Regulation of SOCE by mitochondria

Mitochondria have been recognized as multifunctional organelles that elementary impact multiple signaling pathways, thus, introducing a new complexity on the long-known cellular power houses. For complementation these tasks, mitochondria communicate with other organelles and the plasma membrane in order to regulate the magnitude of the entry of Ca2+, nature`s most versatile and ubiquitous signaling messenger. Particularly mitochondria have been shown to contribute significantly to the activity of the so called store-operated Ca2+ entry (SOCE), whereby mitochondria play multiplex roles in the activation, maintenance and termination of this important Ca2+ entry pathway. However the molecular mechanisms responsible for this apparent contribution of mitochondria to SOCE are far from being understood. Recently the stromal interacting molecule (STIM) and Orai proteins were identified as the long awaited constituents of the Ca2+-selective current in the SOCE phenomenon. In particular, STIM1 works as a Ca2+ sensor, sensing the luminal ER Ca2+ concentration via an EF-hand domain in the N-terminus, while the C-terminal part of this ER membrane spanning protein is located in the cytosol. Upon ER Ca2+ depletion STIM1 proteins oligomerize and redistribute to form clusters of STIM1 multimeres at ER-plasma membrane (ER-PM) junctions. By delivering the message of ER Ca2+ depletion to plasma membrane ion channels, this dramatic realignment of STIM1 proteins represents the essential step in SOCE activation. The most prominent channel protein, which actually accomplishes ICRAC (Ca 2+ release activated Ca2+ current), as the most Ca2+-selective current of the SOCE phenomenon, is the four-membrane spanning protein Orai1. Orai1 assemble to tetramers upon STIM1 clustering at ER-PM junctions, whereby most likely a direct interaction of STIM1 with Orai1 triggers ICRAC activation of . Notably, the role of mitochondria to contribute to SOCE has not been reevaluated in view of the discovery of STIM1/Orai1 as the key protagonists of SOCE. Consequently, in this project the molecular mechanism(s), signaling pathways and molecules that are responsible for a presumable specific contribution of mitochondria to the activity of the STIM1/Orai1-dependent SOCE will be explored. In particular the impact of mitochondrial Ca2+ handling (Aim 1), mitochondrial metabolic functions (Aim 2), and mitochondrial motility and morphology (Aim 3) on distinct steps of STIM1-mediated Orai1 activation will be studied. The findings will enlighten novel aspects of mitochondrial physiology and will be of particular interest in view of the function of the STIM1/Orai1 dependent SOCE in many cell types (lymphocytes, mast cells, platelets and endothelial cells) as this might guide to the development of anti-inflammatory, immune modulating and anti- allergic drugs.

One of the most ambitious approaches to understand the miracles of life is to investigate functional processes in intact living cells: the basic units of all living organisms. In course of this project sophisticated methods in the fields of molecular biology, cell biology and fluorescence microscopy have been developed in order to visualize distinct molecular processes within cells of interest in detail. A main research focus was to examine novel signaling events within the endoplasmic reticulum (ER), a central cellular organelle, and mitochondria, which are often referred to as the cellular power houses. We have been able to develop novel probes, components of imaging systems, and experimental protocols in order to successfully unveil so far unknown molecular processes in cell biology that are related to specific functions of these organelles in health and diseases. A green and red fluorescent probe for imaging specifically the uptake of cytosolic Ca2+ into mitochondria with high spatial and temporal resolution was generated. This novel sensor allows investigating specific subcellular Ca2+ signaling events that are crucial in all known cell types. Using such techniques we have been able to unveil that Ca2+ transfer into mitochondria modulates distinct molecules within the ER so that a certain Ca2+ entry pathway is activated efficiently. This kind of Ca2+ entry is very important for immunologically active cells. Another class of genetically encoded fluorescent probes was developed in order to study for the first time the dynamics of ATP (adenosine triphosphate), the most important cellular energy carrier, within the lumen of the ER. These experiments revealed that cancer cells require huge amounts of ATP within the ER. Further experiments are planned to investigate if Ca2+ dependent ER ATP import is a novel promising molecular target for the development of new anticancer therapies. Very recently differently colored fluorescent proteinbased-sensors for nitric oxide (NO) have been successfully designed, produced and tested (patent pending). NO is one of the most studied molecules as it functions as an important messenger in cells of the cardiovascular system, neurons, cells of the immune system, and cancer cells. The fluorescent NO probes allow real-time imaging of cellular NO dynamics and will soon be used to unveil when and where this reactive molecule is generated under physiological and pathological conditions. We have been able to publish already 18 peer reviewed papers in international journals and 2 patents have been submitted in course of this research project. 6 PhD students and 3 diploma students contributed to this research, whereas already 5 thesis and all 3 diploma works have been successfully completed.