BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.
Speakers
Efstathia Thymiakou
Medical Research Council - Clinical Sciences Centre
Faculty of Medicine
Currently a postdoctoral fellow in the lab of Prof. Dimitris Kardassis (IMBB-FORTH) working on the role of the nuclear receptor HNF4α in lipid metabolism. Work so far, reveals new targets of HNF4α involved in High-density lipoproteins (HDL) metabolism and the development of steatosis and enriches our knowledge on HDL functionality in non-alcoholic fatty liver disease (NAFLD). During her first postdoc in the lab of Prof. Vasso Episkopou (Imperial College London) and Efstathia Thymiakou investigated the role of a newly identified ubiquitin ligase, Arkadia2C, in motor axon growth. The work was published in PLosBiology in 2013, revealing an involvement of BMP-Smad signaling in motor axon advancement. During my Ph.D. in the lab of Prof. Dimitris Kardassis (IMBB-FORTH), I studied the transcriptional regulation of ABCA1, a membrane transporter of cholesterol and phospholipids, which plays important role in reverse cholesterol transport and protection from atherosclerosis (Biochemistry. 2007 Oct 16;46(41):11473-83, Biochim Biophys Acta. 2014 Jun;1839(6):526-36). I am a member of the European and Hellenic Atherosclerosis Society and of the Hellenic Society for Biochemistry and Molecular Biology and have presented my work in many national and international conferences.
Vasso Episkopou, Ph.D.
Faculty of Medicine, Department of Brain Sciences
Professor in Developmental Biology
Ph.D. in Genetics and Development Columbia University, New York , 1980 B.Sc. Biology University of Patras Greece. POSITIONS: 2011-present Professor in Developmental Biology, Faculty of Medicine; 1995-2011 Group Leader MRC CSC Institute, Imperial College London; 1992-1994 Lecturer St. Mary’s Medical School, London. EXTERNAL APPOINTMENTS: 2006-2007 Director Alexander Fleming Institute, Athens Greece (Visiting); 2013 Visiting Professor, Columbia University, New York (Sabbatical)
Protein biology
- Protein and nucleic acid interactions
Duration:13min
Language:English
Session 1:presented May 6, 2020
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