Richard Firtel - US grants

We propose to examine the problem of differential gene expression during the developmental cycle of Dictyostelium discoideum. Specifically, we plan on examining the structure and expression of developmentally regulated genes in this organism including those encoding the carbohydrate binding protein discoidin I, a lectin known to be involved in species-specific cell-cell adhesion in D. discoideum, and a group of genes whose expression is modulated during development by cAMP. Using recombinant DNA methods, we have isolated two groups of genes whose expression in modulated by cAMP. The first group are genes which are activated early in normal development and which can be induced in suspension culture by cAMP. The second is a population of genes which are expressed late in development and require continued levels of cAMP for expresion. We plan on comparing the biological conditions which affect the activity of these genes and to compare the structure of these genes using molecular techniques. We plan on using a DNA mediated transformation system we are developing to investigate the function of sequences flanking the genes in controlling when during the developmental cycles these genes are expressed and the level of expression of these genes when they are activated. These experiments will be aimed at obtaining insight into the molecular mechanisms by which gene expression is regulated in eukaryotic cells during development.

This application consists of two major sections. In the first, we propose experiments directed at understanding the control of cell-type gene expression and differentiation of prestalk and prespore cells. Cis-acting regulating regions will be identified using DNA-mediated transformation combined with in vitro mutagenesis approaches. Sequence-specific DNA binding proteins will be examined with the goal of understanding if common regulating proteins are involved in controlling these gene sets. Potential regulating mutants will also be isolated. Using immunostaining and in situ hybridization we will examine where within the developing aggregate prestalk and prespore genes are first expressed to understand how the spatial patterning of cells is established. The regulation and function of the Dictyostelium ras gene, a prestalk-specific gene, will be examined during development. The cis-acting regulating regions of this gene will be defined and possible functions of the protein in prestalk differentiation will be examined. In the second project, we examine the control of the 17-20 member, developmentally regulated, multigene family encoding actin. The relative expression of the 15 cloned actin genes will be examined. Cis-acting control regions of a few selected actin genes will be identified. The function of identified, conserved GC-rich sequences within the 5' flanking region will be examined to determine if they are important in regulating the expression of specific actin genes. DNA binding proteins specific for identified regulating sequences will be isolated and binding sites mapped. These experiments are aimed at understanding the complex regulating pattern of this essential gene family.

The goal of the proposed work is to gain insight into the function of ras in eukaryotic cells. For this, we will use the relatively simple developmental system Dictyostelium discoideum. We have constructed a missense mutation (Gly12-Thr12) in the Dictyostelium ras gene. When this is transformed into Dictyostelium, the transformed cells develop abnormally and do not form mature fruiting bodies. The abnormalities suggest that the observed phenotypes result from an alteration of the extracellular-cAMP-receptor signal transduction pathway. We propose experiments to examine the function of the ras protein in this pathway during Dictyostelium development. This will involve expressing wild-type ras and the ras-Thr12 missense mutation using promoters from Dictyostelium genes which are expressed at specific developmental stages. This will be combined with a biochemical analysis of the different steps within the signal transduction pathway in normal and transformed cells. We will also select and analyze second site suppressor mutations of the ras-Thr12 gene. It is hoped that the proposed study of ras in Dictyostelium will result in a better understanding of its function both in this organism and in other organisms including mammals. Because ras overexpression or missense mutations have been associated with a large number of human cancers and can transform cells in tissue culture, we hope that the work described in this proposal will also lead to a better understanding of these processes.

This application directs itself at further elucidating the molecular mechanisms regulating prestalk and prespore differentiation in Dictyostelium. The two cell types are spatially localized in the anterior (prestalk) and posterior (prespore) of the migrating slug. Signals of cAMP transduced through cell surface receptors activate the expression of genes unique or predominantly expressed in either one of the two cell types. Using diverse molecular and genetic approaches, we propose to study the cAMP receptor-activated signal transduction pathway. We will purify and clone the transacting factor that controls one class of induced prestalk genes and examine its putative posttranslational modification in response to cAMP receptor activation. The cis-acting sequences and trans-acting factor(s) controlling the expression of the prestalk, cell-type-specific Dd-ras genes and a coordinately regulated, cAMP-inducible class of prespore genes will be further defined. The trans-acting factor associated with a conserved regulatory element in the prespore promoters will be purified and analyzed. Using molecular complementation with genomic libraries, we will rescue existing mutants and mutations we will generate, blocked in the expression of cAMP-induced genes. With the plasmids that provided rescue, we will clone and characterize the wild-type genes. We will investigate the molecular mechanisms regulating spatial localization of the prestalk and prespore gene expression using prestalk and prespore specific promoter/beta-gal fusions. These will be used to examine the molecular basis of an anterior to posterior gradient of prespore gene expression within the prespore zone of the slug and the role of the extracellular morphogens DIF and cAMP in establishing and maintaining this gradient. We will also examine the role of the cell cycle in regulating cell-type differentiation and the ontogeny of prestalk and prespore cells. These diverse approaches will be integrated with the goal of understanding the integrated pathways that regulate cellular differentiation in this organism.

