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AssigneeMemorial Sloan Kettering Cancer Center
InventorsGero Miesenböck, Dino De Angelis, James E. Rothman
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The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.
A nucleic acid molecule encoding a pH sensitive mutant of GFP of Aequora victoria, wherein a change in pH results in an alteration in one or more spectral properties, including intensity, of the excitation and/or emission spectra of said GFP, wherein substitutions on said GFP are made at an amino acid position selected from the group consisting of positions 147, 149, 161, 163, 166, 168, 175 and 202 relative to the sequence of the wild type GFP (SEQ ID NO:2), and wherein no substitutions are made at amino acid position 203 relative to the sequence of said wild type GFP, when there is a substitution at amino acid position 202.
Dependencies are inferred from the claim text.