Under pathological conditions it is functioning as a major activator of tumour-induced angiogenesis and also acts as a survival factor of tumour cells mediated by an autocrine signaling loop suppressing apoptosis. Despite its defined extracellular functions, FGF2 lacks a signal peptide and was shown to get exported from cells by an unconventional, ER/Golgi-independent mechanism of protein secretion. Understanding the molecular mechanism by which FGF2 is secreted from tumour cells may pave the way to develop a new class of anti-angiogenic inhibitors with a high potential for cancer therapy.
The molecular mechanism of unconventional secretion of FGF2 is based upon direct translocation across the plasma membrane. This process is initiated by the recruitment of FGF2 at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P2. This interaction causes FGF2 to oligomerize and, stimulated by phosphorylation of FGF2 at a specific tyrosine residue, results in membrane insertion of a FGF2 oligomer concomitant with the formation of a toroidal membrane pore. This structure has been interpreted as an intermediate in FGF2 membrane translocation. In a final step, cell surface heparan sulfate proteoglycans trap FGF2 molecules at the outer leaflet resulting in translocation of FGF2 molecules into the extracellular space.
The goal of this project will be to analyze how FGF2 transport into the extracellular space is coordinated in space and time in livings cells. In particular, the roles of three kinds of molecular switches governing this process need to be explored. Those include (i) a phosphoinositide switch governed by dynamic pools of PI(4,5)P2 at the inner leaflet, (ii) a tyrosine phosphorylation switch controlled by Tec kinase and (iii) a redox switch coordinating PI(4,5)P2 dependent FGF2 oligomerization and pore formation. The principal aim of this project is to use advanced chemical biology tools to manipulate these switches in living cells, to establish novel experimental systems to synchronize FGF2 membrane recruitment and to use single molecule fluorescence technologies to follow FGF2 transport by super resolution TIRF microscopy. This approach will allow for studying switch-controlled coordination of FGF2 secretion with unmatched spatio-temporal resolution in living cells. As long-term goals, we aim at following single FGF2 molecules throughout their lifetime, i.e. starting from their synthesis at cytoplasmic ribosomes all the way to their final localization on cell surfaces where they are found associated with heparan sulfates. These studies will deliver detailed information on spatio-temporal coordination of FGF2 transport into the extracellular space.
La Venuta G, Zeitler M, Steringer JP, Müller HM, Nickel W (2015) The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells Without a Signal Peptide at Hand? J Biol Chem. 290:27015-20
Steringer JP, Müller HM, Nickel W (2014) Unconventional Secretion of Fibroblast Growth Factor 2-A Novel Type of Protein Translocation across Membranes? J. Mol. Biol. 427:1202-1210
Rabouille C, Malhotra V, Nickel W. (2012) Diversity in unconventional protein secretion.
J Cell Sci. 125:5251-5.
Walter Nickel (2011) The Unconventional Secretory Machinery of Fibroblast Growth Factor 2. Traffic 12:799-805
Walter Nickel and Catherine Rabouille (2009) Mechanisms of Regulated Unconventional Protein Secretion. Nat. Rev. Mol. Cell Biol. 10:148-155
Walter Nickel and Matthias Seedorf (2008) Unconventional Mechanisms of Protein Transport to the Cell Surface of Eukaryotic Cells. Annu. Rev. Cell Dev. Biol. 24:287-308
Methods that will be used:
Single molecule super-resolution TIRF microscopy in living cells
Prof. Dr. Helge Ewers
Institut für Biochemie und Chemie, Freie Universität Berlin
Profile of candidate’s qualification:
We are looking for enthusiastic students with a master degree in the life sciences and a prime interest in advanced live cell imaging techniques along with an interest in interdisciplinary studies at the interface of biochemistry, biophysics, structural biology and cell biology.
Office: +49 6221 54-5425
Lab: +49 6221 54-5427
Fax: +49 6221 54-4366