Profilin From the Cell Edge into the Nucleus Sara Sadi Doctoral Thesis from the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2014 ©Sara Sadi, Stockholm University 2014 ISBN 978-91-7447-822-8 Printed in Sweden by Universitetsservice US-AB, Stockholm 2014 Department of Molecular Biosciences, The Wenner-Gren Institute 2 To my beloved Mother and Father for always encouraging me to follow my passion. 3 4 Summary Internal and external signaling dependent changes in cell behav- ior are directly linked to force-generating remodeling of the actin microfilament system which is juxtaposed to the inside of the plasma membrane. This dynamic filament system is involved in many processes in the cytoplasm and the nucleus of eukaryotic cells. This thesis studies profilin, a regulator of actin filament dynam- ics which functions during incorporation of new actin molecules at growing filament ends at the cell periphery. Profilin is also present in the nucleus but its function is less well understood in this compartment. Here I present results concerning profilin and the activity of the transcription factor SRF, which is known to control the expression of actin and many actin-binding proteins in a process requiring the MRTF-A co-factor. MRTF-A binds monomeric actin and is released upon receptor mediated actin polymerization. Depletion of the two profilin isoforms I and IIa reduced MRTF-A/SRF-dependent transcription, most likely since the lack of profilin enable more MRTF-A to bind actin monomers and thereby prevent SRF-transcription. Interestingly profilin de- pletion also seemed to affect general transcription in the two cell lines investigated. In a separate study, a close connection be- tween profilin, and possibly also profilin:actin, with microtubules was revealed. Microtubules are important for intracellular traf- ficking of vesicles as well as directional cell migration and the observation made here suggests the existence of a microtubule- associated platform for actin filaments formation. In congruence, the microtubule-associated actin nucleation promoting factor WHAMM was found to interact with profilin. Finally, the intra- cellular distribution of profilin was investigated by fluorescence microscopy using different peptide specific antibodies. Since these antibodies showed unique but varying results our work emphasizes common problems connected with this technique. 5 List of Articles Included in This Thesis Paper I: Profilin I and II Influence Migration and Transcription in non- Neuronal Cells Sara Sadi, Michaela Nejedla, Anna-Karin Johnsson and Roger Karlsson (2013). Under Revision Paper II: Profilin is Associated with Microtubules in Cultured Cells Sara Sadi, Michaela Nejedla, Felicia Nunes de Almeida, Pontus Aspen- ström and Roger Karlsson (2013). Under Revision Paper III: Cellular Distribution of Profilin as Determined by Sequence- specific Antibodies Sara Sadi and Roger Karlsson (2013). Manuscript Work not included in this thesis: Nuclear translocation of IGF-1R via p150Glued and an importin- β/RanBP2-dependant pathway in cancer cells Sylvia Packham, Dudi Warsito, Yingbo Lin, Sara Sadi, Roger Karlsson, Bita Sehat and Olle Larsson (2014). Accepted, pending revision 6 Table of Contents Actin-Based Cell Motility .......................................................................... 8 The Actin Microfilament System in non-muscle cells............................ 12 The Actin Molecule and its Polymerization Characteristics ................. 12 Profilin in Cell Motility .......................................................................... 16 Profilin Dependent Processes in the Nucleus ................................... 20 Profilin and post-translational modification .................................... 24 Profilin in the Cytoplasm ................................................................... 26 Other Actin Regulating Proteins of Relevance in the Context of Profilin .................................................................................................... 28 Ena/VASP ........................................................................................... 28 WASP family of proteins .................................................................... 30 The Arp2/3 complex ........................................................................... 32 Formins ............................................................................................... 34 ADF/Cofilin ........................................................................................ 36 Thymosin β4 ........................................................................................ 38 The Microfilament System in Cell Communication ............................. 39 Serum Response Factor and the Microfilament System ....................... 41 Microtubule-Based Cell Motility - the Involvement of Actin .............. 45 Present Investigations ............................................................................ 47 Study I .................................................................................................. 48 Study II ................................................................................................ 54 Study III ............................................................................................... 56 Acknowledgements ................................................................................. 58 References ...............................................................................................60 7 Actin-Based Cell Motility Motility is a major characteristic of all living organisms; it in- cludes movement of individual cells as well as of intracellular components. The mechanisms behind the motility of eukaryotic cells are largely based on the actin microfilament (MF) system. This system is highly dynamic; under certain conditions it un- dergoes remodeling due to tightly regulated polymerizations and depolymerizations of actin coupled with filament reorganiza- tions. Such actin remodeling is often a consequence of signals captured by receptor molecules at the cell surface (Chinkers et al., 1979; Lindberg et al., 2008; Mellstroom et al., 1983; Pollard and Borisy, 2003; Ridley, 2011). My studies have been concentrat- ed on actin in complex with profilin, profilin:actin, which repre- sents a major fraction of polymerization-competent actin in the cell: how profilin and/or profilin:actin influences cell migration and how profilin and/or profilin:actin distribute to different re- gions of the cell are central questions which I have tackled. In the long range, an aim of these studies is to understand how pro- filin:actin accumulates at sites in the cell periphery where actin polymerization is prominent. Furthermore, actin and actin bind- ing proteins are also components of the nucleus where they con- tribute to gene control at different levels (Grosse and Vartiainen, 2013; Percipalle, 2013), and this thesis also addresses the involve- ment of profilin in the regulation of the transcription factor Se- rum Response Factor (SRF). Cell migration is classified according to the character of the movement. One such category of movement is the mesenchymal migration where cells translocate over the substratum by extend- ing a broad and flat leading edge or lamellipodia in conjunction with finger-like protrusions called filopodia (Calvo et al., 2011; Faix et al., 2009; Sixt, 2012; Small et al., 2002) (Fig. 1). These pro- 8 trusions which are formed by actin polymerization, attach to the substratum via specific surface proteins at specific sites, called focal complexes. These contacts are highly dynamic but some matures into more stable adhesions called focal adhesions (Buccione et al., 2004; Rottner et al., 1999b). Transmembrane proteins known as integrins are important receptors which func- tion together with multiple intracellular proteins such as talin, paxillin, tensin and α-actinin and are responsible for mediating adhesion to components of the extracellular matrix (Galbraith et al., 2007). As a consequence of integrin activation the cell extend new protrusions in the direction of migration. The continued forward translocation is also contributed for by acto-myosin contractions that occur internally along the long axis of the cell, in special arrangements of the MF-system, consisting of bundled filaments, among them stressfibers play a central role (Vallenius, 2013) (Fig. 1). Characteristic of many cells moving with the mes- enchymal type of migration such as fibroblasts is the ruffling phenomenon (Abercrombie et al., 1970). Ruffles can be seen at the leading edge when lamellipodia fail to establish stable adhe- sions or detach from the substrate and form wave-like structures that collapse on the cell surface. Cells migrating in vitro in 3D- networks of collagen fibers or similar tissue models also exhibit ruffling phenomena although these activities are less prominent compared to when moving on a flat surface. Mesenchymal mi- gration in tissue is associated with degradation of ECM due to the activity of matrix metalloproteases typically exocytosed by many cells and particularly a prominent feature of metastasizing cancer cells (Friedl and Wolf, 2010). Another major type of mi- gration is often referred to as amoeboid. It is characterized by movement of rounded or ellipsoid cells that lack mature focal adhesions. Instead their interaction with the substrate is charac- terized by many weak contacts. A surface phenomenon often connected with this type of