Principal Investigator: Michael Kotlikoff
Contact Information: E-mail: mik7@cornell.edu - Phone: 607-253-3771
Sponsor: NIH-NIGMS
Grant Number: 1 R01 GM086736-01A1
Title: ARRA: Design of Genetically Encoded Ca2+ Indicators for Vivo Application
Annual Direct Cost: $65,000
Project Period: 09/16/09-08/31/11
DESCRIPTION (provided by applicant): Genetically encoded Ca2+ sensors hold great promise for the dissection of complex physiology in vivo. The ability to make molecular scale measurements in real time in mammals, and to determine lineage –specific signaling events by genetic specification, provides unprecedented experimental power to determine the complex cell-cell communications that underlie normal organ function, and the dysfunction that attends and is the hallmark of disease. A number of laboratories have modified green or yellow fluorescent proteins, producing sensors that provide the ability to examine cell signaling in vivo. Current probes are limited to the green/yellow spectrum and spectrally distinct molecules are needed to enable determination of the critical signals that occur between individual lineages. We have recently developed bright, circularly permutated red fluorescent proteins, based on the monomeric mKate, and have produced initial Ca2+ sensors from these reengineered molecules. The goal of this proposal is to optimize genetically encoded, red Ca2+ sensors that enable studies of cell-cell signaling in vivo through the determination of the structural basis of Ca2+ -dependent fluorescence. The effort represents a collaboration between the laboratories of Dr. Michael Kotlikoff, who has significant experience with the design and function of GECIs, Dr. Holger Sondermann, who is an expert in protein structure and function, and Dr. Warren Zipfel, a biophysicist with expertise in fluorescence photophysics. The development of red fluorescent Ca2+ sensors will both extend the range of physiological studies for which these probes are informative, and enable higher dynamic range ratiometric sensors that incorporate both colors in the same molecule.
Revised Public Health Relevance Section
This project will produce novel molecules that can be used in vitro in cell systems and in vivo in animals to determine cellular function in the context of normal organ function, organ development, and disease and organ repair. These molecules also have the potential to enhance cell based therapies, such as stem cell therapy, by allowing the determination of the function of transplanted or engrafted cells.