Arpita Upadhyaya

University of Maryland, College Park, College Park, MD, United States

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Publications (35)124.53 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Antigen binding to the B cell receptor (BCR) induces receptor clustering, cell spreading, and the formation of signaling microclusters, triggering B cell activation. Although the biochemical pathways governing early B cell signaling have been well studied, the role of the physical properties of antigens, such as antigen mobility, has not been fully examined. We study the interaction of B cells with BCR ligands coated on glass or tethered to planar lipid bilayer surfaces to investigate the differences in B cell response to immobile and mobile ligands. Using high-resolution total internal reflection fluorescence (TIRF) microscopy of live cells, we followed the movement and spatial organization of BCR clusters and the associated signaling. Although ligands on either surface were able to cross-link BCRs and induce clustering, B cells interacting with mobile ligands displayed greater signaling than those interacting with immobile ligands. Quantitative analysis revealed that mobile ligands enabled BCR clusters to move farther and merge more efficiently than immobile ligands. These differences in physical reorganization of receptor clusters were associated with differences in actin remodeling. Perturbation experiments revealed that a dynamic actin cytoskeleton actively reorganized receptor clusters. These results suggest that ligand mobility is an important parameter for regulating B cell signaling.
    Biophysical Journal 01/2014; 106(1):26-36. · 3.67 Impact Factor
  • King Lam Hui, Sae In Kwak, Arpita Upadhyaya
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    ABSTRACT: Contact formation of T cells with antigen presenting cells results in the engagement of T cell receptors (TCRs), recruitment and aggregation of signaling proteins into microclusters and ultimately, T cell activation. During this process, T cells undergo dramatic changes in cell shape and reorganization of the cytoskeleton. While the importance of the cytoskeleton in T cell activation is well known, the dynamics of the actin cytoskeleton and how it correlates with signaling clusters during the early stages of spreading is not well understood. Here, we used total internal reflection fluorescence microscopy to study the dynamics of actin reorganization during Jurkat T cell spreading and the role of integrin ligation by the adhesion molecule, vascular cell adhesion molecule (VCAM), in modulating actin dynamics. We found that when T cells spread on anti-CD3 antibody-coated glass surfaces, the cell edge exhibited repeated protrusions and retractions, which were driven by wave like patterns of actin that emerged from signaling microclusters. Addition of VCAM on the activating substrate altered the dynamics of actin both globally and locally, leading to a smooth expansion of the cell edge and the disappearance of waves. Our results suggest that the actin cytoskeleton in Jurkat cells is capable of organizing into spatial patterns initiated by TCR signaling and regulated by integrin signaling. © 2013 Wiley Periodicals, Inc.
    Cytoskeleton 11/2013; · 2.87 Impact Factor
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    ABSTRACT: Upon recognizing cognate antigen, B cells mobilize multiple cellular apparatuses to propagate an optimal response. Antigen binding is transduced into cytoplasmic signaling events through B-cell antigen receptor (BCR)-based signalosomes at the B-cell surface. BCR signalosomes are dynamic and transient and are subsequently endocytosed for antigen processing. The function of BCR signalosomes is one of the determining factors for the fate of B cells: clonal expansion, anergy, or apoptosis. Accumulating evidence underscores the importance of the actin cytoskeleton in B-cell activation. We have begun to appreciate the role of actin dynamics in regulating BCR-mediated tonic signaling and the formation of BCR signalosomes. Our recent studies reveal an additional function of the actin cytoskeleton in the downregulation of BCR signaling, consequently contributing to the generation and maintenance of B-cell self-tolerance. In this review, we discuss how actin remodels its organization and dynamics in close coordination with BCR signaling and how actin remodeling in turn amplifies the activation and subsequent downregulation process of BCR signaling, providing vital feedback for optimal BCR activation.
    Immunological Reviews 11/2013; 256(1):177-189. · 12.16 Impact Factor
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    ABSTRACT: Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.
