More recently we have initiated a collaboration with the laboratory of Jeremy Thorner (UC Berkeley) to study septin filaments, an additional cytoskeletal element involved in cell division. Septins comprise a discrete family of GTP-binding proteins conserved from fungi to humans. Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7). Only Shs1 is non-essential. These septins form filaments to define a collar at the bud neck during cytokinesis. The collar filaments impose a barrier to diffusion of integral membrane proteins between mother and bud cells, and act as a scaffold to recruit proteins required for bud-site selection and a morphogenesis checkpoint. We carried out single-particle analysis by electron microscopy (EM) and revealed that the hetero-oligomer of the four essential mitotic septins is an octameric linear rod (Bertin et al., 2008, PNAS 105, 8274-8279).

We identified the location of each subunit within the rod by examining complexes lacking a given septin by antibody decoration, and by fusion to marker proteins (GFP or MBP). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments, but not when Cdc11 is absent or its N-terminus altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by hetero-tetrameric coiled-coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures. Some of our major interests now are to characterize the interplay of septins and lipids and to define the structural bases of septin regulation by kinases.