Michael Gold

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Detecting clusters of data points in physical or high-dimensional (HD) space is a common task in biology and biomedicine. Single-molecule localization microscopy (SMLM), a category of super-resolution microscopy, is often used to analyze spatial distributions of proteins on the surface membranes of, or inside, biological cells. Proteins sometimes need to form clusters to surpass a critical signalling threshold for functional activity. Therefore, investigating protein clustering can yield important insights about protein and cell functions in health and disease. Mass cytometry, also called CyTOF, is a high-throughput technique for investigating the abundance of multiple proteins simultaneously in single cells, resulting in HD data in which cells cluster into different phenotypes. Cluster analysis of CyTOF data is important for understanding heterogeneity in biological cell populations, which has clinical implications in cancer biology.This dissertation first describes a new method, called StormGraph, to detect clusters in diverse SMLM data. StormGraph converts 2D or 3D SMLM data to a weighted graph, applies a community detection algorithm to assign localizations to clusters at multiple scales, and includes a new algorithm to generate a single-level clustering from a multi-level cluster hierarchy. Unlike most other clustering algorithms, StormGraph utilizes uncertainties associated with point positions. Results of using SMLM and StormGraph to analyze clustering of B-cell antigen receptors on the membranes of normal and malignant B cells are presented. Next, this dissertation describes a new measure of similarity between clusters in HD data. Computed by a method called ASTRICS, it is based on local dimensionality reduction and triangulation of alpha shapes. A strategy for clustering and visualizing HD data, with ASTRICS used to construct a graph from an initial set of fine-grained clusters, is presented and demonstrated on three very different HD datasets, including public CyTOF data. Finally, new CyTOF experiments were designed and performed to analyze heterogeneity among diffuse large B-cell lymphoma (DLBCL) cell lines. Results of the analysis, including clustering and visualization using the strategy based on ASTRICS, are presented. Most interesting were revelations about signalling dynamics linked to the cell cycle, which differed between DLBCL subtypes.

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The immune synapse is a contact-dependent cellular communication platform that is important for activating, amplifying and executing immune responses. A B cell immune synapse is formed when B cells encounter antigens (Ags) on the surface of an Ag-presenting cell (APC). The binding of the B cell receptor (BCR) to these Ags triggers dynamic, multi-scale reorganization of the BCR and associated signal transduction machinery. Initially, BCRs are gathered into microclusters that are dispersed throughout the B cell-APC contact site. Microclusters are then moved centripetally and coalesce into the central supramolecular activation cluster (cSMAC) of an immune synapse. Although the formation of BCR-Ag microclusters recruits key signaling enzymes and increases BCR signaling efficiency, the mechanisms controlling BCR organization during immune synapse formation are not fully understood. Importantly, how the spatial patterning of the BCR promotes the translation of extracellular information (recognition of membrane-associated Ags) into functional outcomes (B cell activation) is not clear. I showed that the coalescence of BCR microclusters is dependent on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, I showed that this dynamic spatial reorganization of BCR microclusters is critical for amplifying proximal BCR signaling reactions and enhancing the ability of membrane-associated Ags to induce transcriptional responses and proliferation. My finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound Ag, but not for soluble Ags, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound Ags. I also demonstrated that the disassembly of actin networks mediated by the actin severing protein, cofilin, and its regulators is important for cytoskeletal remodeling during immune synapse formation. In addition, I showed that actin dynamics mediated by cofilin activity is important for amplifying BCR signaling in response to membrane- bound Ags. Thus, by controlling the dynamic spatial reorganization of BCR-Ag microclusters, the actin cytoskeleton amplifies APC-induced BCR signaling and B cell activation responses.

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B cells that bind antigens displayed on antigen-presenting cells (APCs) form an immune synapse (IS), a polarized cellular structure that optimizes the dual functions of the B cell receptor (BCR), signal transduction and antigen internalization. Immune synapse formation involves the polarization of the microtubule-organizing center (MTOC) towards the APC. I showed that BCR-induced MTOC polarization requires the Rap1 GTPases (which has two isoforms, Rap1a and Rap1b), an evolutionarily conserved regulator of cell polarity, as well as cofilin-1, an actin-severing protein that is regulated by Rap1. MTOC reorientation towards the antigen contact site correlated strongly with cofilin-1-dependent actin reorganization and cell spreading. I also showed that BCR-induced MTOC polarization requires the dynein motor protein as well as IQGAP1, a scaffolding protein that can link the actin and microtubule cytoskeletons. At the periphery of the immune synapse, IQGAP1 associates closely with F-actin structures and with the microtubule plus-end-binding protein CLIP-170. Moreover, the accumulation of IQGAP1 at the antigen contact site depends on F-actin reorganization that is controlled by Rap1 and cofilin-1. I also demonstrate that the hematopoietic-cell specific cortactin-homologue, HS1, is essential for regulating actin cytoskeletal remodeling during immune synapse formation and acts downstream of Rap to promote BCR-induced antigen gathering. Additionally, inhibiting the Rap1-cofilin-1 pathway, CLIP-170 expression, or cytoskeletal dynamics impairs the ability of B cells to acquire antigens from APCs. Thus, Rap1 coordinates actin and microtubule organization at the IS, facilitating antigen acquisition from APCs.