Development in the cellular slime mold Dictyostelium discoideum is regulated in part by extracellular molecules that interact with cell surface receptors. This application is directed at understanding the molecular and biochemical mechanisms by which signal transduction pathways control both cellular morphogenesis and gene expression during the pre-aggregation and aggregation stages of development. Using molecular techniques, we have identified and cloned genes whose products are essential in controlling this and other stages of development. These include genes encoding four G alpha protein subunits, each with a distinct developmental pattern of expression, a developmentally regulated phosphotyrosine phosphatase, and developmentally regulated putative serine/threonine kinases. Gene disruptions and overexpression studies have indicated that the proteins encoded by each of these genes play essential roles in development. Our analysis indicates that G alpha2 couples to the cAMP receptor cARI and regulates diverse signal transduction pathways that mediate chemotaxis during aggregation and the induction of pulse-induced genes. In this application, we propose to dissect the signal transduction pathways regulated by these proteins using molecular techniques to produce appropriate strains that either do not express the proteins or express modified proteins. In vitro analysis will be used to characterize the effects of the mutations at a biochemical level. Using molecular complementation, we propose to isolate additional genes in the signaling pathways that have been previously identified by in vivo mutations. We will also continue to characterize the function of the cell-type-specific ras gene within the signal transduction pathway. In order to understand the molecular basis of receptor-mediated control of gene expression during aggregation, we plan to further identify the cis-acting regulatory regions of genes regulated by cAMP pulses and to purify the regulatory trans-acting factors that mediate the response at the level of transcription.

Spatial patterning in multicellular eukaryotes involves the interaction of multiple signaling pathways that respond to morphogenetic signals in the organism to control cell-fate decisions and morphogenetic movements. These events lead to the proper positioning and differentiation of the cell types along an anterior-posterior axis. We have identified two pathways that regulate cell-type differentiation and spatially patterning in Dictyostelium. One pathway uses the MEK kinase MEKKalpha that contains an F-box and WD40 repeats. Activity of MEKKalpha is spatially and temporally regulated by protein degradation via a ubiquitination/deubiquitination regulatory network involving the ubiquitin conjugating enzyme UBC1 and the deubiquitinating enzyme UBP1. MEKKalpha is required for cell fate decisions and proper spatial patterning within the multicellular organism. The second pathway includes Spalten, a novel signaling component containing an N-terminal regulatory domain with homology to heterotrimeric G proteins a subunits and alpha C-terminal serine/threonine protein phosphatase PP2C. Spalten phosphatase activity is required to induce cell-type differentiation by dephosphorylating a substrate. Second site suppressor screens have identified a kinase, designated ARCK1, which has structure similarities to mammalian Raf-1. ARCK1 appears to function to phosphorylate the Spalten substrate and to maintain cells in an undifferentiated state. Spalten and ARCK1 thus function on a pathway that regulates cell fate decisions in Dictyostelium. Our goals include the identification of other components of these regulatory cascades and elucidation of the mechanisms by which signaling molecules control their activity and how these pathways regulate cell fate decisions in Dictyostelium. We will employ a combination of molecular, genetic, and biochemical approaches to achieve these goals. Because of the simplicity of the developmental system and the facility with which one can pursue multiple approaches simultaneously to analyze gene function, it is expected that significant progress will be made in achieving these goals. Moreover, as much of our analysis is directed at pathways that have counter parts in other systems, our work should shed light onto how regulatory cascades, such ubiquitin-mediated protein degradation, can be use to control complex developmental pathways, including spatial patterning and cell-type differentiation in eukaryotes in general.

