N OCIETY' - the Society for Reproductive Biology

NEW GENETICALLY TARGETTED SENSORS FOR THE CONFOCAL IMAGING OFINTRACELLULAR Ca 2 + AND pH IN LIVING CELL SYSTEMSDavid A. Williams, David N. Bowser, Angela M. Reilly, Rekha Panchal, Megan L. Smart, RobertNicholls and Steven PetrouConfocal and Fluorescence Imaging Group, Department of Physiology, The University ofMelbourne, Parkville, 3052, Australia.The majority of cellular processes are sensitive to changes in Ca 2 + and/or W ion concentration. As such,study of the regulation of intracellular, particularly intraorganellar, Ca 2 + and pH is of fundamentalimportance to the understanding of most cell systems. Two experimental strategies directly explore theinterorganellar ion concentrations in living cells. The first involves loading of intact cells with membranepermeantfluorophores, with subsequent quenching or removal of potentially confounding cytosolicfluorescence. This strategy has largely focussed on Ca 2 + and the mitochondria and while it has had somesuccess (Bowser et al., 1998), these fluorescence techniques are difficult and the attribution of results tospecific cell locations has not always been unambiguous. The second approach has also focussed on Ca 2 + andinvolves intracellular targeting of the luminescent, Ca 2 +-sensitive protein "aequorin", to specific organellesand has been in use for a number of years. Aequorin is a very sensitive Ca 2 + sensor and its properties havebeen thoroughly categorised well before its use in "targeted mode". However, it has always been difficult toimage (its luminescence producing less that one photon per molecule) and has suffered from difficultiesinvolved in calibrating the light emission in terms of absolute [Ca 2 +J.Developments in molecular biological synthesis have recently provided Ca 2 +and pH sensors, that offer majoradvantages over chemically synthesized Ca 2 + sensors and targeted Ca 2 + sensors with sub-optimalcharacteristics. "Cameleons" are genetically engineered protein sensors of free Ca 2 +comprised of tandemfusions of a blue or cyan mutant of fluorescent protein (GFP), calmodulin, calmodulin-binding protein M13and an enhanced green or yellow emitting GFP. Following the binding of Ca 2 +, calmodulin wraps around theM13 domain, thereby bringing the two flanking GFPs closer together. This proximity allows for Resonance.Energy Transfer (RET) of the emitted energy (from the "donor") directly into the excitation of the second"acceptor" GFP. The degree to which RET occurs represents a direct, ratiometric measure of free Ca 2 +concentration. The Ca 2 + affinity of the fusion protein is not fixed to that of the native calmodulin segment.Point mutations in this sequence with standard molecular genetic techniques have allowed fine-tuning of theCa 2 +affinities from as low as approximately 10- 8 M to up to 10- 2 M, suitable for different locations (i.e. highfor the cytosol, nucleus or mitochondria, and low for ER, Golgi or plasmalemma). The pH dependency ofvarious GFP mutants, in particular, that of enhanced yellow fluorescent protein (EYFP), with the highestpK'a of the mutants, provides exciting possibilities for targeted pH measurements.These sensors can be produced in situ by gene transfer of plasmid DNA into a number of cell types with avariety of well-established transfection techniques. Targeted chameleons can also be packaged intorecombinant adenoviruses (Adeasy system) to allow for infection of primary cultured adult cardiac myocytes.This system has already been used in our laboratory to create a recombinant aden0 viral plasmid containingthe SERCA2A gene and GFP for successful infection of adult cardiac myocytes. Protein-based sensors aredirected to their intended cellular location by 'tar~eting seque.nces' that are encoded within the sequence of aprotein. For nuclear localization the targeting sequence can be placed within a·protein or at the N or Cterminus and directs protein translocation to the nucleus of the cell, Similarly, for mitochondrial targeting aleader sequence directs the translocation of the protein to the mitochondria. ill the case of the endoplasmicreticulum (ER) the actions of a signal peptide and ER retention sequence together act to direct and thenretain a protein within the ER. Proteins lacking a targeting sequence are retained within the cytoplasm and,thus, 'targeted' to the cytoplasm. Our present work is characterising existing protein construct Ca 2 +-sensorsfor the ER and cytosol, and developing alternative versions with modified Ca 2 + affinities and differentlocalisation sequences, particularly for use in the mitochondria and at the plasma membrane.Bowser, D.. Minamikawa, T., Nagley, P & Williams, D. (1998) Biophysical Journal. 75: 2004-2014.TISSUE RECOMBINANT TECHNOLOGYGail P. RisbridgerMonash Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Road,Clayton, Victoria, 3168.The technique of constructing tissue recombinants was pioneered by Kratochwil in the 1970's. Heused TFM mice to demonstrate that regression of mouse mammary glands during embryogenesis, isan androgen regulated process. Since then, a number of studies in the mammary gland, prostate orvagina have used tissue recombinants to reveal. The nature of the cell - cell interactions between theepithelium and mesenchyme and the requirement for receptors on cell type. The purpose of thisworkshop is to describe the technique of constructing tissue recombinants using the prostate as amodel system. The development of the prostate is under androgenic control, but this talk will focuson the use of tissue recombinants to demonstrate a requirement for estrogens. Using the a estrogenreceptor knock-out (a ERKO) mouse model, tissue recombinations demonstrated that the presenceof ER a in the stroma and the epithelium is essential to obtain a full response to estrogeniccompounds such as diethylstilbestrol. The preparation and use of tissue recombinants is a uniqueand powerful method of dissecting the interplay between the stroma and epithelium in mediatinghormonal responses by these organs. As the development of new mouse models of targeteddisruption of steroid receptors and steroidogenic enzymes became available, we can define thecellular mechanisms of all types of hormone responses.3637