Proteolysis as a Regulatory Event in Pathophysiology

Project Z3

Microscopy core facility and monoclonal antibodies:
Analysis of protease structure, cell biology and function

Summary of the project

Cell biological and morphological examinations are a central part for a deeper insight into the regulation, function and subcellular biology of the proteases and their associated co-factors and putative substrates. The majority of SFB projects of this proposal rely on knowledge and equipment allowing a detailed subcellular localization of proteins in isolated cells and tissues. Such analyses will be instrumental for the functional understanding of proteases and their regulated proteins. In addition a detailed (immuno)microscopical analysis of tissues and cells derived from genetically modified mice is required by many groups. A critical evaluation of possible changes in the intracellular composition of the analyzed proteins requires both a sophisticated qualitative but also quantitative microscopical methodology. To secure a high quality and to provide a focus and optimization of resources the microscopy unit will be embedded in the available infrastructure and expertise. The main role of the microscopy core project will be the consultation of members of the consortium as well as providing human resources and infrastructure to apply a wide spectrum of microscopical investigations including preparation of cell and tissue samples and analysis of samples by classical microscopy, TIRF microscopy and advanced confocal laser scanning microscopy (e.g. FLIM, FRAP and live cell imaging). Monoclonal antibodies are ideally suited for the specific detection of macromolecules in tissue sections and isolated cells. Moreover, monoclonal antibodies can be applied for the elucidation of functional enzymatic domains, for a specific blockade of various protease domains as well as for the isolation of interacting molecular partners. The production of new monoclonal antibodies (in addition to commercially available antibodies) to the proteases under study will offer new perceptions of their functional significance. Hence, they are indispensable for an understanding of molecular interactions in inter- and intracellular signaling. The majority of SFB projects of this proposal rely on the application of monoclonal antibodies for subcellular localization of proteases. Especially the microscopical analysis of tissues and cells derived from genetically modified mice will profit form the production of new protease-specific monoclonal antibodies.

Training
Methods in microscopy
Advanced methods in fluorescence microscopy (Helge Schmidt, Olympus) Date: Dezember 02, 2011
Theory and basics of fluorescence microscopy Date: January 27, 2011 Time: 9.30 am - 16.00 pm
Advanced methods in fluorescence microscopy Date: July 1, 2011 Time: 10:00 am - 12:30 am
Generation of monoclonal antibodies Lecture & Practical Course Dr. Ahmad Trad, Biochemisches Institut Date: July 10, 2012 Time: 9:30 a.m. - 5:00 p.m.
Microscopy Workshop Dr. Helge Schmidt, Olympus Date: 25 - 27 July 2012 Part 1: Basics Part 2: Fluorescence microscopy Part 3: Fluorescence microscopy of living cells Part 4: Specific application at the FV1000 System
Lecture: "High Resolution Techniques" Dr. Helge Schmidt, Olympus Date: 9 November 2012