Development in the cellular slime mold Dictyostelium discoideum is regulated in part by extracellular molecules that interact with cell surface receptors. This application is directed at understanding the molecular and biochemical mechanisms by which signal transduction pathways control both cellular morphogenesis and gene expression during the pre-aggregation and aggregation stages of development. Using molecular techniques, we have identified and cloned genes whose products are essential in controlling this and other stages of development. These include genes encoding four G alpha protein subunits, each with a distinct developmental pattern of expression, a developmentally regulated phosphotyrosine phosphatase, and developmentally regulated putative serine/threonine kinases. Gene disruptions and overexpression studies have indicated that the proteins encoded by each of these genes play essential roles in development. Our analysis indicates that G alpha2 couples to the cAMP receptor cARI and regulates diverse signal transduction pathways that mediate chemotaxis during aggregation and the induction of pulse-induced genes. In this application, we propose to dissect the signal transduction pathways regulated by these proteins using molecular techniques to produce appropriate strains that either do not express the proteins or express modified proteins. In vitro analysis will be used to characterize the effects of the mutations at a biochemical level. Using molecular complementation, we propose to isolate additional genes in the signaling pathways that have been previously identified by in vivo mutations. We will also continue to characterize the function of the cell-type-specific ras gene within the signal transduction pathway. In order to understand the molecular basis of receptor-mediated control of gene expression during aggregation, we plan to further identify the cis-acting regulatory regions of genes regulated by cAMP pulses and to purify the regulatory trans-acting factors that mediate the response at the level of transcription.

The cellular slime mold Dictyostelium discoideum provides a genetically, biochemically, and molecularly tractable system in which to study signal transduction pathways controlling temporal and spatial regulation during multicellular development. It has many advantages over other developmental systems in that a combination of biochemistry and molecular genetics can be used to examine processes controlling multicellular development. Analyses of regulatory pathways controlling development in this organism have shown that many have direct parallels with known pathways in more complex systems including C. elegans, Drosophila, and mammals. In this Project, we propose to identify genes essential for development in this organism. For this, we will use REMI (Restriction Enzyme Mediated Integration) combined with restriction endonucleases that are expected to contain sites within all genes that affect, but are not essential for cell growth and multicellular development. The identified genes will be sequenced as part of this Program Project. The genes identified in this manner will act as a resource to be used in other studies aimed at understanding how signaling pathways regulate development in this system. Such studies combined with the sequence information derived from the efforts of this Program should thus provide a sequence data base with insights into the biochemical function of the gene product. This information is expected to provide a basis for understanding how development is regulated in other systems.

Cells must be able to sense and respond to a variety of extracellular signals to fulfill their physiological roles. In eukaryotes, chemotaxis and morphogenetic movements in response to extracellular signals control processes such as migration of leukocytes and macrophage in wound healing, cell movements during embryogenesis, axonal guidance, and aggregation of Dictyostelium cells to form a multicellular organism. These diverse processes all require a reorganization of the cytoskeleton that results pseudopod extension and cell migration. Many of the pathways that mediate these changes are highly conserved and use common factors that function in integrated circuits to control directional cell movement toward a chemoattractant. In this application, we focus on two signaling pathways that are involved in controlling cell movement and chemotaxis during the aggregation and multicellular stages of Dictyostelium development. The first is the pathway regulated by the Dictyostelium homologue of Akt/PKB (pkbA), which we have demonstrated is rapidly and transiently activated in response to chemoattractant signals and is essential for cell polarization and movement. In addition, we have identified a second, highly related gene that functions at a slightly later stage in development and may be required for morphogenetic movements necessary for multicellular differentiation. We propose experiments to (1) dissect the mechanisms by which these genes products are activated, (2) characterize cell movement and gene expression defects in strains with null mutations of these genes, and (3) identify downstream effectors of the pathways in which they function. The second pathway uses a MAP kinase activation cascade that plays a central role in integrating extracellular signals and appears to function as a checkpoint for the ability of the cells to respond to chemoattractants. We have demonstrated that the MAP kinase kinase of this cascade, DdMEK1, is essential for cell movement and that it plays a central role in the regulation of receptor activation of guanylyl cyclase, which in turn generates a key second messenger that promotes chemotaxis, and activation of Akt/PKB. Our goals are to (1) identify the other components of the DdMEK1 MAP kinase activation pathway, (2) identify this pathways's downstream effectors and regulators and (3) elucidate the mechanism(s) by which this pathway controls the ability of Dictyostelium cells to respond to the aggregation-stage chemoattractant cAMP. Understanding how the Akt/PKB and DdMEK1 pathways control cell polarization and movement will provide a new understanding of how signaling circuits are integrated to achieve a coherent physiological response.

