Spacer
PhD student position in Studying the spatio-temporal control of FGF2 membrane translocation in living cells using single molecule TIRF microscopy
Category:
Phd

Description:

Fibroblast Growth Factor 2 (FGF2) is a strong mitogen promoting angiogenesis in health and disease.  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.

 

References:

Reviews:

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

Original articles:

La Venuta G, Wegehingel S, Sehr P, Müller HM, Dimou E, Steringer JP, Grotwinkel M, Hentze N, Mayer MP, Will DW, Uhrig U, Lewis JD, Nickel W (2016) Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2. J. Biol. Chem. 291:17787-803

Zeitler M, Steringer JP, Müller HM, Mayer MP, Nickel W (2015) HIV-Tat Forms Phosphoinositide Dependent Membrane Pores Implicated in Unconventional Protein Secretion. J. Biol. Chem. 290:21976-84

Müller HM, Steringer JP, Wegehingel S, Bleicken S, Münster M, Dimou E, Unger S, Weidmann G, Andreas H, García-Sáez AJ, Wild K, Sinning I, Nickel W. (2015) Formation of disulfide bridges drives oligomerization, membrane pore formation, and translocation of fibroblast growth factor 2 to cell surfaces. J. Biol. Chem. 290:8925-8937

Zacherl S, La Venuta G, Müller HM, Wegehingel S, Dimou E, Sehr P, Lewis JD, Erfle H, Pepperkok R, Nickel W (2014) A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2. J. Biol. Chem. 290:3654-3665

Steringer, JP, Bleicken, S, Andreas, H, Zacherl, S, Laussmann, M, Temmerman, K, Contreras, FX, Bharat, TA, Lechner, J, Müller, HM, Briggs, JA, Garcia, AJ, Nickel, W (2012) PI(4,5)P2 Dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion. J. Biol. Chem. 2012; 287(33):27659-69.

Ebert, A, Laußmann, M, Wegehingel, S, Kaderali, L, Erfle, H, Reichert, J, Lechner, J, Beer, HD, Pepperkok R, and Nickel W (2010) Tec kinase mediated phosphorylation of Fibroblast Growth Factor 2 is essential for unconventional secretion. Traffic 11:813-826

Torrado, LC, Temmerman, K, Müller, HM, Mayer, MP, Seelenmeyer, C, Backhaus, R, and Nickel, W (2009) An Intrinsic Quality Control Mechanism Ensures Unconventional Secretion of Fibroblast Growth Factor 2 in a Folded Conformation. J. Cell Sci. 122:3322-3329

Temmerman, K, Ebert, AD, Müller, HM, Sinning, I, Tews, I, and Nickel W (2008) A Direct Role for Phosphatidylinositol-4,5-bisphosphate in Unconventional Secretion of Fibroblast Growth Factor 2. Traffic 9:1204-1217

Zehe, C, Engling, A, Wegehingel, S, Schäfer, T, and Nickel W (2006) Cell surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2. Proc. Natl. Acad Sci. U.S.A. 103:15479-15484

Seelenmeyer, C, Wegehingel, S, Tews, I, Künzler, M, Aebi, M, and Nickel W (2005) Cell Surface Counter Receptors are Essential Components of the Unconventional Export Machinery of Galectin-1. J. Cell Biol. 172:373-381

Schäfer, T, Zentgraf, H, Zehe, C, Brügger, B, Bernhagen, J, and Nickel W (2004) Unconventional Protein Secretion of Fibroblast Growth Factor 2 Is Mediated by Direct Translocation across the Plasma Membrane of Mammalian Cells. J. Biol. Chem. 279:6244-6251

 

Methods that will be used:

Single molecule super-resolution TIRF microscopy in living cells

 

Collaboration Partner:

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 biochemisty, biophysics, structural biology and cell biology.

 

Keywords:

Fibroblast Growth Factor 2 (FGF2) and tumour-induced angiogenesis; Unconventional protein secretion; Membrane translocation of proteins; phosphoinositide-triggered oligomerization and membrane insertion, live cell imaging of plasma membrane associated processes using super-resolution TIRF microscopy