Septins are proteins that provide structural support for cells as they divide. Yeast cells are known to have seven types of septins, which have been widely studied, and 13 different septins have been identified in human cells, although they all seem similar to those found in yeast. Mutations in the genes that carry the genetic code for septins lead to a range of debilitating conditions in humans, including neurodegenerative diseases and male infertility.

An enzyme called Cdc42 is thought to have a key role in the formation of ring-like structures by septins before a cell divides, and in the subsequent dismantling of these rings after the cell has divided. A pair of proteins, called Gic1 and Gic2, is known to be critical for the formation of the septin rings, but the details of the interactions between these two proteins, Cdc42 and the septins are sketchy.

Now Sadian et al. have used two imaging approaches—electron microscopy and cryo-electron tomography—to scrutinise the role of Gic1 in greater detail in yeast cells. Gic1 interacts with specific subunits within adjacent septins, and these interactions have the effect of crosslinking the septins and stabilizing them in long filaments. However, high concentrations of the enzyme Cdc42 block the interaction between the Gic1 proteins and the subunits, causing the filaments to be dismantled. A future challenge will be to elucidate the interaction of these proteins in molecular detail using other techniques, in particular X-ray crystallography.

Septins are ubiquitous guanine nucleotide-binding proteins that have been implicated in many cellular processes such as cytokinesis, spindle positioning, morphogenesis, and exocytosis, and their mutation or overexpression is associated with neoplasia, neurodegenerative diseases and male infertility (Hall and Russell, 2004).

In yeast, four essential septins (Cdc3, Cdc10, Cdc11 and Cdc12) are found at the bud neck (Haarer and Pringle, 1987; Ford and Pringle, 1991; Kim et al., 1991), where they form an ordered ring composed of membrane-adjacent filaments (Hartwell, 1971; Byers and Goetsch, 1976). In total seven different septins were identified in S. cerevisiae, where they form filaments of variable size and combinations. Whereas the human genome encodes thirteen septins, C. elegans has only two and plants are devoid of septin genes (Hall and Russell, 2004; Ihara et al., 2005; Kinoshita, 2006). Despite the genetic variability, all septins share defined structural features. A recent crystallographic study on the human SEPT2-SEPT6-SEPT7 complex has shed light on the structural organization of human septins at the atomic level, which differs profoundly from that of other cytoskeletal structures (Sirajuddin et al., 2007, 2009). Septins interact via their central guanine nucleotide-binding domains (G-domains) and/or the N- and C-terminal extensions forming oligomers and non-helical filaments.

The basic structural unit of the yeast septin complex is an octamer, composed of four subunits, namely Cdc10, Cdc3, Cdc12 and Cdc11, arranged into two tetramers with two-fold rotational symmetry (Bertin et al., 2008).

Cdc42 has been identified as a central regulator of septin ring assembly and disassembly during different stages of the cell cycle (Gladfelter et al., 2002; Kozminski et al., 2003). Mutations that affect the GTPase activity of Cdc42 impair the initial assembly of septin rings, while after bud emergence, septin rings are maintained independently of Ccd42 (Gladfelter et al., 2002). It was also reported that the activity of Cdc42’s guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) are required for proper septin ring formation and localization, implying that one or more cycle(s) of nucleotide binding and hydrolysis are required for Cdc42 at the beginning of budding (Gladfelter et al., 2002; Caviston et al., 2003).

Among the essential effectors of Cdc42 in yeast are the two structurally homologous proteins Gic1 and Gic2, which are functional homologues of the human Borg protein (Joberty et al., 2001; Sheffield et al., 2003). It has been shown that Gic1 and Gic2 play an essential and overlapping role in cytoskeletal polarization (Brown et al., 1997; Hall and Russell, 2004) and septin recruitment (Iwase et al., 2006). However, the complex interplay between Cdc42, Gic1 and septins at the molecular level and its role during the cell cycle is not yet understood.

In this study, we have used electron microscopy and cryo electron tomography (cryo-ET) to describe the structural basis for the direct interaction of Gic1 and Cdc42 with septin filaments. Gic1 interacts with Cdc10 subunits of adjacent septin filaments and cross-links them. Because of this scaffolding, septin filaments are stabilized and form long railroad-like ordered filament cables. Cdc42-GTP directly binds to Gic1 and at higher concentrations inhibits the Gic1 interaction with Cdc10, resulting in the dissociation of the Gic1-septin complex. In its GDP-state, however, in absence of Gic1 Cdc42 interacts directly with Cdc10 and thereby mechanically disassembles septin filaments. Gic1 and Cdc42-GDP therefore compete for the same septin subunit. Finally, based on our results we propose a novel regulatory mechanism for septin ring formation and dissociation involving Cdc42 and Gic1.

