Septins are key organisers of cellular architecture that assemble into heterooligomeric complexes and higher-order filamentous structures, particularly at membrane interfaces. Through these assemblies, they contribute to processes such as cytokinesis, cell polarity, and membrane compartmentalisation. Despite their central role in cell biology, how septin complexes form, how they assemble into filaments, and how these processes are regulated remain incompletely understood, especially for human septins. This thesis investigates septin assembly as a dynamic and regulable process using a reconstitution-based strategy. By combining biochemical characterisation with membrane- based assays, it links the behaviour of septin complexes in solution to their organisation into higher-order structures at membranes. In solution, recombinant human septin complexes display intrinsic heterogeneity. While defined hexameric complexes are formed, smaller subcomplexes such as dimers and trimers are consistently observed. These observations suggest that dimers may contribute to hexamer assembly, whereas trimers do not appear to be required for this process. Upon membrane binding, septin behaviour undergoes fundamental changes. Dynamic mass photometry on supported lipid bilayers enabled direct observation of septin complexes diffusing, interacting, and assembling. Filament formation proceeds via both annealing of membrane-bound complexes and direct incorporation from solution. At the same time, septin filaments remain highly dynamic, undergoing continuous assembly and fragmentation, predominantly involving hexameric units, resulting in a dynamic steady state. BORG proteins, known cellular regulators of septins, modulate septin organisation. Both the isolated BD3 domain and full-length BORG2 induce a reorganisation of septin complexes, promoting septin complex pairing. The BD3 domain forms stoichiometric complexes with septins that are detectable in solution, whereas full-length BORG2 does not, suggesting more transient interactions. Despite these differences, both lead to septin complex pairing. Collectively, these findings demonstrate that septin organisation arises from the interplay of intrinsic assembly properties, physical constraints imposed by membranes, and regulatory interactions. This work provides a mechanistic framework linking molecular-level interactions to the dynamic organisation of septin structures.
