CA2+ IS A UNIVERSAL AND VERSATILE INTRACELLULAR MESSENGER ABLE TO REGULATE MANY DIFFERENT CELLULAR FUNCTIONS WITHIN A CELL. THIS IS DUE TO BOTH COMPARTMENTALIZATION OF CELL FUNCTIONS AND TO THE GENERATION OF SUBCELLULAR MICRODOMAINS WITH DIFFERENT CYTOSOLIC CA2+ CONCENTRATIONS. CA2+ HANDLING BY INTRACELLULAR ORGANELLA (ENDOPLASMIC RETICULUM (ER), GOLGI APPARATUS, MITOCHONDRIA OR NUCLEUS) IS ESSENTIAL FOR THE FORMATION OF SUCH MICRODOMAINS. ON THE OTHER HAND, CA2+ CONCENTRATION INSIDE ORGANELLA IS ESSENTIAL FOR VARIOUS PHYSIOLOGICAL FUNCTIONS SPECIFIC OF EACH ORGANELLE. THUS, IT IS KNOWN THE DIRECT ROLE OF CA2+ IN PROTEIN SYNTHESIS IN THE ER, PORTEIN SORTING BY GOLGI, APOPTOSIS OR CONTROL OF RESPIRATION IN THE MITOCHONDRIA OR GENE EXPRESSION IN THE NUCLEUS. IN FACT, CA2+ DISHOMEOSTASIS SEEMS TO PLAY AN IMPORTANT ROLE IN THE PATHOGENESIS OF NEURODEGENERATIVE DISEASES. RECENTLY, WE HAVE DEVELOPED A NEW FAMILY OF FLUORESCENT CA2+ INDICATORS THAT WE DUBBED GAP (FOR GFP-AEQUORIN PROTEIN) AND THAT CAN BE TARGETED TO THE MATRIX OF INTRACELLULAR ORGANELLES OF LIVING CELLS ALLOWING REAL-TIME IMAGING OF LUMINAL CHANGES OF [CA2+], BOTH AT REST AND DURING PHYSIOLOGICAL ACTIVATION. IN OUR PREVIOUS PROYECT WE FOCUSED ON THE HIGH CA2+ CONTENT ORGANELLA, THE ER AND GOLGI. WE DESIGNED AND PRODUCED A NEW GAP VERSION WITH THE OPTIMAL CA2+ AFFINITY TO CONFORM TO THE EXPECTED LUMINAL CA2+ CONCENTRATIONS. BESIDES, WE HAVE GENERATED NEW TRANSGENIC ANIMALS, FLIES AND MICE, EXPRESSING FUNCTIONAL GAP INSIDE ER THAT ALLOWS IMAGING IN VIVO CA2+ DYNAMICS IN THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE. THE NEW TRANSGENIC MICE ARE AN EXCELLENT TOOL TO STUDY THE CA2+ DYNAMICS IN A VARIETY OF ORGANS OR TISSUES WITH HIGH EXPRESSION OF THE INDICATOR. IN THE PRESENT PROJECT WE WILL EXPLOTE THIS TOOL BY ADDRESSING STRUCTURAL, PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL STUDIES GROUPED INTO THREE GLOBAL GOALS:_x000D_ 1. NEW METHODOLOGICAL DEVELOPMENTS AND GENERATION OF NEW TOOLS. WE PROPOSE TO GENERATE EXPRESSION VECTORS TO ACHIEVE TISSUE SPECIFIC EXPRESSION OF THE INDICATOR (NEURONS, GLIA AND PANCREATIC CELLS), AS WELL AS NEW TRANSGENIC LINES, BOTH IN FLY AND MOUSE. IN COLLABORATION WITH OTHER GROUPS WE INTEND TO UNDERGO MECHANISTIC AND STRUCTURAL STUDIES ABOUT THE GAP-CA2+ INTERACTION. _x000D_ 2. STUDY OF CA2+ HOMEOSTASIS IN ER AND ITS PARTICIPATION IN VARIOUS PHYSIOLOGICAL FUNCTIONS. IN COLLABORATION WITH OTHER LABORATORIES WE WILL ADDRESS A FUNCTIONAL SCREENING OF THE LEAK CHANNEL BASED ON COMPARATIVE PHYSIOLOGY AND EVOLUTIONARY GENETICS. WE PROPOSE COLLABORATIONS WITH OTHER GROUPS TO STUDY THE INVOLVEMENT OF THESE ORGANELLES IN VARIOUS PHYSIOLOGICAL FUNCTIONS (HORMONE SECRETION, GLIAL AND RETINA CA2+ OSCILLATIONS AND ACTIVATION OF SENSORY NEURONES)._x000D_ 3. WE FINALLY PROPOSE TO STUDY POSSIBLE ALTERATIONS OF CA2+ HOMEOSTASIS IN ER AND GOLGI APPARATUS IN VARIOUS CELLULAR MODELS OF AGING (NEURONS AND GLIA), BOTH IN VITRO AND IN VIVO. WE WILL COMPARE THE LEVELS OF CA2+, AS WELL AS THE ENTRY AND EXIT IN ER, AND GOLGI IN YOUNG AND OLD CELLS.