Micro- and Nanoscale Studies |
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Ethan Chiang Reorganization of Membrane by Activated IgE Receptors Observed with SEM |
We utilize high resolution scanning electron microscopy (SEM) to directly visualize sub-micron membrane domains in intact cell membranes. In our experiments, the distribution of gold-labeled proteins and lipids is analyzed at the surface of intact fixed cells using backscattered electron detection. In parallel, we also observe membrane topography using secondary electron detection. We use a pair-correlation function analysis to quantify protein distributions and parameterized domain size. We have mapped the distribution of a variety of proteins, both related and non-related to the IgE signaling pathway. Using this experimental and quantitative method, we observe dramatic changes in the nano-scale membrane distribution of IgE due to stimulation with multivalent ligands. Following receptor cross-linking, receptors are rapidly redistributed into large domains which are correlated at long length-scales. Additionally, we observe cross-linking dependent rearrangement of several inner leaflet-associated proteins that are implicated in early signaling events. In contrast, outer leaflet GPI-linked proteins are not affected. These findings demonstrate selective nanoscopic reorganization during the initiation of receptor signal transduction. (below) 5nm gold labeled IgE-FcεRI (red) and 10nm gold labeled Lyn (green) rapidly co-redistribute after receptor cross-linking with varying degrees of co-clustering at longer stimulation time points. Scale bar is 100nm. Figure is currently missing |
Figure is currently missing (above) Gold labeled protein distributions on the top surface of intact cells are visualized with high resolution SEM. (Left) Secondary electron detection of top cell surface. (Inset) Magnified view of 10nm gold particles labeling IgE-FcεRI imaged with back scattered electron detection (false colored). |
(above) IgE-FcεRI complexes redistribute into large clusters after 1 minute of receptor cross-linking. (a,b,c) Gold particle distributions of surfaceimmobilized IgE (yellow) and unstimulated (red) and stimulated (green)cell surface IgE-FcεRI are quantified using correlation functions(d). Scale bar is 500nm. |
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Alexis Torres Micro-patterned ligand arrays for the study of molecular interactions in immune cell signaling |
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| Spatial targeting and compartmentalization are common mechanisms of regulation in receptor mediated signaling. However the tools for studying these dynamic interactions are limited. We have established the use of micro-patterned lipid bilayers that contain specific ligands as a tool for visualizing spatial distribution of signaling molecules, and this approach also provides new insights into the structural and functionally relevant membrane components. We are investigating fundamental mechanisms in immune cell signaling, specifically IgE receptor (FcεRI) signaling in mast that is involved in allergic responses. | Figure is currently missing (above) Figure 1. Cartoon showing the interactions between the IgE on the cell surface and the patterned antigen. |
(below) Figure 2. Vinculin and Talin, both actin binding protein known to link paxillin and the actin cytoskeleton, are recruited to the activated IgE receptors. Figure is currently missing |
Previously, we showed that early signaling components such as Lyn kinase selectively co-redistribute with IgE-FcεRI that are clustered specifically over the patterned ligands. Currently we are studying the dynamics of actin cytoskeleton rearrangement that accompany FcεRI mediated activation and its role in the regulation in both signaling and internalization of the receptor complexes, and we have identified possible new adapter proteins that may be involved in linking F-actin to other signaling molecules. We found that F-actin and actin binding proteins such as vinculin, talin and paxillin are recruited to the clustered receptor sites and this local recruitment may be mediated by interactions with Lyn kinase. Further biochemical characterization revealed a role for paxillin in the negative regulation of FcεRI receptor signaling. Micropatterned ligand arrays combined with more standard biochemical and biophysical analysis are proving valuable for untangling the complex reorganization of the actin cytoskeleton during FcεRI receptor signaling. |
Deepti Gadi and Alice Wagenknecht-Wiesner Elucidating the role of protein kinase C in stimulated degranulation in mast cells |
Protein Kinase C (PKC) is a family of Ser/Thr kinases which play multiple roles in cell signaling. Previous studies showed that the PKC βI isoform plays an important role in granule exocytosis in mast cells but the molecular mechanism for this role is not yet clearly understood. In our lab, we are carrying out real time fluorescence imaing studies with PKCβI tagged with GFP and substrate, MARCKS-effector domaim tagged with RFP to elucidate their roles in degranulation. A hallmark of PKCβI activation is its oscillatory association and dissociation from plasma membrane upon cell stimulation that temporally correlates with calcium oscillations. Its substrate, MARCKS-ED is bound to polyphosphoinositides at the plasma membrane in the resting state of the cells and upon PKCβI activation, MARCKS-ED is phosphorylated, causing its dissociation from the plasma membrane. Interestingly, MARCKS-ED also shows oscillatory association with the plasma membrane upon stimulation by antigen. We are currently testing the hypothesis that PKCβI regulates access of secretory vesicles to PIP2 through phosphorylation of MARCKS-ED. |
(above) PKC βI-EGFP in the cytosol(green) and mRFP-MARCKS-ED (red) at the plasma membrane prior to stimulation. |
(above) PKC βI-EGFP (green) at the plasma membrane and mRFP-MARCKS-ED (red) in the cytosol following stimulation by antigen. |
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