Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.:Summary 1 List of publications 3 1 Introduction 9 Aims of the thesis 15 2 Generation of a local BMP signal in testis and wing disc 17 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.1.1 Stem cells and stem cell niches . . . . . . . . . . . . . . 19 2.1.2 The Drosophila testis stem cell niche . . . . . . . . . . 20 2.1.3 BMP signalling in the fly . . . . . . . . . . . . . . . . . 23 2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2.1 The BMP niche signal is transduced locally at adherens junctions 25 2.2.2 Generation of the local BMP niche signal . . . . . . . . 30 2.2.3 Exocyst involvement in long-range BMP signalling . . 34 2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3 Hedgehog pathway overactivation in the testicular niche 41 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.1.1 The role of Hedgehog in the fly . . . . . . . . . . . . . 43 3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.2.1 Overexpression of Hh increases the CySC number and expands their range 45 3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4 Visualization of Smo phosphorylation and biophysical detection of Smo clustering 49 4.1 Introduction (part I) . . . . . . . . . . . . . . . . . . . . . . . 51 4.1.1 Hedgehog signalling in the fly . . . . . . . . . . . . . . 51 4.1.2 Reception and transduction of the Hh signal by Ptc and Smo 54 4.2 Results (part I) . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.2.1 A fluorescent reporter for Drosophila Smo tail phosphorylation 56 4.2.2 Smo phosphorylation and localisation in the salivary gland 61 4.2.3 Smo localisation in cultured insect cells . . . . . . . . . 63 4.2.4 Smo membrane localisation and phosphorylation . . . . 65 4.3 Introduction (part II) . . . . . . . . . . . . . . . . . . . . . . . 67 4.3.1 Fluorescence correlation spectroscopy (FCS) . . . . . . 67 4.3.2 Dual-color fluorescence cross-correlation spectroscopy (FCCS) 72 4.3.3 Artefacts in FCS/FCCS . . . . . . . . . . . . . . . . . 73 4.4 Results (part II) . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.4.1 Smo clustering measured by FCCS . . . . . . . . . . . 79 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82