Fast laser scanning confocal microscope for real-time investigation of subcellular immunological and cell biological processes

Scientific catalyst for the Canton of Ticino, maintaining close contact and having produced collaborative, high-impact publications with the Oncology Institute of Southern Switzerland (IOSI, Bellinzona), the San Giovanni Cantonal Hospital (Bellinzona), the Cantonal Pathology Institute (Locarno), the Cantonal Microbiology Institute (Bellinzona), Universita’ della Svizzera Italiana (Lugano), the Scuola Universitaria Professionale della Svizzera Italiana (Lugano), and the Centro Svizzero di Calcolo Scientifico (Manno). We requested funds to assist with the purchase of a state-of-the-art confocal microscope, capable of multi-color, high-speed imaging of live cells, with maximal 3D resolution. Advanced imaging technology has become essential for timely, competitive biomedical research and the lack of such equipment in Ticino has blocked development of numerous projects. Our application supports 6 independent research projects of relevance to Immunology, Hematology, AIDS, and Alzheimer's disease, each from a different SNF-funded Group Leader of the IRB. In particular, the new instrument will be used for time laps video microscopy to resolve spatio-temporal signaling events in leukocytes, tumors and HIV infected cells. Activation internalization and destiny of receptors and associated proteins are important steps for the regulation of cell migration and metastasis formation. The quantitative tracking of HIV-1 subvirion particles at distinct subcellular compartments of the host cell will provide new insights on the early steps of the retroviral life-cycle. Disassembly of the viral capsid and reverse transcription of the viral RNA are highly regulated processes which must precede integration of virus-derived DNA into the host genome. Interactions of T cells with antigen presenting dendritic cells are critical for the development of a specific adaptive immune response. The immunological synapse which represents a highly ordered and regulated interface between the two cells harbors key regulatory proteins and is the site of secretory events. A project aims to portray with 3D resolution the immunological synapse in live cells and to track the temporary association of its components. Proteolytic cleavage of the b-amyloid precursor protein (APP) by b- and g-secretases generates a highly fibrillogenic peptide, the amyloid b-peptide (Ab). Aggregation and deposition of Ab oligomers and fibers initiates a variety of toxic insults leading to the vast neurodegenerative processes observed in the brain of Alzheimer’s disease patients. We propose to covalently conjugate the antibody species with a fluorescent moiety to decorate APP and at the same time follow the internalization process in the living cell. In addition we will perfom 3D reconstructions of the position of APP and subcellular markers at they travel in real time within the cell. These projects are but the “tip of the iceberg”, when one considers the enormous potential utilization of state-of-the-art microscopy by investigators from IRB its collaborating institutions.