Penn IMIG

Description of Penn IMIG Expertise

Research:

Dynamic regulation of intracellular calcium levels dictates immunological and physiological responses of lymphocytes to pathogens and endogenous antigens.  Tight regulation of intracellular Ca2+ levels is critical for lymphocyte effector responses and its dysregulation contributes to immunodeficiency as well as autoimmune and inflammatory diseases. Work in the Freedman lab focuses on the mechanisms of macrophage and lymphocyte Ca2+ signaling in normal and pathological responses.  High resolution imaging of living cells combined with Fluorescence Resonance Energy Transfer (FRET) microscopy is used to obtain quantitative measurements of the localization and interactions of proteins such as the endoplasmic reticulum (ER) transmembrane protein STIM1 and Orai1 which encodes the CRAC channel known to mediate sustained Ca2+ entry in lymphocytes. Optical activation of caged compounds such as IP3 using a UV laser or flash lamp allows us to directly examine the requirement for various antigen receptor linked upstream regulatory molecules in steps distal to IP3-mediated Ca2+ release from ER stores. The recent advent of a commercially available super high resolution Stimulated Emission Depletion (STED) microscope from Leica Microsystems provides us with the unique capability of visualizing subdiffraction resolution of structures as small as 30nM in size, and is being used to visualize the formation and localization of single CRAC channel complexes in lymphocytes.  Efforts underway at Penn to obtain a STED microscope will enable us to directly visualize interactions between CRAC channels and regulatory proteins that control the activity of these channels.

Lab expertise and resources:

• Single cell calcium imaging of live cells


• Regulation of calcium signaling in lymphocytes, macrophages and dendritic cells.


• Widefield and confocal fluorescence microscopy of live cells


• Spinning disk confocal microscopy of living cells


• Widefield and confocal visualization of cell interactions (lymphocytes and DC’s)


• Analysis of protein trafficking and interactions within living and fixed cells by FRET and FLIM microscopy.


• Analysis of T and B lymphocyte and macrophage pathogen-mediated signal transduction pathways


• Role of calcium in regulation of cytoskeletal proteins and cell motility


• Photo-activation of “caged” signaling molecules (e.g. IP3) in living cells to study signal transduction.

IMIG Collaborations:

• Freedman and Burkhardt: analysis of the role of actin regulatory proteins in controlling Ca2+ signaling in lymphocytes.


• Freedman and Hunter – calcium flux imaging

Lab members:

• Xiaohong Liu – Research Associate
  
  
  
  • Calcium signaling in lymphocytes and macrophages.  Single cell calcium imaging, live cell FRET, confocal imaging, photoactivation.
  
  
  • liux3@vet.upenn.edu
  
  
  
  

  Selected Publications

  Babich Alexander, Li Shuixing, O'Connor Roddy S, Milone Michael C, Freedman Bruce D, Burkhardt Janis K: F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation. The Journal of cell biology 197(6): 775-87, Jun 2012.
  
  Carrizosa Esteban, Gomez Timothy S, Labno Christine M, Klos Dehring Deborah A, Liu Xiaohong, Freedman Bruce D, Billadeau Daniel D, Burkhardt Janis K: Hematopoietic lineage cell-specific protein 1 is recruited to the immunological synapse by IL-2-inducible T cell kinase and regulates phospholipase Cgamma1 Microcluster dynamics during T cell spreading. Journal of immunology (Baltimore, Md. : 1950) 183(11): 7352-61, Dec 2009.
  
  Zhu Peimin, Liu Xiaohong, Treml Laura S, Cancro Michael P, Freedman Bruce D: Mechanism and regulatory function of CpG signaling via scavenger receptor B1 in primary B cells. The Journal of biological chemistry 284(34): 22878-87, Aug 2009.
  
  Shaffer Meredith H, Dupree Renell S, Zhu Peimin, Saotome Ichiko, Schmidt Richard F, McClatchey Andrea I, Freedman Bruce D, Burkhardt Janis K: Ezrin and moesin function together to promote T cell activation. Journal of immunology (Baltimore, Md. : 1950) 182(2): 1021-32, Jan 2009.
  
  Masek K S, Zhu P, Freedman B D, Hunter C A: Toxoplasma gondii induces changes in intracellular calcium in macrophages. Parasitology 134(Pt.14): 1973-9, Dec 2007.
  
  Nolz, Jeffrey C. Medeiros, Ricardo B. Mitchell, Jason S. Zhu, Peimin. Freedman, Bruce D. Shimizu, Yoji. Billadeau, Daniel D.: WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse. Molecular & Cellular Biology 27(17): 5986-6000, Sep 2007.
  
