HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: jimmunol.0901615v1 183/10/6478    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Cohen, R. Articles by Baird, B. PubMed PubMed Citation Articles by Cohen, R. Articles by Baird, B.

Ca²⁺ Waves Initiate Antigen-Stimulated Ca²⁺ Responses in Mast Cells¹

Roy Cohen,^* Alexis Torres,^* Hong-Tao Ma, David Holowka,^2* and Barbara Baird^*

^*Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850; and Laboratory of Molecular Immunology, National Heart, Lungs and Blood Institute, National Institute of Health, Bethesda, MD 20892

Ca²⁺ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca²⁺ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca²⁺ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca²⁺ oscillations throughout the cell that usually follow the initiating Ca²⁺ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca²⁺ puffs at the region of cell contact. Initiating Ca²⁺ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca²⁺ release from endoplasmic reticulum stores and to extracellular Ca²⁺, but they do not depend on store-operated Ca²⁺ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca²⁺ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca²⁺ response.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant AI022449 and by the Nanobiotechnology Center at Cornell (NSF:ECS-9876771). H.-T.M. is supported by the International Program of the National Heart, Lung, and Blood Institute, National Institutes of Health.

² Address correspondence and reprint requests to Dr. David Holowka, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850. E-mail address: dah24{at}cornell.edu

³ Abbreviations used in this paper: SOCE, store-operated Ca²⁺ entry; BMMC, bone marrow-derived mast cell; BSS, buffered salt solution; CRAC, Ca²⁺ release-activated Ca²⁺; DNP, 2,4-dinitrophenyl; PLC, phospholipase C; RBL, rat basophilic leukemia; ROI, region of interest; shRNA, small hairpin RNA; 2-APB, 2-aminoethyldiphenyl borate; TG, thapsigargin; TRPC, transient receptor potential channel.

⁴ The online version of this article contains supplemental material.