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In Vivo Imaging of Cell-Cell Signaling
Michael Kotlikoff, Professor of Biomedical Sciences, Cornell University.
Austin O. Hooey Dean, College of Veterinary Medicine
Funding: NIH R01DK07227 (03/01/06-06/30/11).

The combination of transgenic, molecular engineering, and imaging technologies provides remarkable opportunities to design and target the expression of molecules that can be used to examine intercellular signaling in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Thus, purposely designed, genetically-encoded reporters of cell signaling hold great promise for the study of in vivo mammalian physiology. The Kotlikoff laboratory has developed several lines of transgenic mice in which G-CaMP is targeted to specific cell lineages, and they have exploited these mice for physiological studies. The smGC1 mice line robustly expresses the Ca2+ reporter in smooth muscle, and this genetically encoded sensor can be used to monitor physiological increases in [Ca2+] during single cell contraction bronchoconstriction, and arteriospasm. They have made transgenic mice expressing G-CaMP2 in smooth muscle (smGC2) and in cardiac myocytes (cmGC2), the latter under temporal control using a tetracycline controlled transcriptional activation-off (tet-off) double transgenic system. The cmGC2 mice line is being used to examine the development of Ca2+ signaling in the embryonic heart. They are also developing separate lines of mice in which this sensor is targeted for expression in neurons, including postsynaptic autonomic nerves, interstitial cells of Cajal, and pancreatic b cells. The Kotlikoff-DRBIO collaboration has two basic directions (project 1) calcium signaling and transgenic mouse phenotyping, and (project 2) probe development.

Project 1: We use confocal microscopy and two-photon flash photolysis of caged calcium to examine the mechanisms of Ca2+ release from the sarcoplasmic reticulum (SR) of urinary bladder smooth muscle (Ji, et al 2006). Movement of calcium from intracellular stores into the cytosol is an essential component of excitation-contraction coupling in all types of muscle. In cardiac and smooth muscle, Ca2+ is released from the SR in response to Ca2+ influx. Sarcoplasmic release of Ca2+ through ryanodine receptors (RyR) can be either spontaneous SR Ca2+ release events known as calcium sparks, or it can be triggered by the influx of Ca2+ through sarcolemmal ion channels (i.e. CICR). CICR in smooth muscle is a graded process that requires sufficient Ca2+ flux to activate release and is “loosely coupled”.  In smooth muscle RyR gating is coupled to Ca2+ channel activity through increases in cytosolic Ca2+ that must extend over a mean path length on the order of 100 nm, unlike in cardiac muscle where a cluster of RyR2 channels sense Ca2+ in the microdomain of L-type Ca2+ channels. However, no studies have established the relationship between a rise in intracellular Ca2+ independent of L-type Ca2+ channel activity and SR release. Using localized two-photon uncaging, we were able to make three novel observations regarding the mechanisms of SR Ca2+ release in smooth muscle where we found that local photorelease of Ca2+ evokes SR Ca2+ release in the form of Ca2+ sparks and Ca2+ waves throughout the cell, that the loss of FK506-binding protein 12.6 expression lowers the threshold and increases the activation of RyRs by local increases in Ca2+ in smooth muscle; and that SR Ca2+ release in response to local photorelease of Ca2+ requires activation of RyRs and IP3 receptors. We have also collaborated in imaging experiments of Dr. Kotlikoff’s transgenic lines to assist in phenotyping (Tallini et al, 2006).

Project 2:  This project focuses on the development of several new genetically encoded probes for cell and live animal imaging.  One strategy involves the use of a GFP binding aptamer for labeling and sensing tool for GFP fusion proteins in cells.  GFP fluorescence decreases by ~75% when the aptamer binds due a shift back to the 390 nm state (Figure 1A,B). We are developing a set of aptamer based labeling strategies will provide a basis for a new intracellular sensor mechanism (see Figure C-9C).  This aptamer (isolated from the Lis lab aptamer library by the Kotlikoff group) also pays an important role in the Lis/DRBIO probe developments. Another sensor under development is a GCaMP-mCherry calcium indicator, where the red protein serves a reference signal.  This construct will greatly advance intravital imaging by providing a genetically encodable absolute calcium sensor.  Initial results indicate a change of ~4 in green emission between the Ca-bound and Ca-free forms, while the mCherry signal remains constant (Figure 1D).  We also found that a small amount of the Ca-dependent FRET occurs (higher FRET for calcium free form) and the slightly decreased dynamic range (~20%) of GCaMP2-mCherry compared to GCaMP2 alone is due to differential FRET.  

Enlarge This Image

To further enhance our ability to image in vivo signaling events in the various transgenic models produced by the Kotlikoff lab, our long term goal is to utilize the high speed MPM systems.  Initially however, we image 3d-resolved calcium signaling in heart tissue by triggering synchronized line scans from the EKG.  Kotlikoff project 2 (sensors) uses our FCS instrumentation; improved cross-correlation of GCaMP and dual fluorescent protein binding apatamers are critical to a good understanding of the probe photophysics. We will determine the mode of inhibition and whether the fluorescence quenching is dynamic or static and due to differences in GFP structure (i.e. about 25% of the GFP are not affected by the aptamer).  These experiments are being carried out using single molecule TIRF imaging of single (his-tagged) GFPs on a nickel coated coverslip in the presence of added aptamer.

References

Ji G, Feldman M, Doran R, Zipfel W, Kotlikoff MI. 2006. Ca2+-Induced Ca2+ Release through Localized Ca2+ Uncaging in Smooth Muscle. J Gen Physiol. 127(3):225-35.

Tallini YN, Shui B, Greene KS, Deng KY, Doran R, Fisher PJ, Zipfel W, Kotlikoff MI. 2006. BAC Transgenic Mice Express Enhanced Green Fluorescent Protein in Central and Peripheral Cholinergic Neurons. Physiol Genomics. 27(3):391-7.

 

 



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