We have developed an accurate and practical method for measuring cytoplasmic [Ca2+] in the light microscoope. The method involves the use of the calcium-sensitive fluorescent dye Quin 2. The ratio of fluorescence intensities with excitation at 340 nm and 360 nm (I340/I360) provides a measure of [Ca2+] which is independent Quin 2 concentration. Using image intensification fluorescence microscopy and digital analysis of video images we are able to measure I340/I360 (and cytoplasmic [Ca2+]) throughout the cell. The video image analysis system provides enough temporal resolution, spatial resolution, and sensitivity to detect [Ca2+] gradients within a single cell using Quin 2. First, we will refine methods for measurement of localized [Ca2+]. We will then use the method to determine whether significant [Ca2+] gradients are developed in various cell types under a variety of physiological conditions. We have already found that mitotic cells have a considerable local variation in cytoplasmic [Ca2+]. Cell types to be studied including macrophages during phagocytosis, rat basophilic leukemia cells with clustered IgE receptors, and neutrophils exposed to chemotactic peptide. We will examine the contributions of ion pumping, diffusion, and buffering in dissipating [Ca2+] gradients, and we will try to obtain data on the sites of Ca2+ release into the cytoplasm. The role of intracellular messengers for the release of Ca2+ will be studied by micro-injecting putative messengers into the cytoplasm of Quin 2-loaded cells and observing changes in I340/I360. These studies will complement a large number of studies from several laboratories which are providing evidence for overall changes in cytoplasmic [Ca2+] throughout the cells. Local [Ca2+] gradients may be important for localized cell responses to external stimuli or for local control of the cytoskeleton.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM034770-04
Application #
3286322
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1987-09-26
Project End
1988-11-30
Budget Start
1987-09-26
Budget End
1988-11-30
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Schools of Medicine
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Recht, Phoebe A; Tepedino, Gerard J; Siecke, Neil W et al. (2004) Oxalic acid alters intracellular calcium in endothelial cells. Atherosclerosis 173:321-8
Pierini, Lynda M; Eddy, Robert J; Fuortes, Michele et al. (2003) Membrane lipid organization is critical for human neutrophil polarization. J Biol Chem 278:10831-41
Eddy, Robert J; Pierini, Lynda M; Maxfield, Frederick R (2002) Microtubule asymmetry during neutrophil polarization and migration. Mol Biol Cell 13:4470-83
Seveau, S; Eddy, R J; Maxfield, F R et al. (2001) Cytoskeleton-dependent membrane domain segregation during neutrophil polarization. Mol Biol Cell 12:3550-62
Seveau, S; Keller, H; Maxfield, F R et al. (2000) Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule. Blood 95:2462-70
Pierini, L M; Lawson, M A; Eddy, R J et al. (2000) Oriented endocytic recycling of alpha5beta1 in motile neutrophils. Blood 95:2471-80
Eddy, R J; Pierini, L M; Matsumura, F et al. (2000) Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration. J Cell Sci 113 ( Pt 7):1287-98
Mandeville, J T; Lawson, M A; Maxfield, F R (1997) Dynamic imaging of neutrophil migration in three dimensions: mechanical interactions between cells and matrix. J Leukoc Biol 61:188-200
Mandeville, J T; Maxfield, F R (1997) Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices. J Cell Physiol 171:168-78
Mandeville, J T; Maxfield, F R (1996) Calcium and signal transduction in granulocytes. Curr Opin Hematol 3:63-70

Showing the most recent 10 out of 27 publications