Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
School Of Veterinary Medicine
Non Technical Summary
Lymphocytes are cells of the immune system that circulate in the blood and reside in lymph nodes. They are essential elements in the defense against pathogenic organisms including bacteria and viruses. Lymphocytes recognize and kill these pathogens. When pathogens interact with the external membranes of lymphocytes through specific receptors, they trigger a sequence of events in the cells (signals), which initiate functions of lymphocytes involved in killing the pathogen These signals cause opening and closing of ion channels which let ions (e.g. calcium) move across the external (plasma) cell membrane of lymphocytes, and these ions regulate the immunological functions of lymphocytes. The goal of this project is to define the mechanisms of pathogen-induced calcium signaling and to immune function of these signals in lymphocytes. Calcium signaling pathways, including membrane entry channels are targets for drugs that can support positive functions, but also may prevent abnormal or undersirable responses of lymphocytes. It is our expectation that by defining the complex mechanisms which regulate intracellular calcium in lymphocytes, we will gain insight into how lympocyte functions are regulated, including beneficial activities and aberrant or destructive actions. Specific benefits of this work may include defining new strategies to inhibit the undesirable immune responses that lead to organ rejection following transplantation or autoimmune diseases (e.g. degenerative arthritis, multiples sclerosis, and systemic lupus erthematosis). The results of these studies should also help us identify new strategies to modify, enhance, or prevent aberrant immune responses, but also to redirect insufficient or inappropriate responses in vivo, for example in the defense against serious pathogens like HIV-1
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Calcium is a multifunctional second messenger that regulates nearly every aspect of lymphocyte differentiation and function. Consequently, pathways that control intracellular calcium levels play a primary and fundamental role in immune regulation. The molecular mechanisms that initiate antigen receptor mediated calcium signaling in B cells are well delineated. For example, antigen receptor (BCR) engagement signals via tyrosine kinases Lyn/Syk to activate PLC gamma 2, which produces the messenger IP3, which binds to and activates channels (IP3 receptors [IP3R]) on the endoplasmic reticulum (ER) membrane. IP3-induced depletion of ER Ca2+ stores promotes relocalization of STIM1, the ER calcium sensor, to membrane domains in close proximity to the plasma membrane. Following stimulation, Orai1, the recently identified CRAC channel pore located in the plasma membrane, also relocalizes, bringing it into apposition with STIM1, allowing interactions between these proteins that trigger activation of "store-operated" CRAC channels (1). While it has long been thought that IP3-mediated store depletion is both necessary and sufficient for maximal CRAC activation, our recent work clearly challenge this notion. The first suggestion that ER store depletion was, by itself, insufficient to activated CRAC wa revealed in studies demonstrating grossly deficient CRAC channel activation in mice deficient for the kinases Lyn and Syk and the adaptor SLP-65. These studies suggested that Lyn and/or Syk participates in CRAC activation, an idea supported by our studies demonstrating that CRAC activation by intracellular IP3 is abrogated by either Lyn and Syk inhibition or suppression. We also find that ORAI physically interacts with phospho-Syk leading us to hypothesize that Syk and Lyn directly regulate CRAC channel activity. A primary objective of our efforst is to understand how Lyn and Syk regulate calcium in lymphocytes. Our preliminary data also indicates that the critical kinase PLCgamma2 directly interacts with ORAI and that such interactions can be inhibited by the tyrosine phosphorylated from of the transcription factor TFII-I (p-TFII-I), a known target of the activation-induced kinase, Btk. As PLC has previously been shown to regulate activation of the non-selective cation channel TRPC3 and it has been demonstrated that such interactions can be inhibited by p-TFII-I, we hypothesize that p-TFII-I-mediated regulation of PLCgamma-2/ORAI complex formation plays an analogous role in the regulation of CRAC channel activity. Finally, our recent work has revealed yet another critical regulator of calcium entry in B cells, the cytoskeletal adaptor protein WAVE2. A critical third objective of this work is to explore the mechanistic role of WAVE2 in post store regulation of BCR-induced CRAC activation and calcium signaling. Thus, the central hypothesis of this proposal is that ER calcium store depletion by IP3 is necessary but not sufficient for CRAC channel activation, and that channel activation and the resulting calcium signal is also tightly regulated by distal actions of Lyn, Syk, TFII-I, PLCgamma-2 and WAVE2.
Project Methods
Dr. Freedman's lab focuses on mechanisms of pathogen induced calcium signaling within lymphocytes and macrophages in normal and pathological responses of these cells utilizing a range of optical, electrophysiological, and molecular approaches. High resolution imaging of living cells combined with Fluorescence Resonance Energy Transfer (FRET) microscopy is used to obtain quantitative measurements and localization 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 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 the capability for subdiffraction resolution of structures as small as 30nM in size. STED microscopy provides us with the unique capability of visualizing the formation and localization of single CRAC channel complexes in lymphocytes. Efforts underway at Penn to obtain a STED microscope which will enable us to directly visualize CRAC channel complexes and resolve the interactions of regulatory proteins with these complexes. These high end optical imaging approaches are combined with functional studies and molecular techniques to manipulate the expression of proteins implicated in the regulation of calcium and immune cell fates and functions.