    PLoS Biology 11/2013; 11(11):e1001704. · 12.69 Impact Factor
  • Wenxia Song, Chaohong Liu, Arpita Upadhyaya
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    ABSTRACT: The actin cytoskeleton is a dynamic cellular network known for its function in cell morphology and motility. Recent studies using high resolution and real time imaging techniques have revealed that actin plays a critical role in signal transduction, primarily by modulating the dynamics and organization of membrane-associated receptors and signaling molecules. This review summarizes what we have learned up to now about a regulatory niche of the actin cytoskeleton in the signal transduction of the B cell receptor (BCR). The activation of the BCR is initiated and regulated by a close coordination between the dynamics of surface BCRs and the cortical actin network. The actin cytoskeleton is involved in regulating the signaling threshold of the BCR to antigenic stimulation, the kinetics and amplification of BCR signaling activities, and the timing and kinetics of signaling down regulation. Actin exerts its regulatory function by controlling the kinetics, magnitude, subcellular location, and nature of BCR clustering and BCR signaling complex formation at every stage of signaling. The cortical actin network is remodeled by initial detachment from the plasma membrane, disassembly and subsequent reassembly into new actin structures in response to antigenic stimulation. Signaling responsive actin regulators translate BCR stimulatory and inhibitory signaling into series of actin remodeling events, which enhance signaling activation and down-regulation by modulating the lateral mobility and spatial organization of surface BCR. The mechanistic understanding of actin-mediated signaling regulation in B cells will help us to explore B cell-specific manipulations of the actin cytoskeleton as treatments for B cell-mediated autoimmunity and B cell cancer. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters.
    Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
  • King Lam Hui, Arpita Upadhyaya
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    ABSTRACT: T cell activation is a key process in cell-mediated immunity, and engagement of T cell receptors by peptides on antigen presenting cells leads to activation of signaling cascades as well as cytoskeletal reorganization and large scale membrane deformations. While significant advances have been made in understanding the biochemical signaling pathways, the effects imposed by the physical environment and the role of mechanical forces on cell activation are not well understood. In this study, we have used anti-CD3 coated elastic polyacrylamide gels as stimulatory substrates to enable the spreading of Jurkat T cells and the measurement of cellular traction forces. We have investigated the effect of substrate stiffness on the dynamics of T cell spreading and cellular force generation. We found that T cells display more active and sustained edge dynamics on softer gels and that they exert increased traction stresses with increasing gel stiffness. A dynamic actin cytoskeleton was required to maintain the forces generated during activation, as inferred from small molecule inhibition experiments. Our results indicate an important role for physical properties of the antigen presenting cell as well as cytoskeleton-driven forces in signaling activation.
    03/2013;
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    ABSTRACT: B cells are activated upon binding of the B cell receptor (BCR) with antigen on the surface of antigen presenting cells (APC). Activated B cells deform and spread on the surface of APCs which may comprise of complex membrane topologies. In order to model the diverse range of topographies that B cells may encounter, substrates fabricated with vertical ridges separated by gaps ranging from hundreds of nm to microns were coated with activating antigen to enable B cell spreading. Simultaneous imaging of actin and BCR shows that the organization of both depends profoundly on the ridge spacing. On smaller ridge spacing (2 microns), actin forms long filopodial structures that explore the substrate parallel to ridges while the BCR clusters accumulate linearly along the direction of the ridges with limited ability to escape these channels. Cells on larger ridge spacing (2 microns) exhibit central actin patches and peripheral actin waves and form semi-stable polymerization zones at ridges, while BCR distribution is more homogeneous. Our results indicate that surface topography may be a critical determinant of cytoskeletal dynamics and the spatiotemporal organization of signaling clusters.
    03/2013;
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    ABSTRACT: Actin reorganization has been shown to be important for lymphocyte activation in response to antigenic stimulation. However, methods for quantitative analysis of actin dynamics in live lymphocytes are still underdeveloped. In this study, we describe new methods to examine the actin dynamics in B cells induced by antigenic stimulation. Using the A20 B cell line expressing GFP-actin, we analyzed in real time the redistribution of F-actin and the lateral mobility of actin flow in the surface of B cells in response to soluble and/or membrane associated antigens. Using fluorescently labeled G-actin, we identified the subcellular location and quantified the level of de novo actin polymerization sites in primary B cells. Using A20 B cells expressing G-actin fused with the photoconvertible protein mEos, we examined the kinetics of actin polymerization and depolymerization at the same time. Our studies present a set of methods that are capable of quantitatively analyzing the role of actin dynamics in lymphocyte activation.
