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Imaging the Translocations of CLIC4 and Epac1 Ponsioen, B

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Imaging the Translocations of CLIC4 and Epac1 Ponsioen, B Imaging the translocations of CLIC4 and Epac1 Ponsioen, B. Citation Ponsioen, B. (2009, May 12). Imaging the translocations of CLIC4 and Epac1. Retrieved from https://hdl.handle.net/1887/13784 Version: Corrected Publisher’s Version Licence agreement concerning inclusion of doctoral License: thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/13784 Note: To cite this publication please use the final published version (if applicable). Imaging the translocations of CLIC4 and Epac1 Bas Ponsioen Imaging the translocations of CLIC4 and Epac1 Proefschrift ter verkrijging van de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof.mr. P.F. van der Heijden, volgens besluit van het College voor Promoties te verdedigen op dinsdag 12 mei 2009 klokke 15.00 uur door Bas Ponsioen geboren te Woerden in 1977 Promotiecommissie Promotor Prof.dr. W.H. Moolenaar Co-promotor Dr. K. Jalink Nederlands Kanker Instituut Overige leden Prof.dr. J.J. Neefjes Prof.dr. J.L. Bos Universiteit Utrecht Dr. R. Michalides Nederlands Kanker Instituut © Bas Ponsioen Cover by Suus van den Akker (www.suusvandenakker.com) The work described in this thesis was performed at the Divisions of Cellular Biochemistry and Cell Biology of the Netherlands Cancer Institute, Amsterdam, the Netherlands. This work was supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds), grant nr. NKB 2003-2964 and the Centre for Biomedical Genetics (CBG). Publication of this thesis was financially supported by the Dutch Cancer Society and the Netherlands Cancer Institute. Printed by Wöhrmann Print Service, Zutphen. Contents Chapter 1 Introduction 7 Chapter 2 Spatiotemporal regulation of CLIC4, a novel player in the Gα13-RhoA 23 signaling pathway Chapter 3 Direct spatial control of Epac1 by cAMP 39 Chapter 4 ERM proteins recruit Epac1 to the plasma membrane and facilitate 55 Epac1-induced cell adhesion Chapter 5 Detecting cAMP-induced Epac activation by fluorescence resonance 69 energy transfer: Epac as a novel cAMP indicator Chapter 6 Direct measurement of cyclic AMP diffusion and signaling through 81 connexin43 gap junctional channels Chapter 7 Regulation of connexin43 gap junctional communication by 93 phosphatidylinositol 4,5-bisphosphate Chapter 8 A Comparison of Donor-Acceptor Pairs for Genetically Encoded 111 FRET Sensors: Application to the Epac cAMP Sensor as an Example Chapter 9 8-pCPT-2’-O-Me-cAMP-AM: an improved Epac-selective cAMP analogue 125 Summary 134 Samenvatting 136 Curriculum Vitae 140 List of publications 141 Introduction 7 Introduction 1 Prelude Regulation of specific interactions often results in the redistribution of molecules within the cell (see section This thesis comprises a set of explorations into vari- 3). Such ‘translocations’ can result in strong enrich- ous intracellular and intercellular signaling pathways. ment at intracellular compartments where their action Throughout these explorations, ‘translocations’ have is required. Therefore, translocations are intrinsic to been a constant theme. Scattered across various cell signaling cascades. biological topics, this thesis reflects the discovery of Translocations studied in this thesis are based on three translocations and the experimental work aimed passive diffusion. Passive transport is distinct from ac- to unravel their molecular mechanisms and cell bio- tive transport by its use of energy. Passive transport logical relevances. At first glance, the word ‘transloca- is thermodynamically ‘downhill’, whereas active trans- tion’ is self-explanatory; moving from one place (‘lo- port requires input of energy to go ‘up the hill’. The cus’) to the other (‘trans’ is across). Circulating red latter can, for instance, be provided by coupling the blood cells can be considered to undergo continuous transport process to the dissipating energy of adenos- translocation, but the work presented here focuses on ine triphosphate (ATP) hydrolysis. molecular movements within the micrometer-sized di- Active mechanisms enable transport of large mensions of the cell. cargo, such as that of vesicles along the microtubule The interior of a cell is a tangle of continuous ac- network. Also transport against the chemical gradient tivity. On the to-do-list: thousands of simultaneous is ATP-consuming, e.g. the strong enrichment of cal- interactions and enzymatic conversions that cooper- cium in the endoplasmic reticulum (105 over cytosolic ate to maintain healthy cellular physiology and, in concentrations). Other examples of active transport addition, to accomplish cell type-specific functions. are the transfer of newly synthesized polypeptides The immense ensemble of molecules that constitute