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Interactions with the extracellular matrix (ECM) are critical for tumor cell survival and dissemination. Cell-ECM interactions are mediated primarily by integrins, cell surface receptors that nucleate the formation of adhesomes. Adhesome complexes contain signaling proteins as well as proteins that link integrins to the cytoskeleton, thereby transducing extracellular forces into the cell. Both altered ECM composition and altered adhesome signaling can contribute to cancer progression. In this thesis I tested the hypothesis that ECM-integrin interactions drive cancer progression and that this depends on the adhesome proteins FAK and talin, and on mechanobiological tension.In chapter 3, I show that in B16F1 melanoma cells, the expression of three cancer signature genes, Cyr61, MUC18 and TRPM1, is strongly regulated by cell-ECM adhesion, independently of cytoskeletal tension. In chapter 4, I used global transcriptome profiling to compare gene expression changes caused by increased ECM ligand density versus cytoskeletal tension. These two perturbations regulated genes that belong to distinct pathways. Increasing ECM ligand density upregulated genes that are associated with adhesion, migration and ECM remodelling pathways, as well as the Hippo pathway, whereas applying mechanical stretch to the cells upregulated genes associated with metabolic pathways and the HIF-1 pathway. In chapter 5, I show that the regulation of the cancer signature genes is dependent on the adhesome proteins talin and FAK. Consistent with a role for talin in adhesome signaling, loss of talin had the same effect on MUC18 and TRPM1 mRNA levels as forcing B16F1 cells into suspension. Knocking down FAK however, regulated Cyr61 and MUC18 differently than knocking down talin.In chapter 6, I show that the two isoforms of talin, talin 1 and talin 2, differentially regulate expression of the three cancer signature genes and have different effects on B16F1 spreading, migration and in vivo tumor growth. Together, my findings illustrate the complexity of how changes in ECM-cell interactions, and subsequent adhesome signaling, influence processes that are critical for cancer progression.

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The nucleation, polymerization, and depolymerization of actin filaments is spatially and temporally controlled in order to regulate cell motility, cell morphology, and protein organization within the plasma membrane. By limiting receptor diffusion, the submembrane actin cytoskeleton modulates the signaling output of receptors such as the B-cell antigen (Ag) receptor (BCR) that are activated by clustering. Restricting BCR mobility limits BCR-BCR collisions and the resultant ‘tonic’ signaling. Conversely, more dynamic actin filaments or F-actin clearance promotes BCR-BCR collisions and leads to a ‘primed’ state where the threshold for Ag-induced activation is reduced. In chapter 2, I show a mechanism of receptor cross-talk where microbial danger signals (TLR ligands) prime B cells for Ag-induced activation by enhancing actin dynamics. TLR signaling reduced BCR confinement, promoted BCR-BCR collisions and potentiated responses to low densities of membrane-associated Ags.The interaction of B-cells with antigen-presenting cells displaying membrane-Ags results in initial BCR signaling that promotes cell spreading and increases the probability of BCRs encountering Ag. This is coupled with increased BCR mobility and the formation of BCR microclusters that recruit and activate signaling enzymes. Cell spreading and BCR microcluster mobility require severing of cortical submembrane actin, a precursor to F-actin branching that drives cell spreading. In chapter 3, I show that BCR signaling increases actin dynamics and BCR microcluster formation by activating the actin-severing protein cofilin via a signaling pathway involving Rap GTPases.