    Biochemical and Biophysical Research Communications 09/2012; 427(1):202-6. · 2.41 Impact Factor
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    ABSTRACT: The morphology and duration of contacts between cells and adhesive surfaces play a key role in several biological processes, such as cell migration, cell differentiation, and the immune response. The interaction of receptors on the cell membrane with ligands on the adhesive surface leads to triggering of signaling pathways, which allow cytoskeletal rearrangement, and large-scale deformation of the cell membrane, which allows the cell to spread over the substrate. Despite numerous studies of cell spreading, the nanometer-scale dynamics of the membrane during formation of contacts, spreading, and initiation of signaling are not well understood. Using interference reflection microscopy, we study the kinetics of cell spreading at the micron scale, as well as the topography and fluctuations of the membrane at the nanometer scale during spreading of Jurkat T cells on antibody-coated substrates. We observed two modes of spreading, which were characterized by dramatic differences in membrane dynamics and topography. Formation of signaling clusters was closely related to the movement and morphology of the membrane in contact with the activating surface. Our results suggest that cell membrane morphology may be a critical constraint on signaling at the cell-substrate interface.
    Biophysical Journal 04/2012; 102(7):1524-33. · 3.67 Impact Factor
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    ABSTRACT: B cells encounter both soluble Ag (sAg) and membrane-associated Ag (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg- and sAg-stimulated B cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAg-stimulated B cells are greater than those in sAg-stimulated B cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg- and sAg-stimulated B cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but with much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg.
    The Journal of Immunology 03/2012; 188(7):3237-46. · 5.52 Impact Factor
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    ABSTRACT: Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with α-actinin in stress fibers and focal adhesions and binds to both actin and α-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and α-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of α-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and α-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.
    PLoS ONE 01/2012; 7(8):e42773. · 3.73 Impact Factor
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    ABSTRACT: The activation of the BCR, which initiates B cell activation, is triggered by Ag-induced self-aggregation and clustering of receptors at the cell surface. Although Ag-induced actin reorganization is known to be involved in BCR clustering in response to membrane-associated Ag, the underlying mechanism that links actin reorganization to BCR activation remains unknown. In this study, we show that both the stimulatory Bruton's tyrosine kinase (Btk) and the inhibitory SHIP-1 are required for efficient BCR self-aggregation. In Btk-deficient B cells, the magnitude of BCR aggregation into clusters and B cell spreading in response to an Ag-tethered lipid bilayer is drastically reduced, compared with BCR aggregation observed in wild-type B cells. In SHIP-1(-/-) B cells, although surface BCRs aggregate into microclusters, the centripetal movement and growth of BCR clusters are inhibited, and B cell spreading is increased. The persistent BCR microclusters in SHIP-1(-/-) B cells exhibit higher levels of signaling than merged BCR clusters. In contrast to the inhibition of actin remodeling in Btk-deficient B cells, actin polymerization, F-actin accumulation, and Wiskott-Aldrich symptom protein phosphorylation are enhanced in SHIP-1(-/-) B cells in a Btk-dependent manner. Thus, a balance between positive and negative signaling regulates the spatiotemporal organization of the BCR at the cell surface by controlling actin remodeling, which potentially regulates the signal transduction of the BCR. This study suggests a novel feedback loop between BCR signaling and the actin cytoskeleton.