EGFP-labeled septins without Gic1 (Cdc3-EGFP, Cdc10, Cdc11 and Cdc12) form relatively short and straight filaments (Figure 1A). Interestingly, when Gic1 is added during septin polymerization, long filaments that cluster together in large bundles are formed (Figure 1B). Studying the same but not EGFP-labeled samples using electron microscopy (EM), we found that in contrast to blank septin polymers that form long, often pairwise arranged filaments (Figure 1C), Gic1-septin complexes display a regular railroad-like structure with many cross-linked filaments bundled together (Figure 1D). Gic1 forms cross-bridges between at least two filaments, keeping them at a distance of about 20 nm (Figure 1E). At each cross-bridge, Gic1 binds to at least two adjacent septin subunits on each filament (Figure 1F), leaving a gap of six free subunits between individual Gic1 molecules (Figure 1G). The structure of Gic1 is not well defined (Figure 1F) indicating that Gic1 is flexible and oriented differently at each cross-bridge.

Figure 1

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To determine which septin subunit interacts with Gic1, we labeled septin-Gic1 complexes with antibodies against Cdc11 and observed that it sits exactly in the middle of a septin filament between two Gic1 cross-bridges (Figure 2A,B). Based on the known sequential order of septin filaments, this suggests that Gic1 binds to Cdc10 (Figure 2C).

Figure 2

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However, previous yeast two-hybrid, in vitro interaction and coimmunoprecipitation data indicated that Gic1 directly interacts with Cdc12 (Iwase et al., 2006). Therefore, we performed additional experiments to further corroborate our findings and prepared septin-Gic1 complexes devoid of either Cdc10 or Cdc11 (Figure 2D–I).

As expected from the results of antibody labeling, septin-Cdc10Δ, which formed short filaments, did not bind to Gic1 (Figure 2D–E). Septin-Cdc11Δ, however, which in accordance with previous studies (Bertin et al., 2010) does not polymerize (Figure 2F) produced a similar track-like structure as the wildtype when Gic1 was added (Figure 2G). Thus, addition of Gic1 resulted not only in the binding of the protein to the septin complex but also induced septin polymerization. We then performed single particle analysis (SPA) of the septin filaments and observed that only four subunits were located between Gic1 cross-bridges of Cdc11Δ filaments, ruling out an interaction with Cdc12 (Figure 2H–I).

In addition, yeast two-hybrid studies supported our in vitro data, showing that Gic1 only interacts with Cdc10 (Figure 2J). Notably, Cdc10 is mainly responsible for the specific interaction of septin filaments with PIP2, localizing them to membranes and promoting filament polymerization and organization (Bertin et al., 2010). For Gic2, which is a homologue of Gic1, a direct interaction through its polybasic region with PIP2 was also reported (Orlando et al., 2008). Together with our findings, this suggests that the organization and polymerization of septin filaments is controlled by the interaction of Gics with Cdc10 and the interaction of both proteins with PIP2.

Bertin et al. reported that septin filaments are cross-linked by overlapping C-terminal extensions of Cdc3 and Cdc12 (Bertin et al., 2008). Depending on the stain thickness we also observed these thin cross-links between bare septin filaments. However, in septin-Gic1 complexes, the large Gic1 cross-bridges are very prominent, causing stain to accumulate between the Gic1 cross-bridges. This makes it impossible to visualize the thin coiled-coils between Cdc3 and Cdc12, although they are probably still there.

To study the native three-dimensional structure of the filaments, we vitrified septin-Gic1 complexes and determined their structure using cryo electron tomography (cryo-ET) (Figure 3; Videos 1–4). We observed that Gic1, instead of cross-bridging only two filaments, simultaneously interacted with up to six septin polymers, forming long filament cables (Figure 3E–G). The organization of these cables is such that Gic1 forms a central scaffold to which septin filaments attach. Remarkably, individual septin filaments do not start at the same position and run along in parallel rigid lines. The septin-Gic1 cables are rather composed of many filaments of variable lengths that start at random positions, sometimes bypassing a Gic1 cross-bridge or changing place with another filament (Figure 3H–I). Filaments are often only attached to adjacent filaments for several nanometers. This gives the septin-Gic1 cables a certain flexibility that on the one hand allows them to bend and adjust to membrane curvature (Figure 3C). On the other hand it enables the interaction between cables, resulting in mesh-like structures (Figure 3D). Because of the limited resolution of the reconstructions, the additional thin connections described by Bertin et al. (Bertin et al., 2008) are not visible.

