Thomas Jefferson University

Er-mitochondrial Communication in Calcium Signaling, Energy Metabolism and Liver Disease - Abstract the Effects of Various Hormones on Oxidative Metabolism and Other Mitochondrial Functions, in the Liver and Other Tissues, Are Mediated by Cytoplasmic [CA2+] Oscillations Propagated to the Mitochondria. CA2+ Is Released to the Cytoplasm From the Endoplasmic Reticulum (ER) Through the IP3 Receptors (IP3RS), Which, Based on Findings From Us and Others, Expose Mitochondria at Er-mitochondria Contact Sites (ERMCS) to High [CA2+] Nanodomains to Attain Activation of the Low-affinity Mitochondrial CA2+ Uptake Sites. Ermcs Were Also Recognized in Other Processes Including Lipid Metabolism, Organelle Dynamics and Autophagy. Our Work Has Revealed the Physical Support of Ermcs by Tethering Proteins. We Have Created Synthetic Membrane Linkers to Measure and Perturb Ermcs and Local Inter-organelle Communication in Live Cells, and Provided Clues to Local Calcium and Reactive Oxygen Species (ROS) Signaling. We Have Also Provided These Reagents to Several Hundred Laboratories Worldwide, Which Together Have Showed the Role of Interorganellar Contacts in a Range of Paradigms Including Metabolism, Vesicle Dynamics, Neuronal and Immune Functions and Linked Structural or Functional Impairments of the Er-mitochondrial Coupling to an Array of Disorders Across Organs Including the Liver (e.g. Fatty Liver). However, Fundamental Questions Remain Unanswered. Ermcs Are Dynamically Restructured to Meet the Continuously Changing Demands of the Cell, But How Ermcs Are Formed and Dissolved Is Yet to Be Determined. IP3R-MEDIATED Fluctuations in [CA2+] Might Provide a Means to Control Contact Formation, Given That Elevations of Cytoplasmic [CA2+] Stop Mitochondrial Movements Close to the Er Through the CA2+-SENSING Miro Proteins, and Both the IP3R and Miro Proteins, Have Been Implicated as Components of Interorganellar Complexes. However, the Interaction Partners and Mechanisms Are Elusive. We Hypothesize That IP3RS and Miros Mediate Ermc Formation in an Isoform-specific and CA2+-DEPENDENT Manner to Regulate Physiological Functions of Hepatocytes. AIMS#1&2 Will Test This Hypothesis Using Novel Genetic and Microscopic Imaging Toolkits That Will Enable Us to Specifically and Systematically Measure and Perturb Ermc Forming Elements. the Effect of Genetic Perturbations in the Liver Will Be Tested by Novel, in Vivo Imaging Approaches. the Interactomes of IP3R and Miro Will Be Evaluated Primarily by Unbiased Proteomics, But Bcl-xl Will Also Be Specifically Tested as a Tether Forming Partner for the IP3R. Finally, a Major Limitation in the Field of Inter-organellar Communication Research Is That Quantitative Evaluation of the Geometry of Nanometer Scale Membrane Contacts Remains Difficult. in AIM#3 We Will Develop and Characterize Methods of Measuring and Describing Organelle Interface Geometry in 2-AND 3D Electron Microscopy Data and Uncover the Structural Features Most Relevant to Calcium Transfer. the Proposed Work Will Explore the Molecular Mechanisms of Ermc Dynamics and Their Physiological Relevance and Will Continue Our Efforts in Creating Molecular Tools and Methods That Allow Many Investigators to Explore the Local Communications of Er and Mitochondria or Other Organelles.