Mechanisms of assembly and function of the septin cytoskeleton in Drosophila

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Mechanisms of assembly and function of the Drosophila septin cytoskeleton. Background and Preliminary Data Septins form a family of highly conserved, cytoskeletal proteins that act at the interface of membranes and the actin- and microtubule-based cytoskeletons. Septins form palindromic, hetero-oligomeric rods, or "complexes", that can polymerize end-on-end into filaments. These filaments can assemble into higher-order structures such as bundled filaments, rings, gauzes and as we have found, tubes. Septins can act as scaffolds and as membrane diffusion barriers in diverse cellular structures such as the base of the primary cilium, the sperm annulus, and the cleavage furrow during cell division. However, it is not clear how higher order assemblies form, are regulated or how they impact cellular homeostasis. Septins share a conserved organization including a central GTPase domain that is essential for complex formation and more variable N- and C-termini that contain polybasic regions (N-termini) and coiled-coil domains (C-termini) whose roles are poorly understood. We have identified three prominent structures comprising higher-order assemblies of septins in cultured Drosophila Schneider's S2 cells: cytoplasmic rings, cytoplasmic tubes and the plasma membrane-anchored cytokinetic ring. Preliminary experiments show that these structures have differential requirements for their formation. Hypothesis, Aims and Approach Our long-term aim is to define the molecular basis of the full complement of septin higher-order assemblies and to understand their specific functions in both cellular and organismal contexts. We hypothesize that specific interaction partners collaborate with specific septin domains to produce different higher-order assemblies and that these differentially regulate cellular architecture and function. Using a combination of biochemical and cell biological approaches we will perform a comprehensive analysis of Drosophila septins to define the molecular determinants of each type of higher-order structure. We propose the following specific aims: 1) Define the requirements for the assembly of septin-based structures in Drosophila S2 cells. 2) Define septin assembly mechanisms and interactions with actin in vitro. 3) Define the roles of the septin cytoskeleton in vivo in Drosophila. These studies will elucidate the molecular mechanisms by which septins can assemble into different higher-ordered structures in living cells, and will shed light on the undoubtedly complex interplay between septins, other cytoskeletal elements and the plasma membrane. This will greatly advance our understanding of the molecular events underlying the multitude of cellular processes in which the septins participate.