  Masek, Katherine S. Fiore, Jim. Leitges, Michael. Yan, Shi-Fang. Freedman, Bruce D. Hunter, Christopher A.: Host cell Ca2+ and protein kinase C regulate innate recognition of Toxoplasma gondii. Journal of Cell Science 119(Pt 21): 4565-73, Nov 1 2006.
  
  Gomez, Timothy S. McCarney, Sean D. Carrizosa, Esteban. Labno, Christine M. Comiskey, Erin O. Nolz, Jeffrey C. Zhu, Peimin. Freedman, Bruce D. Clark, Marcus R. Rawlings, David J. Billadeau, Daniel D. Burkhardt, Janis K.: HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse. Immunity 24(6): 741-52, Jun 2006.
  
  Wu, Jennifer N. Gheith, Shereen. Bezman, Natalie A. Liu, Qing-Hua. Fostel, Lindsey V. Swanson, Andrew M. Freedman, Bruce D. Koretzky, Gary A. Peterson, Erik J.: Adhesion- and degranulation-promoting adapter protein is required for efficient thymocyte development and selection. Journal of Immunology 176(11): 6681-9, Jun 1 2006.
  
  Myung, P.S., Jordan, M.S., Punt, J.A., Liu, Q-H, Judd, B.A., Sigmund, C.D., Freedman, B.D., Koretzky, G.A.: Differential requirements for SLP-76 domains in T cell development and function. Immunity 15: 1011-26, 2001.
  
  Collman, R.G., Yi, Y., Liu, Q-H, and Freedman, B.D.: Chemokine signaling and HIV-1 fusion mediated by macrophage CXCR4: Implications for target cell tropism. Journal of Leukocyte Biology 68: 318-23, 2000.
  
  Freedman, B.D., Liu Q.H., Gaulton, G., Kotlikoff, M.I., Heschler, J., and Fleischmann, B.K.: ATP-evoked Ca2+ transients and currents in murine thymocytes: Possible role for P2X receptors in death by neglect. European Journal of Immunology 29: 1635-1646, 1999.
  
  Freedman, Bruce D.: Mechanisms of calcium signaling and function in lymphocytes. [Review] [123 refs] Critical Reviews in Immunology 26(2): 97-111, 2006.
  
  Liu, Xiaohong. Zhu, Peimin. Freedman, Bruce D.: Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands. American Journal of Physiology - Cell Physiology 290(3): C873-82, Mar 2006.
  
  Nolz, Jeffrey C. Gomez, Timothy S. Zhu, Peimin. Li, Shuixing. Medeiros, Ricardo B. Shimizu, Yoji. Burkhardt, Janis K. Freedman, Bruce D. Billadeau, Daniel D.: The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation. Current Biology 16(1): 24-34, Jan 10 2006.
  
  Liu, Qing-Hua. Liu, Xiaohong. Wen, Zhiyun. Hondowicz, Brian. King, Leslie. Monroe, John. Freedman, Bruce D.: Distinct calcium channels regulate responses of primary B lymphocytes to B cell receptor engagement and mechanical stimuli. Journal of Immunology 174(1): 68-79, Jan 1 2005.
  
  Liu, Q H. Williams, D A. McManus, C. Baribaud, F. Doms, R W. Schols, D. De Clercq, E. Kotlikoff, M I. Collman, R G. Freedman, B D.: HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation. Proceedings of the National Academy of Sciences of the United States of America 97(9): 4832-7, Apr 25 2000.
  
  Freedman, B D. Liu, Q H. Somersan, S. Kotlikoff, M I. Punt, J A.: Receptor avidity and costimulation specify the intracellular Ca2+ signaling pattern in CD4(+)CD8(+) thymocytes. Journal of Experimental Medicine 190(7): 943-52, Oct 4 1999.
  
  Biswas, G. Adebanjo, O A. Freedman, B D. Anandatheerthavarada, H K. Vijayasarathy, C. Zaidi, M. Kotlikoff, M. Avadhani, N G.: Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter-organelle crosstalk. EMBO Journal 18(3): 522-33, Feb 1 1999.
  
  Adda, S. Fleischmann, B K. Freedman, B D. Yu, M. Hay, D W. Kotlikoff, M I.: Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle. Journal of Biological Chemistry 271(22): 13239-43, May 31 1996.
  
  Freedman, B D. Fleischmann, B K. Punt, J A. Gaulton, G. Hashimoto, Y. Kotlikoff, M I.: Identification of Kv1.1 expression by murine CD4-CD8- thymocytes. A role for voltage-dependent K+ channels in murine thymocyte development. Journal of Biological Chemistry 270(38): 22406-11, Sep 22 1995.
  
  Freedman, B D. Price, M A. Deutsch, C J.: Evidence for voltage modulation of IL-2 production in mitogen-stimulated human peripheral blood lymphocytes. Journal of Immunology 149(12): 3784-94, Dec 15 1992.
  
  
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  Last updated: 04/10/2013
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