Proposed work will continue our long-term efforts to understand molecular mechanisms by which crosslinking of IgE receptors on mast cells triggers complex cellular signaling processes that lead to important functional responses in immune host defense. Central to mast cell and other leukocyte signaling responses is mobilization of intracellular Ca2+, and we will investigate this spatio-temporally orchestrated process and its functional roles in mast cell exocytosis, cytokine production, and dynamic interactions with epithelial tissue.
Specific Aim 1 focuses on elucidating molecular mechanisms of IgE receptor-activated Ca2+ responses, including novel antigen-stimulated Ca2+ waves that precede store-operated Ca2+ influx and impart spatial direction to this signaling response. The mechanism for activation of store-operated Ca2+ influx via recently identified Orai1/CRACM1 calcium channels will be characterized using fluorescence resonance energy transfer imaging for measurements of molecular proximity between this channel and the calcium sensor, STIM1. We will utilize this method to characterize the mechanism for regulation of Ca2+ influx by phosphatidylinositol 4,5- bisphosphate (PIP2) that we recently identified. Our evidence that different family members of phosphatidylinositol 4-phosphate 5-kinases synthesize functionally separate pools of PIP2 involved in Ca2+ mobilization leads us to propose that these PIP2 pools are spatially segregated in the plasma membrane, and we will test this hypothesis using biochemical, molecular genetic and imaging approaches. We established the participation of Ca2+ mobilization in IgE-receptor stimulated trafficking from recycling endosomes, and in Aim 2 we will test the role of this process in stimulated cytokine secretion important for adaptive immune responses. We will also test our hypothesis that protein kinase C regulates the access of secretory granules to Ca2+/PIP2/synaptotagmin complexes at the plasma membrane.
In Aim 3 we will characterize cellular mechanisms for mucosal mast cell motility, chemotaxis, and dynamic interactions with intestinal epithelial cells, both in cell culture and in live tissue. Mast cell motility and interactions within physiological tissue are regulated by changes in Ca2+ levels, and our proposed work will extend our general understanding of Ca2+ mobilization as a fundamental yet complex regulator of hematopoietic cellular homeostasis and response.

Public Health Relevance

Mast cells play a central role in allergic immune responses, and their significant participation in innate as well adaptive immunology is increasingly appreciated. Many aspects of mast cell function depend on highly orchestrated changes in intracellular Ca2+ in response to stimulation through the IgE receptor and by other means. Our proposed research draws from a broad range of experimental approaches to investigate molecular mechanisms for Ca2+-dependent signaling in key responses, including degranulation to release chemical mediators in allergies, cytokine production to recruit other cells in inflammation, and dynamic interactions of mast cells with epithelial cells as a first line of defense to invading pathogens. Elucidation of mast cell mechanisms will provide new opportunities for intervention in therapeutic applications and a clearer understanding of hematopoietic cell biology that is seminal to regulation of immune responses in health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022449-23
Application #
8204988
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Dong, Gang
Project Start
1985-08-01
Project End
2014-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
23
Fiscal Year
2012
Total Cost
$386,481
Indirect Cost
$138,981
Name
Cornell University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Holowka, David; Korzeniowski, Marek K; Bryant, Kirsten L et al. (2014) Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca(2+) sensor STIM1 and the Ca(2+) channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization. Biochim Biophys Acta 1841:1210-6
Smith, Norah L; Abi Abdallah, Delbert S; Butcher, Barbara A et al. (2013) Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase C?-mediated Ca(2+) mobilization. Front Microbiol 4:179
Bryant, Kirsten L; Antonyak, Marc A; Cerione, Richard A et al. (2013) Mutations in the polybasic juxtamembrane sequence of both plasma membrane- and endoplasmic reticulum-localized epidermal growth factor receptors confer ligand-independent cell transformation. J Biol Chem 288:34930-42
Santos, Marcela de Souza; Naal, Rose Mary Zumstein Georgetto; Baird, Barbara et al. (2013) Inhibitors of PI(4,5)P2 synthesis reveal dynamic regulation of IgE receptor signaling by phosphoinositides in RBL mast cells. Mol Pharmacol 83:793-804
Gadi, Deepti; Wagenknecht-Wiesner, Alice; Holowka, David et al. (2011) Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells. Mol Biol Cell 22:4908-17
Torres, Alexis J; Holowka, David; Baird, Barbara A (2011) Micropatterned ligand arrays to study spatial regulation in Fc receptor signaling. Methods Mol Biol 748:195-207
Calloway, Nathaniel; Owens, Tristan; Corwith, Kathryn et al. (2011) Stimulated association of STIM1 and Orai1 is regulated by the balance of PtdIns(4,5)P? between distinct membrane pools. J Cell Sci 124:2602-10
Calloway, Nathaniel; Holowka, David; Baird, Barbara (2010) A basic sequence in STIM1 promotes Ca2+ influx by interacting with the C-terminal acidic coiled coil of Orai1. Biochemistry 49:1067-71
Holowka, David (2009) Biomembrane liquid-liquid phase separation and detergent resistance: a relationship strengthened. Biochem J 424:e5-6
Cohen, Roy; Torres, Alexis; Ma, Hong-Tao et al. (2009) Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells. J Immunol 183:6478-88

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