Septin Filament Self-Assembly and Architecture

We are currently in 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.

Structure and Assembly of Yeast Septins

Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1/Sep7) during vegetative growth. 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).

septin filament

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.

Interplay between Septins and Membranes

Phosphoinositides are essential signaling lipids, enriched at the bud neck in yeast. We have shown that PIP2 promotes septin assembly, and now hypothesize that septin filaments may constrain PIP2 mobility within the membrane bilayer. By binding to PIP2 and self-assembling (and, thereby, bringing yet more PIP2-binding sites into the local area), septins could restrict PIP2 diffusion and/or induce formation of a PIP2-enriched domain, which might, in turn, recruit to the same region more septins, and/or other PIP2-binding factors that may influence septin structure and/or function, generating a positive feedback loop.

septin filament

Left, Shs1-containing complexes are rod-shaped in high salt conditions. Middle, Shs1-containing rods assembly into rings in low salt conditions. Right, A phosphomimetic mutation in Shs1 (S259D) causes formation of gauzes in low salt conditions.

Effect of Isoforms and Modifications on Septin Assembly and Function

In addition we have discovered that Shs1 can replace Cdc11 at the ends of octameric rods, and at low ionic strength Shs1-containing rods assemble to form rings (Garcia et al. JCB 2011). Phosphomimetic mutation of Shs1 residues known to be phosphorylated in vivo either inhibit self assembly of Shs1-containing rods or result in the formation of a novel ultrastructure, septin gauzes. Deletion of SHS1 in the cell results in the formation of incomplete septin collars and the disorganization of the bud neck filaments, which indicates that Shs1 organizes septin filaments into circumferential rings in vivo as well as in vitro. Our results suggest that alternate septin subunits can modify the self-assembly properties of septin complexes and thereby confer functional specificity. This is especially relevant in light of the fourteen septin genes present in mammalian genomes which cell type-specific expression. 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.

Interaction of Septins with Cellular Partners

Among the cellular factors that interact directly with septins is the protein kinase Hsl1, an essential component of a cell cycle checkpoint that becomes activated when the collar of septin filaments at the bud neck is properly formed. We have developed a pelleting assay for assembled septins that we can utilize to test the binding of septin-interacting proteins in vitro using highly purified proteins. We have found that the truncation Hsl1(1245-1518) co-pellets with septin filaments and rings, while size exclusion chromatography shows that it does not bind to free septin octamers. Thus, Hsl1 interact with septins in an assembly-dependent fashion. We have also analyzed the biophysical properties of the septin-binding protein Bni5 and how its association with septin filaments affects their organization. We found that the interaction of Bni5 with the terminal subunit (Cdc11) at the junctions between adjacent hetero-octamers in paired filaments is highly cooperative. Both the C-terminal end of Bni5 and the C-terminal extension of Cdc11 make important contributions to their interaction. Moreover, this binding may stabilize the dimerization of Bni5, which, in turn, forms cross-filament braces that significantly narrow, and impose much more uniform spacing on, the gap between paired filaments.

Booth, E.A, Sterling, S.M., Dovala, D., Nogales, E. and Thorner, J. (2016) Effects of Bni5 Binding on Septin Filament Organization. J.Mol. Biol 428, 4962-4980.

Finnigan, G., Sterling, S., Duvalyan, A., Liao, E., Sargsyan, A., Garcia, G., Nogales, E. and Thorner, J. (2016) Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae MBoC 27, 2213-2233.

Garcia G 3rd, Finnigan, G.C., Heassley, L.R., Sterling SM, Aggarwal A, Pearson CG, Nogales E, McMurray MA, Thorner (2016). Assembly, molecular organization and membrane-binding properties of developmental-specific septins J Cell Biol 212, 515-29.

de Val, N., McMurray, M.A, Lam, L.H., Hsiung, C. C.-S., Bertin, A., Nogales, E. and Thorner, J. (2013) Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins. Proteins 81, 1964-1979

Bertin, A., MacMurray, M., Pierson, J., Thai, L., MacDonald, K., Zerh, E., Peters, P., Garcia III, G., Thorner, J. and Nogales, E. (2012) Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae. MBoC 23, 423-432. Selected “Highlight” from MBoC by the ASCB.

Garcia, G.III, Bertin, A., Li, Z., McMurray, M., Thorner, J. and Nogales, E. (2011) Subunit- dependent modulation of septin assembly: budding yeast septin Shs1 promotes ring and gauze formation. J.Cell Biol 195, 993-1004. Focus article on the same issue.

McMurray, M.A., Bertin, A., Garcia, G.III, Lam, L., Nogales, E. and Thorner, J. (2011) Plasticity in Higher-order Septin Assembly: Evidence that Septin Filament Formation is Essential in Budding Yeast. Dev. Cell, 20, 540-549.

Bertin, A., Thai, L., McMurray, M., Garcia, G., Votin, V., Grob, P., Allyn, T., Thorner, J. and Nogales, E. (2010) The phosphoinositide PI(4,5)P2 promotes budding yeast septin filament assembly and organization. J. Mol. Biol 404, 711-731.

Bertin, A., McMurray,M.A., Grob,P., Park, S-S., Garcia, G. III, Patanwala, I., Ng, H-L., Alber, T.C., Thorner, J. and Nogales, E. (2008). Saccharomyces cerevisiae: Supramolecular organization of hetero-oligomers and the mechanism of filament assembly. PNAS 105, 8274- 8279.