Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. In mammalian cells Spir proteins are targeted towards intracellular membranes by a modified FYVE zinc finger motif and an adjacent potential Rab GTPase binding sequence, the Spir-box. Although a role for mammalian Spir-1 in vesicle transport processes of the exocytic pathway and the transport beyond early endosomes has been described, the function and regulation of the actin organizers at vesicular membranes is unknown. One way Spir function is regulated is by a direct interaction with the distinct actin nucleation factors of the formin subgroup of formins. The interaction is mediated by the Spir KIND domain and the formin Spir interaction (FSI) sequence. Several members of the formin superfamily have been shown to contain at the corresponding position an autoregulatory peptide (DAD, diaphanous autoregulatory domain), that is responsible for binding of a N-terminal autoinhibitory domain (DID, diaphanous inhibitory domain), which causes an autoinhibited backfolded conformation of the formin proteins. It is intriguing to speculate that the FSI sequence acts like a DAD domain and mediates an autoinhibited conformation, which can be released by a competing interaction with the Spir KIND domain. By addressing the question if Spir proteins are autoregulated, we found that the N-terminal Spir KIND domain can pull down the Spir C-terminal part from cell lysates. An autoinhibitory backfolding mechanism including KIND/Spir-CT could regulate both, the membrane targeting and actin nucleation activity. In order to address the regulation of the Spir/formin complex in the context of subcellular localization and actin nucleation, we propose experiments to functionally reconstitute the Spir/formin actin nucleator complex at model membrane systems. We will employ the most recent advances in fluorescence microscopy and lipid membrane technologies, to learn outside of the overwhelming complexity of a cell, how Spir proteins are targeted towards membranes and if and how membrane targeting influences the nucleation activity of the Spir/formin complex. We will address questions such as the membrane docking and lipid specificity of the Spir FYVE module, the potential interaction of Spir with Rab GTPases, a possible dimerization of Spir proteins at membranes and the influence of membrane targeting or dimerization on actin nucleation activity. By incorporating the formin subgroup proteins into our studies, we will analyse how these processes are influenced by the Spir/formin interaction.
