burdens, infecting over 200 million and killing nearly 700,000 people annually. Of several possible disease manifestations, cerebral malaria has the worst survival outcomes. Cerebral malaria, characterized by coma and neurological deficits, ensues when parasitized red blood cells (pRBCs) sequester in the cerebral vasculature. Cytoadherence can contribute to disease by obstructing peripheral blood vessels, limiting oxygen delivery to tissue, and inducing significant inflammation. Cerebral pRBC sequestration is linked to breakdown of the blood brain barrier, yet the molecular mechanisms mediating vascular damage in cerebral malaria are poorly understood. The host inflammatory response plays a major role, but a more complete understanding of sequestration-related pathophysiology is necessary to develop adjunct therapies for cerebral malaria. Recent in vitro findings demonstrate that human brain endothelial cells (ECs) take up pRBCs via formation of an apical cup resembling endothelial protrusions known to mediate leukocyte transendothelial migration (TEM). ICAM1 binding in TEM activates signaling that induces cytoskeletal remodeling and junction opening to allow for migration either through or between ECs. Since pRBCs also bind ICAM1 and the cup- formation described in pRBC uptake resembles that associated with transcellular TEM, we hypothesize that pRBC cytoadherence can mistakenly induce uptake via TEM pathways and that this contributes to the pathology of cerebral malaria. Our aims seek: 1) to explore the relationship between cup formation and pRBC uptake by ECs, investigating the receptors involved and comparing different clinical isolates of P. falciparum for their ability to induce cup formation; and 2) to determine how pRBC-induced cups and uptake contribute to disruption of the EC barrier, whether this occurs by signaling pathways that open junctions or by inducing cell death. Our long term goal is to understand how pRBCs induce endothelial activation and blood brain barrier breakdown in order to improve therapeutic development. Not only is characterizing endothelial pRBC uptake in the context of leukocyte TEM pathways a novel idea, but exploring pRBC uptake has significant implications for understanding and controlling cerebral malaria pathology.
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0.988 |