DESCRIPTION (provided by applicant): Chemotaxis plays a central role in diverse biological processes, including metastasis of cancer cells, inflammatory responses involving movement of neutrophils and macrophage, migration of neural crest cells, embryonic morphogenesis, and aggregation in Dictyostelium. The underlying processes that control chemotaxis in eukaryotic cells are highly conserved between Dictyostelium and man and utilize common factors in integrated circuits to control directional cell movement toward a chemoattractant. The key component of this process is the ability of cells to produce a pseudopod at the leading edge in the direction of the chemoattractant source and contraction of the posterior of the cell. Our goal is to understand the mechanisms by which cells sense the direction of the chemoattractant gradient and utilize downstream signaling pathways to control this reorganization of the cytoskeleton. In this application, we focus on the role of the Dictyostelium PAK/Ste2O family member PAKa, which we have demonstrated is essential for the regulation of myosin assembly during chemotaxis, and the Rac family of small GTPases. We propose to elucidate the mechanisms by which PAKa is activated in response to chemoattractant signaling and understand the molecular interactions that regulate its subcellular localization and function in the posterior of chemotaxing cells. In addition, we propose experiments to examine the mechanism of activation of Rac1, which we have demonstrated is a key player in the control of the actin cytoskeleton during chemotaxis. These studies include investigating the kinetics and regulation of Rac1 activation and determining the possible changes in the subcellular localization of Rac1 in cells responding to a chemoattractant gradient. To understand the upstream regulation of Rac activation, we propose to examine the role of putative Rac exchange factors (GEFs) that control the activation of Rac proteins in response to various cellular stimuli. Using cell biological and molecular genetic approaches in Dictyostelium, we will define the role of Rac GEFs and how they are regulated, with a specific focus on examining changes in their subcellular localization and the role of such changes in regulating directional responses. Our proposed studies should also help elucidate how PAKa and Rac integrate into other signaling pathways that are essential for controlling the ability of Dictyostelium cells to directionally sense and respond to chemoattractant gradients. Understanding the mechanisms that regulate PAKa, Rac1, and RacGEFs in Dictyostelium will help define general mechanisms controlling cell polarization and movement that should be applicable to determining how chemotaxis is regulated in a broad range of cells.

DESCRIPTION (provided by applicant): Chemotaxis, or directed cell movement toward a small molecule ligand, plays a key role in many cellular and physiological responses, including metastasis of cancer cells, movement of neutrophils and macrophage in immunity, migration of embryonic cells during development, and aggregation of Dictyostelium during development. Cells must be able to respond to a shallow extracellular chemoattractant gradient and convert this into a steep intracellular gradient of signaling components, which controls the spatially restricted polymerization of F-actin at the leading edge (and to a lesser degree the cell's posterior) and assembly and contraction of myosin II at the cell's posterior. This requires the integration of several signal transduction pathways, many of which are conserved between Dictyostelium and man. Recent findings have established that phosphatidylinositol 3-kinase (PI3K) and MAP kinases cascades as key regulators of a cell's responses to directional signals. This proposal focuses on the further analysis of the Dictyostelium MEK1/ERK1 MAP kinase cascade and its role in controlling chemotaxis. The proposal takes advantage of the biochemical, genetic, and cell biological approaches available to dissect regulatory pathways in this experimental system. We have demonstrated that MEK1 and its downstream MAP kinase ERK1 are required for proper chemotaxis and that both components localize to the leading edge in chemotaxing cells. We have discovered that the control of the subcellular localization of these components requires the reversible SUMOylation of MEK1 and that adaptation of this pathway, which is essential for proper development, involves feedback regulation by the ERK1 and PI3K pathways. Our goal is to elucidate how MEK1 and ERK1 regulate chemotaxis by determining which chemotaxis functions are defective in mek1/erk1 null cells. We will identify and examine the function of ERK1 substrates through two-hybrid screens and then examine their function through biochemical approaches, and mutant screens. In addition, we will elucidate the mechanisms that control MEK1 deSUMOylation and pathway adaptation. Lastly, we will determine the role of SMEK1, an evolutionarily-conserved, second-site suppressor of MEK1 that was identified in my lab in regulating the MEK 1/ERK1 pathway and chemotaxis. The work proposed in this application should provide new and important insights into mechanisms that control this highly evolutionarily conserved cell biological process, and thus provide the needed background to elucidate the cellular basis underlying a variety of human diseases, including those affecting innate immunity and metastasis of cancer cells.

