Michael Gold
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
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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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The immune synapse (IS) is a special polarized structure that optimizes the functions of the B cell receptor (BCR), signal transduction and antigen internalization. The B cell IS is formed when B cells bind antigens (Ags) that are displayed on the surface of Ag-presenting cells (APCs), creating an area of contact between the two cells. The binding of the BCR to the Ag on the APC triggers rapid and dynamic reorganization of the BCRs and the cytoskeleton. This allows the cell to spread across the surface of the APC and induces the formation of BCR microclusters throughout the contact site. The microclusters then move centripetally and coalesce into the central supramolecular activation cluster (cSMAC) of an IS. A major signaling pathway initiated by the binding of the BCR to Ags is the Ras/MEK/ERK pathway. Although ERK activity has been implicated in B cell survival and proliferation, the role of ERK in regulating BCR-induced cytoskeletal reorganization, IS formation, and APC-induced BCR signaling has not been investigated. I showed that ERK activity is important for B cells to spread on immobilized Ag. Inhibiting ERK resulted in decreased spreading and altered actin organization. By imaging B cell-APC interactions, I also showed that inhibiting ERK activity impaired IS formation, resulting in decreased APC-induced BCR signaling and delayed cSMAC formation. Thus, ERK may regulate actin-dependent processes that are important for B cell responses to immobilized and APC-bound Ags.
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Mast cells are central players in inflammatory responses, as they secrete a number of factors that regulate the responses of other immune cells and they participate in pathogen sensing and clearance. However, mast cells are best known for their role in allergic diseases. Mast cells express the cell surface receptor, Fcε Receptor I (FcεRI), which binds the constant region of IgE antibodies. Binding of multivalent antigens to FcεRI-bound IgE initiates intracellular signaling cascades that lead to effector functions such as degranulation and migration. During degranulation, storage granules containing pre-synthesized bioactive molecules such as histamine fuse with the plasma membrane and release their contents. Secretion of granule contents requires dynamic reorganization of the submembrane actin cytoskeleton. Actin dynamics are also required to generate protrusive forces at the leading edge of the cell during antigen-induced mast cell migration. Despite the importance of the actin cytoskeleton for mast cell-mediated immune responses, how actin dynamics are regulated during mast cell activation is poorly understood. Although the actin severing protein, cofilin, is activated by FcεRI signaling, the role of cofilin in actin-dependent mast cell functions has not been investigated. By using siRNA to deplete cofilin in a murine bone marrow-derived mast cell line, I found that cofilin is important for FcεRI-induced actin breakdown as well as mast cell degranulation, cell spreading, and migration. Potential upstream regulators of cofilin activity and actin remodeling are the Rap GTPases but their role in mast cells has not been investigated. The Rap GTPases are members of the Ras family of GTPases that are important for actin reorganization, integrin activation, cell polarity, and proliferation. I showed for the first time that the Rap1 and Rap2 GTPases are activated by FcεRI signaling. Furthermore, I found that Rap1 and Rap2 were important for FcεRI-induced actin breakdown and migration towards antigen but not for degranulation or cell spreading. Interestingly, Rap1 and Rap2 acted independently of cofilin to regulate actin turnover. This work has identified two novel regulatory pathways that facilitate actin-dependent FcεRI-induced mast cell effector functions and broadens our understanding of the signaling pathways that regulate mast cell activation in allergy and inflammation.
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Processing bodies (P-bodies) are cytoplasmic aggregates that contain translationally-repressed mRNAs in complex with repressor proteins (GW182, RCK/p54, and DCP1a), facilitate mRNA storage or degradation, and can be identified by the αGW-body (GWB) serum that detects several P-body proteins. The partitioning of mRNAs between a translationally-competent cytoplasmic pool and a translationally-repressed P-body pool could be an important mechanism for dynamically controlling the synthesis of key proteins. Memory CD8⁺ T lymphocytes contain translationally-repressed RANTES and IFN-γ mRNAs, enabling the secretion of these cytokines within 30 minutes of T cell receptor (TCR) engagement. Although P-bodies have not been characterized in lymphocytes, I hypothesized that storage of RANTES and IFN-γ mRNAs in P-bodies could contribute to the ability of memory CD8⁺ T cells to mount rapid recall responses. Using immunoblotting, flow cytometry, and confocal microscopy, I established that T and B lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a, which are concentrated in cytoplasmic granules. Co-localization analysis identified multiple subsets of P-bodies, raising the possibility that P-bodies with different protein compositions have distinct functional properties. Moreover, I found that P-bodies partially dissociate and move towards the model immune synapse in both T and B lymphocytes. To explore the role of P-bodies in the recall response, I utilized the OT-I model to generate effector and memory CD8⁺ T lymphocytes in vitro. Compared to naïve CD8⁺ T cells from OT-I mice, effector T cells had elevated levels of P-body proteins and a greater number of P-bodies. In contrast, memory T cells had similar numbers of P-bodies as naïve T cells, but contained larger GWBs and RCK/p54 granules. Remarkably, RANTES mRNA did not co-localize with P-bodies in memory T cells, but was distributed diffusely in the cytoplasm. Conversely, IFN-γ mRNA co-localized with GWBs and RCK/p54 granules in memory T cells. The abundance of P-body-targeting AU-rich elements (AREs) in IFN-γ mRNA and the absence of AREs in RANTES mRNA suggests that IFN-γ mRNA transcribed following activation of naïve T cells, is directed for storage into GWB⁺ RCK/p54⁺ P-bodies to be reused during the recall response, whereas RANTES mRNA is stored by an undefined P-body-independent mechanism.
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Naïve resting B cells circulate throughout the body and enter secondary lymphoid organs (SLOs) in response to chemical cues known as chemokines. Within SLOs, B cells scan for foreign antigens, and antigen-induced clustering of the B cell receptor (BCR) initiates B cell activation. This clustering is enhanced through the formation of an immune synapse between the B cell and antigen presenting cell, which greatly amplifies BCR signaling and B cell activation. We have previously shown that activation of the Rap GTPases is important for the cytoskeletal changes that underlie B cell spreading, migration, and immune synapse formation. We have also shown that stimulating B-lymphocytes with a model particulate antigen, anti-BCR-coated polystyrene beads, causes cells to polarize and form actin-rich cups at sites of cell:bead contacts, and that the formation of these cups enhance B cell activation. Although a number of downstream targets of activated Rap (Rap-GTP) have been identified, the mechanisms by which Rap-GTP promotes cell polarization, actin polymerization, reorganization of the actin cytoskeleton, and changes in cell morphology are not completely understood.The establishment of cell polarity often involves complexes of evolutionarily conserved polarity proteins. One such polarity complex includes the Scribble protein. In this thesis, I show that Rap-GTP is important for generating an asymmetric distribution of Scribble when B cells encounter particulate antigens and form cups. The ability of leukocytes to assume a polarized morphology also requires that the cortical F-actin cytoskeleton be disassembled so that new F-actin filaments that contribute to the formation of a leading edge, F-actin-rich cup, or immune synapse can be assembled. Since cofilin plays a major role in severing and depolymerizing F-actin filaments, I tested the hypothesis that the Rap GTPases regulate changes in cell shape by controlling the activation of cofilin, a process that requires its dephosphorylation by the Slingshot phosphatase. I show that BCR clustering leads to cofilin dephosphorylation, and hence activation, which is dependent on activation of the Rap GTPases. This suggests that an early step underlying BCR-induced changes in B cell morphology that involve reorganization of the actin cytoskeleton is the Rap-dependent activation of the actin-severing protein cofilin.
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