Abstract

Mechanisms of transmembrane signaling, particularly non-excitable electromagnetic responses of vertebrate cells to external stimuli, are to be studied. Recording of single molecular channel conductances in situ in cell membranes and also as reconstituted into liposomes is to be used to discover and to measure properties of known and anticipated channels. Micropipette gigaohm seals are to be used for whole vesicle recording for direct measurements of currents generated by reconstituted pumps. Electro-optical image enhanced video microscopy with digital image analysis are to be used with flourescent indicators to search for electrogenic responses in nonexcitable cells, to measure spatial distributions of membrane potentials and ligands on surface receptors, and to detect signals associated with surface receptor cross linking, electromigration in applied electrochemical fields and in endocytotic processes. Technical developments designed to enhance the power of fluorescence micrography for investigation of previously inaccessible properties of transmembrane signaling will be pursued. Fluorescent membrane potential pH and calcium ion indicating dyes will be applied to mapping of the spatial distribution of membrane potentials and ion activities in differentiated cells. Large lipid vesicles bearing reconstituted protein channels will be manipulated in order to develop morphologies suitable for whole cell patch recording, as well as our presently available single channel recording. New strategies to take advantage of our simultaneous sensitive optical and electrical recording capability in cell biology will be explored.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033028-03
Application #
3282345
Study Section
(SSS)
Project Start
1984-09-01
Project End
1989-02-28
Budget Start
1986-09-01
Budget End
1989-02-28
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850

  Publications

Denk, W; Webb, W W (1992) Forward and reverse transduction at the limit of sensitivity studied by correlating electrical and mechanical fluctuations in frog saccular hair cells. Hear Res 60:89-102
Denk, W; Webb, W W; Hudspeth, A J (1989) Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer. Proc Natl Acad Sci U S A 86:5371-5
Millard, P J; Ryan, T A; Webb, W W et al. (1989) Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells. J Biol Chem 264:19730-9
Chandra, S; Gross, D; Ling, Y C et al. (1989) Quantitative imaging of free and total intracellular calcium in cultured cells. Proc Natl Acad Sci U S A 86:1870-4
Ryan, T A; Myers, J; Holowka, D et al. (1988) Molecular crowding on the cell surface. Science 239:61-4
Millard, P J; Gross, D; Webb, W W et al. (1988) Imaging asynchronous changes in intracellular Ca2+ in individual stimulated tumor mast cells. Proc Natl Acad Sci U S A 85:1854-8
Del Priore, L V; Lewis, A; Tan, S et al. (1987) Fluorescence light microscopy of F-actin in retinal rods and glial cells. Invest Ophthalmol Vis Sci 28:633-9
Webb, W W (1986) Light microscopy--a modern renaissance. Ann N Y Acad Sci 483:387-91
Menon, A K; Holowka, D; Webb, W W et al. (1986) Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization. J Cell Biol 102:541-50
Menon, A K; Holowka, D; Webb, W W et al. (1986) Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells. J Cell Biol 102:534-40

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