across the ER membrane or import/export through a living cell requires strict regulation of all possible in- the nuclear pore complex. However, active transport teractions and enzymatic activities. We can formulate is relatively slow. In contrast, the ´downhill´ diffu- two basic principles that are prerequisites to control sional redistributions are generally rapid processes. such ‘organized complexity’. First, interactions among Obviously, fluorescence microscopy is the meth- constituents, be it proteins, lipids, ions or small mol- od of choice to reveal and study rapid translocations in ecules, are to be specific. It is not surprising that cell living cells. It allows visualization with high spatial and biology has described a vast amount of conserved rec- temporal resolution. Furthermore, it enables compari- ognition domains that confer specificity on molecular son of pre- and post-stimulus conditions and cell-to- interactions. The specificity of a recognition domain is cell differences can be appreciated without disturbing accomplished by the unique combination of its three- averaging effects. Not surprisingly, the use of fluores- dimensional structure and charge distribution, fitting cence microscopy has been a common denominator to that of its binding partner following the principle throughout the studies presented in this thesis. Thus, of key-and-lock. Second, most enzymatic conversions before advancing to mechanisms (section 3) and ex- are regulated. In other words, they can be ‘switched amples (section 4) of translocations in cell signaling, on and off’. For example, many enzymes undergo the employed microscopic techniques will be briefly conformational changes that either silence or release introduced. their catalytic activities. Box 1 Potential pitfalls of fluorescent tagging A potential pitfall in fluorescence microscopy is the use of fusion proteins. Most fluorophores have molecular weights of ~30 kD. Thus, in the case of CLIC4 (29kD), fusion with a fluorophore doubles the molecular weight. Such bulky tags may steri- cally hinder protein-protein interactions. In line with this, the choice to tag either the N- or C-terminus could influence subcellular localization of the fusion construct. We have observed the latter for both Epac1 and CLIC4 and used the tagged version whose localization was most comparable to that of the endogenous counterparts. 1 nm In the case of gap junctional protein connexin43, C-terminal fusion of GFP even disrupted the formation of proper gap junctions. Therefore, a major challenge in fluorescence microscopy is to identify a genetically encoded fluorescent tag of mini- mal size. The lab of Roger Tsien (Noble Prize, 2008) seemed to catch a glimpse of the holy grail when they introduced the tetracysteine-system [1]. The concept was to insert six residues (Cys-Cys-Pro-Gly-Cys-Cys) into a protein of interest and sub- sequently label this motif specifically with a fluorescent dye (for comparison: GFP consists of ~240 amino acids). Unfortunately, the labeling conditions await further 1 nm optimization, since the labeling step seriously compromises cellular viability. 9 Chapter 1 2 Measurement of translocations 2.1 Fluorescence Resonance Energy in living cells using Transfer (FRET) fluorescence microscopy FRET is the radiationless transfer of energy from an excited donor to an acceptor fluorophore. It occurs under stringent conditions. First, the donor emission To monitor a protein of interest by fluorescence mi- spectrum must overlap with the absorption spectrum croscopy, it is genetically fused to a fluorescent pro- of the acceptor. Second, donor and acceptor fluoro- tein moiety. The first, green fluorescent protein (GFP), phores must be in close proximity, i.e. < 8 nm when was derived from the jellyfish Aequorea victoria and using fluorophores in the visible spectrum. A third de- multiple others have been cloned from other sea or- terminant is formed by the relative orientations of the ganisms. Subsequent mutational modifications have fluorophore dipoles. FRET is evident from quenched led to optimizations in terms of spectral properties, donor fluorescence and gained acceptor fluorores- brightness and stability (reviewed in [2]). The color cence. Thus, (a change in) FRET can be registered palette spans the spectral range from blue (BFP) to as (a shift in) relative donor and acceptor emission red (mRFP). As described in Box 1, caution is de- intensities. manded when tagging a protein of interest with a fluo- The distance constraint is most important for rescent moiety. the application of FRET in cell biology. When using Four different techniques have been employed in common fluorophore combinations (e.g. CFP/YFP or this thesis: GFP/mRFP), FRET is strictly limited to distances of 1) Confocal laser scanning microscopy (CLSM),
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