    The Journal of Immunology 07/2011; 187(1):230-9. · 5.52 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100. · 3.67 Impact Factor
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    ABSTRACT: We have studied the spreading of Jurkat T-cells on anti-CD3 antibody-coated substrates as a model of immune synapse formation. Cell adhesion area versus time was measured via interference reflection contrast microscopy. We found that the spread area exhibited a sigmoidal growth as a function of time in contrast to the previously proposed universal power-law growth for spreading cells. We used high-resolution total internal reflection fluorescence microscopy of these cells transfected with GFP-actin to study cytoskeletal dynamics during the spreading process. Actin filaments spontaneously organized into a variety of structures including traveling waves, target patterns, and mobile clusters emanating from an organizing center. We quantify these dynamic structures and relate them to the spreading rates. We propose that the spreading kinetics are determined by active rearrangements of the cytoskeleton initiated by signaling events upon antibody binding by T-cell receptors. Membrane deformations induced by such wavelike organization of the cytoskeleton may be a general phenomenon that underlies cell movement and cell-substrate interactions.
    03/2009;
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    ABSTRACT: Vorticella convallaria is one of the fastest and most powerful cellular machines. The cell body is attached to a substrate by a slender stalk containing a polymeric structure-the spasmoneme. Helical coiling of the stalk results from rapid contraction of the spasmoneme, an event mediated by calcium binding to a negatively charged polymeric backbone. We use high speed imaging to measure the contraction velocity as a function of the viscosity of the external environment and find that the maximum velocity scales inversely with the square root of the viscosity. This can be explained if the rate of contraction is ultimately limited by the power delivered by the actively contracting spasmoneme. Microscopically, this scenario would arise if the mechanochemical wave that propagates along the spasmoneme is faster than the rate at which the cell body can respond due to its large hydrodynamic resistance. We corroborate this by using beads as markers on the stalk and find that the contraction starts at the cell body and proceeds down the stalk at a speed that exceeds the velocity of the cell body.
    Biophysical Journal 02/2008; 94(1):265-72. · 3.67 Impact Factor
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    ABSTRACT: We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.
    Journal of Leukocyte Biology 04/2006; 79(3):539-54. · 4.57 Impact Factor
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    Arpita Upadhyaya, Alexander van Oudenaarden
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    ABSTRACT: Actin polymerization has been shown to be sufficient to propel curved objects, for example beads and vesicles coated with the Listeria monocytogenes protein ActA. Recent studies suggest that actin polymerization on flat surfaces can also provide the propulsive force to push them forward.
    Current Biology 07/2004; 14(12):R467-9. · 9.49 Impact Factor
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    Arpita Upadhyaya, Michael P Sheetz
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    ABSTRACT: The endoplasmic reticulum (ER) and Golgi have robust bidirectional traffic between them and yet form distinct membrane compartments. Membrane tubules are pulled from large aggregates of ER or Golgi by microtubule motors to form ER tubulovesicular networks or Golgi tubules both in vivo and in vitro. The physical properties of membranes are critical for membrane traffic and organelle morphology. For example, tension applied to membranes can create tethers, drive membrane flow, and set the diameter of the tubules. Here, we formed ER and Golgi membrane networks in vitro and used optical tweezers to measure directly, for the first time, the membrane tensions of these organelles to clarify the possible role of tension in membrane flow. We report that higher forces are needed to form tethers from ER (18.6 +/- 2.8 pN) than from Golgi (11.4 +/- 1.4 pN) membrane tubules in vitro. Since ER tubules are smaller in diameter than Golgi tubules, it follows that Golgi networks have a lower tension than ER. The lower tension of the ER could be an explanation of how Golgi tubules can be rapidly drawn into the ER by tension-driven flow after fusion, as is observed in vivo.
    Biophysical Journal 06/2004; 86(5):2923-8. · 3.67 Impact Factor

Publication Stats

656 Citations
124.53 Total Impact Points

Institutions

  • 2011–2013
    • University of Maryland, College Park
      • Department of Cell Biology & Molecular Genetics
      College Park, MD, United States
  • 2003–2008
    • Massachusetts Institute of Technology
      • • George R. Harrison Spectroscopy Laboratory
      • • Department of Physics
      Cambridge, MA, United States
  • 2001
    • University of Notre Dame
      • Department of Physics
      United States
    • Claude Bernard University Lyon 1
      • Laboratoire de physique de la matière condensée et nanostructures (LPMCN)
      Villeurbanne, Rhone-Alpes, France