Accounting; Address; Back; base; Biochemical; cell motility; Cells; Chemotactic Factors; Chemotaxis; Communication; Complex; Computer Simulation; Cyclic AMP; Data; Devices; Dictyostelium; Diffusion; Event; Experimental Designs; Experimental Models; Individual; inhibitor/antagonist; Kinetics; Ligand Binding; Ligands; mathematical model; Measures; Methods; Microfluidic Microchips; Modeling; Motion; mutant; neutrophil; novel; Pathway interactions; Phase; Positioning Attribute; Process; programs; receptor; receptor binding; Reporter; Research Personnel; research study; Resolution; response; Role; Side; Signal Pathway; Signal Transduction; simulation; Source; Staging; Stimulus; Techniques; Testing; three dimensional structure; Time; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ras is a key regulator of directional sensing during chemotaxis and has been proposed to be a key component of the cell's directional compass. We have demonstrated that the RasGAP (GTPase activation protein) controls the spatiotemporal regulation of Ras activity and the cell's ability to sense the chemoattractant gradient. What is not clear is where NF1 resides in the cell. We propose to use TIRF to localize NF1 in response to chemoattractant stimulation as this will help us model the signaling pathway.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ras is a key regulator of directed cell migration, controlling directional sensing, cell motility as well as signal relay in Dictyostelium. We showed that Ras is preferentially activated at the plasma membrane at the leading edge of migrating cells, resulting in the localized activation of downstream effectors that regulate the actin cytoskeleton and thereby promote directed cell movement. We recently identified a protein complex made of two Ras guanine exchange factors (RasGEFs), which are activators of Ras, a scaffold protein, and the protein phosphatase 2A (PP2A). We found that this RasGEF complex selectively controls an important Ras signaling pathway that regulates both chemotaxis and signal relay. Since activated Ras is locally enriched at the front of migrating cells, we hypothesized that the RasGEFs would be similarly localized. We tested this hypothesis by fusing GFP to different components of the RasGEF complex and assessing their localization in chemotaxing cells under a confocal microscope. We now have evidence that upon sudden uniform chemoattractant stimulation that the RasGEF complex is transiently translocated from the cytoplasm to the plasma membrane. However, because of the strong cytosolic signal, we were unable to determine if the RasGEF complex is present at the plasma membrane in polarized cells that are migrating in a chemoattractant gradient. We were previously faced with a similar problem when studying the localization of a Ras GTPase activating protein (RasGAP), a Ras inhibitor in this case, and TIRF microscopy allowed us to detect plasma membrane localization of this protein when other types of microscopy did not. We are thus confident that TIRF microscopy would also provide us with an answer concerning any plasma membrane localization of the RasGEF complex in chemotaxing cells. All the conditions to look at chemotaxing cells in TIRF microscopy have been optimized already so we expect we will be able to get all of the needed data in only a few sessions. We propose to look at two different cell lines: one expressing a GFP-fused protein from the RasGEF complex and a control cell line expressing soluble, cytosolic GFP.

