Head of Advanced Microscopy and Imaging Core, TIGEM
Molecular Mechanisms of Membrane Sorting and Biogenesis in health and disease.
Over the last decade, our studies have made substantial contributions to the understanding of cellular trafficking pathways at the morpho-functional level. This has provided us with a solid temporal and topographical framework for the study of the molecular machineries implicated in membrane sorting and trafficking processes. Therefore, our goal is to identify and characterize new molecular players and pathways operating in sorting and transport events under pathological and normal conditions. Currently, the lab’s researchers are focused on the role of membrane trafficking in copper homeostasis
In recent years, it has become clear that several genetic disorders are caused by mutations that disrupt the ability of gene products to reach the specific targets, where they would be expected to execute their functions. Among these genes, ATP7A and ATP7B encode their corresponding proteins, also known as copper (Cu) ATPases, proteins with an important role in regulating copper levels in the body. Newly synthesized Cu ATPases travel through membrane organelles constituting the membrane secretory pathway to arrive at their functional sites. More specifically ATP7A allows dietary Cu to be transported from intestinal cells towards the other organs, while ATP7B removes the excess of Cu from the body by pumping it across the membrane of the liver cell and into biliary flow. Mutations do not allow aberrant Cu ATPases to move properly through the secretory pathway to the specific sites in which Cu transport is needed, resulting in either ATP7A-dependent Cu deficiency (Menkes disease) or ATP7B-dependent Cu toxicosis (Wilson disease), which are fatal if the appropriate treatment is not executed in time.
Given that mis-localization inhibits multiple Cu ATPase mutants from localizing, correcting trafficking and targeting the mutants to appropriate functional sites should help in the the majority of Wilson and Menkes disease patients. However, the question as to which mechanisms drive Cu ATPase transport and to which extent they are affected by mutations remains to be answered.
The aim of our studies is to identify regulatory mechanisms that are involved in Cu ATPase localization and trafficking and to understand their importance for the maintenance of Cu homeostasis and for the development of Menkes and Wilson disease. Therefore, we intend not only to provide answers for the above questions, but also to detect molecules that can be used for the correction of Cu ATPase transport, and therefore, for the development of novel strategies to cure Menkes and Wilson disease.
Advanced Microscopy Core
Over the past decades, the rapid pace of gene discovery has lead to a significant effort in the study of gene function at molecular, biochemical and cellular levels. At this stage, microscopy has gained a fundamental role because it provides high-resolution mapping and dynamics of normal and anomalous gene products and reveals the structural changes induced by the expression of the latter. High-resolution microscopy has become an essential component of all research programs at TIGEM that are aimed at the characterization of gene and protein functions at cellular and sub-cellular levels.
The main objectives of the Advanced Microscopy and Imaging Core are to provide technical assistance, expertise, and qualitative and quantitative evaluation of microscopy data for research projects at TIGEM. The Core is composed of two separate units, the Light Microscopy Division and the Electron Microscopy Division.
The Light Microscopy Division provides users with the equipment and know-how to monitor cell behavior in real time and visualize proteins inside cells with high spatial and temporal resolution. The division is equipped with essential imaging technologies, such as Bright-field microscopy, fluorescence and confocal microscopy. In addition the facility is equipped with instrumentation for various types of live-cell imaging comprising spinning disk microscopy, TIRF, FRET, photobleaching and photoactivation methods. Moreover, the Light Microscopy staff provides TIGEM researchers with optical instrumentation, consultation and training in techniques related to light and fluorescence microscopy, image analysis and data transfer and storage.
The Electron Microscopy Division allows researchers to analyze the ultrastructure (with resolution up to a few nanometers) in cells and tissues and determine the localization of proteins at the sub-organelle level. Therefore the approaches of the Electron Microscopy Division complement those of the Light Microscopy Division. The Core’s expertise in routine and immune-electron microscopy (IEM), advanced EM techniques, correlative light-electron microscopy and EM tomography, allows researchers to analyze detailed the ultrastructures of organelles observed in living cells before fixation and subcellular structures in cells expressing or lacking the gene product of interest.
Chesi G, Hegde R, Iacobacci S, Parashuraman S, Concilli M, Festa BP, Polishchuk EV, Carissimo A, Montefusco S, Canetti D, Monti M, Amoresano A, Pucci P, van de Sluis B, Lutsenko S, Luini A, Polishchuk RS (2016) Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants. Hepatology. 63: 1842-1859
Polishchuk EV, Concilli M, Iacobacci S, Chesi G, Pastore N, Piccolo P, Paladino S, Baldantoni D, Chan J, Chang CJ, Amoresano A, Pane F, Pucci P, Tarallo A, Parenti G, Brunetti-Pierri N, Settembre C, Ballabio A, Polishchuk RS. (2014) Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis. Dev Cell. 29(6):686-700. doi: 10.1016/j.devcel.2014.04.033.
Capestrano M, Mariggio S, Perinetti G, Egorova A, Iacobacci S, Di Pentima A, Iurisci C, Egorov M, Di Tullio G, Buccione R, Luini A, Polishchuk RS (2014). Cytosolic phospholipase A2ε drives recycling in the clathrin-independent endocytic route. J Cell Sci. 127(Pt 5):977-93. doi: 10.1242/jcs.136598.
D’Agostino M, Lemma V, Chesi G, Stornaiuolo M, D’Ambrosio C, Scaloni A, Polishchuk RS, Bonatti S (2013) The cytosolic chaperone α-Crystallin B rescues appropriate folding and compartmentalization of misfolded multispan transmembrane proteins. J Cell Sci. 126(Pt 18):4160-72. doi: 10.1242/jcs.125443.
Egorov MV, Capestrano MG, Vorontsova OA, Di Pentima A, Mariggiò S, Ayala MI, Tetè S, Gorski JL, Luini A, Buccione R, Polishchuk RS (2009). Faciogenital Dysplasia Protein (FGD1) Regulates Export of Cargo Proteins from the Golgi Complex to the Cell Surface. Mol Biol Cell. 20(9):2413-27. doi: 10.1091/mbc.E08-11-1136.