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The ability of the B cell receptor (BCR) to stimulate integrin-mediated adhesion, and induce cytoskeletal reorganization and cell spreading enhances the ability of B cells to bind and respond to antigens (Ag). The proper localization and trafficking of B cells in the secondary lymphoid organs are also critical for B cells to encounter Ags and to be activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that have been shown to regulate cell adhesion, morphology, and migration. However, their functions in B cells are not clear. The overall hypothesis of this thesis was that Pyk2 and FAK are downstream targets of BCR, integrin, and chemokine receptor signaling, and that they are involved in B cell morphological regulation, migration, and adhesion. I showed that the BCR and integrins collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, modifications that increase the kinase activity of Pyk2 and FAK. Activation of the Rap1 GTPase is critical for BCR-induced integrin activation and for BCR-induced reorganization of the actin cytoskeleton and I showed that inhibition of Pyk2 and FAK function by either gene knockdown or the use of chemical inhibitors impaired B cell spreading. Marginal zone (MZ) B cells are innate-like B cells that are responsible for T cell-independent responses to microbial pathogens. The proper localization of MZ B cells is dependent on integrated migration and retention signals provided by the stromal cells in the spleen. Because MZ B cells are not found in Pyk2-/- mice, I hypothesized that Pyk2 and FAK are involved in MZ B cell retention in the spleen. I showed that Pyk2 and FAK are required for MZ B cell migration and that Pyk2 is required for integrin-dependent adhesion in response to chemoattractant stimulation. Moreover, I found that FAK is involved in chemokine-induced Akt phosphorylation in MZ B cells. In summary, Pyk2 and FAK are downstream targets of the Rap GTPases and play a key role in regulating B cell morphology, migration, and adhesion.

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B cells eliminate pathogens by producing antibodies, activating other immune cells, and secreting cytokines. To carry out these functions, B cells must traffic from the bone marrow into the blood and then into secondary lymphoid organs to encounter antigens and become activated. As a result, B cell trafficking is highly regulated and critical for the activation of self-reactive B cells that contribute to autoimmunity and the spread of malignant B cells. The Rap GTPases regulate integrin activation and in chapter two I showed that Rap1 activation is required for B cell migration and adhesion. Because, lipid mediators including Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are potent regulators of cell adhesion and migration, I asked whether LPA regulates B cell adhesion and migration. I found that LPA reduces B cell migration by favoring strong integrin-mediated adhesion.Phosphatidylinositide 3-kinase (PI3K) controls multiple proteins that regulate cell motility, survival and activation. Therefore in the third chapter I investigated the role of p110δ, the major isoform of PI3K in B cells, in the activation, migration, and function of conventional and innate-like B cells. B-2 cells are involved in T cell-dependent antibody responses while B-1 and marginal zone (MZ) B cells are innate-like lymphocytes that mediate T cell-independent responses to microbial antigens. Importantly, these cells are also responsible for many antibody-mediated autoimmune diseases. I showed that p110δ is needed for the activation and chemotaxis of B-2, B-1 and MZ B cells. I also showed that the in vitro adhesion and in vivo localization of MZ B cells is dependent on p110δ activity. Interestingly, I found that p110δ activity is needed for Rap1 activation, making p110δ a key regulator of B cell trafficking. B-1 and MZ B cells produce natural antibodies in the absence of immunization that often recognize self-antigens. We showed that the production of natural antibodies, both protective and pathogenic, depends on p110δ activity and that p110δ inhibition can reduce the levels of pathogenic auto-antibodies in collagen-induced arthritis.This work suggests that by regulating Rap, p110δ, or LPA, it may be possible to control B cell-mediated diseases including inflammation, autoimmunity, and cancer.

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B-lymphocytes rearrange their cytoskeleton and undergo dramatic morphological changes when searching for antigens and when forming immune synapses upon contacting cells that display antigens on their surface. Although these morphological changes are essential to B cell function, the signaling pathways underlying these processes are not fully understood. The aim of this thesis is to investigate how B cell receptor (BCR) and integrin signaling regulate B cell morphological changes. The Rap GTPases (Rap) are molecular switches that regulate integrin activation, adhesion, migration in B cells and other cell types. I hypothesize that activation of the Rap GTPases is important for regulating changes in B cell morphology. Indeed, in this thesis I showed that activation of Rap is essential for B cell cytoskeletal rearrangements. I found that Rap activation is important for BCR- and lymphocyte function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens leading to B cell activation. Consistent with Rap activation being important for B cell adhesion and migration, I showed that Rap activation is important for the dissemination of B cell lymphomas in vivo. B cell lymphomas are common malignancies in which transformed B cells enter the circulation, extravasate into tissues, and form tumors in multiple organs. Lymphoma cells are thought to exit the vasculature and enter tissues via the same chemokine- and adhesion molecule-dependent mechanisms as normal B cells. Using A20 murine B lymphoma cells, I showed that Rap activation is important for circulating lymphoma cells to invade tissue and form tumors in vivo in syngeneic mice. Moreover, using in vitro models I showed that Rap activation is required for these cells to extend membrane processes between vascular endothelial cells and undergo transendothelial migration. Thus, by controlling B cell morphology and cytoskeletal organization, the Rap GTPases play a key role in both malignant and normal B cell functions, and may be a potential therapeutic target for treatment of B cell-related diseases.

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