DESCRIPTION (provided by applicant): Eukaryotic cell motility is essential for many physiological processes such as embryonic development, tissue renewal, and the function of the immune system. Dictyostelium discoideum has proven to be an excellent model for the chemotactic migration of amoeboid cells such as leukocytes. Amoeboid migration is the result of the sequential repetition of pseudopod protrusions and retractions and is driven by the generation of traction forces. The strength and spatiotemporal organization of traction forces is determined by the coordinated interactions of actin-directed motors, F-actin regulation, actin crosslinking, motor protein-mediated contractility, and cell adhesions. However, precise knowledge of the biophysical coordination of these processes has been limited by the lack of quantitative information and analysis. We and others have observed that a considerable portion of the changes in cell shape occurring during amoeboid migration are due to periodic repetitive events, which enables the use of conditional statistics methods to analyze the network of biochemical processes involved in cell motility. The primary goal of this research is to determine in a statistically-robust, quantitative manner the biochemical basis for the spatiotemporal distribution of traction forces and the duration of each phase of the motility cycle by studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the different stages of the motility cycle. We hypothesize that Myosin II is essential not only to the contractility phase of the motility cycle but also to the pseudopod protrusion phase. The generation of the traction forces depends not only on the contractile action of Myosin II, but also in its actin crosslinking effect. Based on preliminary results, we further hypothesize that the spatiotemporal distribution of traction forces and the average distance a cell travels per cycle depend on actin polymerization. To test the above hypotheses, we propose three Specific Aims. Specific Aim 1 is to apply our new 3D force cytometry method to measure the three components of the forces exerted by the cells on the substrate. The second and third aims are aimed at studying the role of candidate cytoskeletal and regulatory molecules with known or suspected involvement in the motility by undertaking systematic comparison of wild type cells and mutant strains with actin crosslinking or motor protein contractility defects (Specific Aim 2), and F-actin regulation defects (Specific Aim 3). Our method consists of simultaneously measuring the spatial and temporal changes in the distribution of fluorescently tagged signaling (or cytoskeletal) proteins and the 3D traction forces that mediate each stage of the cell motility cycle, while also recording the changes in cell shape. We will apply conditional statistics and Principal Component Analysis (PCA) to connect specific biochemical processes to each of the physical events in the motility cycle. Our studies will provide the necessary building blocks to begin constructing the complex network of biochemical processes controlling cell migration. PUBLIC HEALTH RELEVANCE: Motility of eukaryotic cells is essential for many physiological processes such as embryonic development, and tissue renewal, as well as for the function of the immune system. Incorrect regulation of motility plays an important part in many diseases (cancer, destructive inflammation, osteoporosis, mental retardation, etc.), and therefore, future therapeutic approaches will benefit from a precise quantitative understanding of the biophysical processes controlling cell motility. The aim of this study is to establish the mechanisms whereby each individual stage of the motility cycle is related to specific biochemical signaling events, and to elucidate the effects that the regulation of these signaling pathways has on cell motility, with the ultimate goal of developing a level of understanding of the biomechanical processes sufficient to predict and control cell motility.

Address; Affect; Algorithms; Back; Binding; Caliber; Cells; Chemotactic Factors; Chemotaxis; Computer Simulation; Cyclic AMP; Data; Data Analyses; Dictyostelium discoideum; Exhibits; experience; Kinetics; Ligands; Measures; Membrane; Microfluidic Microchips; Modeling; novel; Pathway interactions; Phase; Process; Protocols documentation; Ras Signaling Pathway; receptor; Reporter; research study; response; Role; Signal Pathway; Signal Transduction; Source; Spatial Distribution; Stimulus; temporal measurement; Testing; Time; Work;

? DESCRIPTION: The innate and adaptive immune response involves the recruitment of leukocytes from the blood stream to the site of infection and inflammation. Upon reaching the location, leukocytes clear invaders and begin the process of digesting and repairing damaged tissues. However, when the body fails to properly regulate the recruitment of leukocytes, the inflammation can become chronic, resulting in irreversible tissue injury and loss of functionality. Rheumatoid arthritis, inflammatory bowel disease, type-1 diabetes, and multiple sclerosis are all examples of autoimmune diseases caused by the uncontrolled recruitment of leukocytes. While much research has been dedicated to the identification of the cascade of specific biochemical processes involved in the recruitment of leukocytes, much less is known about the mechanical events driving their migration, in particular how they generate the necessary traction forces to cross the vascular wall and further traverse the three- dimensional (3-D) extravascular space. Thus, the main objective of this study is to provide the much needed complementary information connecting specific cell molecular processes (i.e., adhesion dynamics, actin turnover, and myosin II contraction) to the generation of cellular forces that regulate leukocyte extravasation and their subsequent directional migration in 3-D extravascular tissues through the use of novel 3D Fourier Traction Force Microscopy (3DFTFM) techniques and genetic and pharmacological manipulations. To achieve this objective, we propose three Specific Aims. We will first characterize the temporal and spatial generation of 3-D traction forces exerted by leukocytes crawling on flat surfaces (Aim 1); we will then investigate the mechanical processes regulating transmigration across the vascular endothelial monolayer and the basement membrane (Aim 2); and finally, we will develop a novel Elastographic 3DTFM to determine both traction stresses and the non-linear material properties of the Extra Cellular Matrix to elucidate the molecular mechanisms regulating the mechanics of leukocytes' chemotactic migration in 3-D environments (Aim 3). The proposed in vitro approach overcomes a number of existing challenges to measuring the 3-D traction forces driving leukocyte extravasation and migration and builds on the extensive experience accumulated by our multidisciplinary team of biologists and engineers who have been studying the mechanics of amoeboid cell migration for the last seven years. The outcome of this research will result in a far more comprehensive understanding of the mechanics of leukocyte motility than that available to date and will have the potential to aid the development of new approaches that could target specific mechanical processes to inhibit (or slow down) leukocyte motility and help in the design of complementary regimens to treat inflammatory diseases.