Septin assemblies form by diffusion-driven annealing on membranes

Abstract

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.cytoskeleton | biophysics S eptin filaments form rings, bars, and gauzes that serve as a scaffold at cell division sites; act to retract blebbed regions of membrane; and restrict diffusion between cell compartments (1-4). Septin function is required for cell division and viability in many eukaryotes whereas misregulation is associated with cancers and neurodegenerative disorders (5-8). Furthermore, septins mediate entry of both bacterial and fungal pathogens into host cells (9-11). In vivo, septin assembly is restricted both in time and in space through local activation of small GTPases such as Cdc42. Localized signaling leads to higher-order septin structures forming closely apposed to the plasma membrane at the plane of division, sites of polarity, and curved membranes (10,(12)(13)(14). Notably, eukaryotic cells of different geometries build higher-order septin assemblies of various shapes, sizes, and functions (4, 15, 16). Although septins are critical for spatial organization of cell plasma membranes, their assembly and disassembly dynamics are not understood (15).Electron microscopy (EM) studies of recombinant and immunoprecipitated Saccharomyces cerevisiae septins have shown that septins form nonpolar hetero-octameric rod-shaped complexes in high-salt buffers (>300 mM) and elongated filaments when dialyzed into low-salt buffers (<100 mM) (17, 18). Structural analyses of worm and mammalian septins have revealed that the heteromeric, rod-shaped complex is conserved (19-21). Thus, septin rods characterized to date contain two copies of each septin subunit assembled into a nonpolar, heteromeric complex (Fig. S1). Association of purified septin proteins with phosphoinositide-containing membrane monola...

Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 inSaccharomyces cerevisiae

“…Strikingly, retention of a0, but substitution of all 12 Lys and Arg residues in Cdc11 and Shs1 combined, caused the most severe reduction in the efficiency of their incorporation at the bud neck ( Figure 3E). This latter finding suggests that recruitment of Cdc11-and Shs1-containing complexes to the cytosolic side of the PM is a prelude to their assembly into filaments and higher-order structures in vivo, consistent with recent imaging studies in vivo (Bridges et al 2014) and with in vitro studies wherein association with lipid monolayers promoted septin filament formation (Bertin et al 2010). Thus, the importance of positive charge in a0, at least for Cdc11 and Shs1 (and, likely, Cdc10; Figure S2) (Figures 3 and Figure S3), seems primarily to be in concentrating septin complexes at the PM, thereby increasing the rate of their incorporation into polymers.…”

“…Work both in vitro (Bertin et al 2010) and in vivo (Bridges et al 2014) supports the notion that the presence of membranes promotes filament assembly and the formation of higher-order septin structures. The lipid responsible for high-affinity binding in vivo and in vitro is the highly anionic species PtdIns4,5P 2 (Zhang et al 1999;Bertin et al 2010).…”

“…We were somewhat surprised, therefore, when we observed that a Cdc11(D2-18 R19A)-mC, a mutant that should mimic Cdc11-NT-A-mC with regard to its loss of positive charge, was able to support growth ( Figure S3B, compare third line to first line). In this case, however, we suspected that the positive residues in Shs1 might serve a partially redundant function for recruitment of septin complexes to the cytosolic side of the PM (Bertin et al 2010;Bridges et al 2014). Consistent with this view, cells expressing Cdc11(D2-18 R19A)-mC were inviable when Shs1 was absent ( Figure S3B, compare fourth line to third line) or carried the equivalent alterations in its N-terminal region ( Figure S3B, compare fifth line to third line), even though Shs1(D2-18 K19A R20A) was produced, folded, and able to associate with Cdc12, because like WT Shs1 or Shs1-NT-A, its expression blocked growth of Cdc11-deficient cells ( Figure S3B, compare seventh line to sixth line).…”

Sep(t)arate or not – how some cells take septin-independent routes through cytokinesis

“…In addition, association of septins with membrane lipids and other cytoskeletal elements can influence filament formation (Kinoshita et al, 2002;Tanaka-Takiguchi et al, 2009;Bertin et al, 2010). A recent study clearly shows that cytosolic oligomeric complexes of yeast septins elongate after annealing to the plasma membrane, suggesting that lipid layers act as supports for the formation of higher-order septin structures (Bridges et al, 2014). yeasts (Balasubramanian et al, 2004;Pringle, 2008).…”

Filaments and fingers: Novel structural aspects of the single septin from Chlamydomonas reinhardtii

“…The localization of CrSEPT at the base of the flagella suggests that it could be acting as a membrane-diffusion barrier and, therefore, performing a role similar to that Structure of C. reinhardtii septin described for septins found in cilia and flagella in other organisms. The plasma membrane has been implicated in driving septin filament elongation (44) and thus could play a role at the base of the flagella in controlling polymerization for diffusion barrier formation. This could represent an original ancestral function for septins, which has been preserved throughout evolution.…”