Micro- and Nanoscale Studies
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Dwaipayan Sil Studies with Oligovalent Ligands Domains on RBL Mast Cells with Yougen Li and Dan Luo |
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Schematic diagram of Ydna-3dnp |
The project involves the study of the effect of antigen (ligand) structure on the signaling pathway in mast cells. Mast cells are hematopoetic cells which contain granules that are rich in mediators such as histamine, which are primarily responsible for the allergic reactions in our body. Once these mast cells are stimulated by antigens, these mediators are released from the granules to the outside of the cell, the process being termed as degranulation. On being released in the blood stream, these mediators then move to different connective tissues and cause the various symptoms of allergy, such as running nose, watery eyes etc. |
Towards this goal, we use branched-shape DNA structures with well-defined size and shape, as scaffolds to initiate the signaling pathway. The defined shape and size of these rigid DNA templates provide a way to control the distance between the antigen binding sites of the antibody IgE as well as the level of IgE-receptor clustering due to antigen binding, to a certain extent. These templates thus provide a mechanistic tool to investigate signaling events individually on a molecular level. They provide a way to understand how an extra cellular event such as the antigen-antibody binding, regulates the intracellular functioning of these cells. At the same time, these ligands can be used for the diagnosis and prevention of allergic responses. |
Predicted model of antibody-receptor clustering through engagement by Ydna-3dnp ligands |
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Ethan Chiang Probing the Nano-scale Spatial Organization of IgE Mediated Signaling Pathway Associated Membrane Proteins via Patterned Polymer Brush Surfaces with Rong Dong of the Ober Group |
Upon IgE Receptor mediated activation and various membrane interactions, lipids and proteins can undergo a complex rearrangement and redistribution. We use a combination of fluorescence and electron microscopy to study the membrane heterogeneities and microdomains that result from stimulation of RBL cells via functionalized patterned surfaces. Although fluorescence microscopy gives us the ability to look at membrane organization at larger spatial dimensions, in order to study smaller nano-scale protein distributions we utilize electron microscopy. This requires the preparation of very thin cell membrane sheets that can be preferentially stimulated via patterned surfaces. In collaboration with Christopher Ober’s research group, we are developing polymer brush surface and lithographic techniques that will allow us to probe the spatial and temporal organization of proteins involved in the IgE mediated cell signaling pathway. (below)Thin plasma membrane sheets after stimulation by a patterned polymer brush surface (dim circles) and subsequent membrane disruption, leaving varying amounts of membrane attached to patterned surface.
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(above) Alexa488-IgE sensitized plasma membrane sheets after stimulation by a 2um patterned poly(acrylic acid) DNP-functionalized polymer brush surface |
(above) Nano-scale clustering of Alexa488-IgE labeled with 15nm gold particles on plasma membrane sheets within a single 2 mm polymer brush. |
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Alexis Torres Studies of Spatial Signaling Events and Adhesion on RBL-2H3 Mast Cell with the Use of Micro-fabricated Surfaces |
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| We are interested in the dynamics of the actin cytoskeleton following FcεRI mediated activation. Previous studies have shown that pretreatment of RBL-2H3 cells with inhibitors of actin polymerization, such as cytochalasin D and latrunculin B, enhances FcεRI-IgE mediated degranulation, calcium influx and even tyrosine phosphorylation of the FcεRI β subunit. These accumulating evidence supports the hypothesis that filamentous actin negatively regulates the interactions between activated FcεRI receptor and Lyn kinase and hence the early events of signaling. However, the mechanisms of these interactions are far from understood. |
Figure 1 (above). Cartoon showing the interactions between the IgE on the cell surface and the patterned antigen. |
Figure 2 (below). RBL-2H3 Cells interacting with the patterned antigen. A) Cells showing IgE (green) clustered over the antigen patterns and labeled for F-actin (red). B) Same cell showing only the F-actin accumulation under the clustered receptors.
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To understand these interactions we are using patterned surfaces previously developed in our lab to spatially control receptor activation. These surfaces containing patterned antigens on the micron scale are microfabricated using standard photolithography techniques. By spatially clustering the IgE receptors (FcεRI) we can use fluorescence microscopy to probe the recruitment of other signaling molecules (figure 1). We have found with this approach that F-actin polymerizes under these clustered receptors (figure 2). Our current efforts are concentrated in studying the mechanisms for these interactions by using fabricated surfaces and other more standard biochemical approaches. |
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