Figure 3

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Both in three-dimensional reconstructions and two-dimensional class averages, the density corresponding to Gic1 is poorly defined, suggesting a flexible structure of Gic1. Gic1, which based on its sequence has a molecular weight of 23 kDa (Gic1(104-314)) (Figure 4A), elutes at about 49 kDa from a gel filtration column corresponding roughly to a dimer (Figure 4B). In a typical Gic1 cross-bridge up to 12 Cdc10 subunits are involved (two per filament) (Figure 3E). If we assume that each of them binds independently to a Gic1 dimer, we would expect that 12 Gic1 dimers assemble into a large cross-bridge of 600 kDa. Although we cannot exclude that the density corresponding to Gic1 appears more spread out due to missing wedge artifacts in our tomograms, the average large volume of the Gic1 density in the electron tomograms (Figure 3E–I) indicates that it must contain multiple Gic1 molecules. In addition, the heterogeneity of the Gic1 cross-bridges suggests that the number of Gic1 molecules varies between cross-bridges.

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It was previously reported that Gic1 and its homologue Gic2 are effectors of Cdc42, which bind via their CRIB motif to the GTP-bound form of the Rho GTPase in cells (Brown et al., 1997; Chen et al., 1997). We made similar observations when studying the interaction of purified Gic1 and Cdc42 in vitro. Gic1 formed a stable complex with Cdc42-GppNHp (non-hydrolysable GTP analogue), but did not bind to Cdc42 in the GDP-state (Figure 5). On producing septin-Gic1 complexes in the presence of Cdc42-GppNHp, we found the same railroad-pattern as for the septin-Gic1 complexes, however, in this case with much bulkier cross-bridges (Figure 6A).

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SPA and cryo-ET of the Gic1-Cdc42-septin complex revealed that the cross-bridges were almost one and a half times larger in size, spanning not only two but four septin subunits, leaving only four septins unoccupied (Figure 6B–I, Video 5). This suggests a direct interaction of Cdc42-GppNHp with Gic1 on septin filaments (Figure 6J).

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Unexpectedly, an increase of the Cdc42-GppNHp concentration resulted in dissociation of the septin-Gic1-Cdc42 complex (Figures 6D and 7). Smaller in size, but still filamentous, the septin filament structure deteriorated somewhat as a result of this dissociation. Gic1 and Cdc42, however, formed protein aggregates (Figure 7C,F). A possible explanation for this observation is that full decoration of Gic1 with Cdc42 destabilizes the Gic1 interaction with septins, resulting in dissociation of the septin-Gic1-Cdc42 complex. In contrast, Cdc42-GDP did not bind to septin-Gic1 complexes even at 10 times higher concentrations (Figure 8). Similar behavior was reported for the functionally related human protein Borg (Joberty et al., 1999; Sirajuddin et al., 2007). Borg binds with its BD3 domain to SEPT7/SEPT6 dimers and induces the formation of long and thick septin filament fibers (Joberty et al., 2001; Sheffield et al., 2003). In accordance with our observations with Cdc42-GTP, Gic1 and septin, the human Cdc42-GTP negatively regulates this process and after binding to Borg inhibits the Borg–septin association.

Figure 7

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Figure 8

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To determine whether Cdc42 interacts directly with septins in the absence of Gic1, we produced septin filaments in the presence of Cdc42-GppHNp and Cdc42-GDP, respectively. Although Cdc42 in its GTP-state bound to septin filaments, no regular pattern as in the case of Gic1 was observed, indicating non-specific interaction (Figure 9A). Surprisingly, Cdc42-GDP bound more specifically to septin filaments, and formed, in contrast to Cdc42-GppNHp, railroad track-like structures (Figure 9B). Even more surprising was that Cdc42-GDP completely dissociated septin filaments at higher concentrations in less than an hour (Figures 9C and 10). We then analyzed the oligomers by size-exclusion chromatography, which showed that Cdc42-GDP binds to septin complexes (Figure 9D). SPA of the dissociated septins clearly identified either octamers with an extra density at their ends (90%) or hexamers without additional density (10%) (Figure 9E–F). Cdc3 sits at the ends of the hexamers, and at the second outer position of the octamers (Figure 9G–H). Considering the sequential order of septin oligomers, this indicates that Cdc42-GDP directly binds to Cdc10 (Figure 9I), which is also the binding partner of Gic1 (Figure 2), thereby causing dissociation of the filaments. Consequently, the resulting septin octamers differ from the ones observed under high-salt conditions (Figure 11). Instead of Cdc11, which is now in the center, Cdc10 forms the cap of the octamers at both ends. In addition, binding of Cdc42-GDP to Cdc10 probably influences its interaction with Cdc3 at the G interface resulting in the formation of Cdc42-GDP-Cdc10 dimers and hexameric (Cdc3-Cdc12-Cdc11) complexes. A direct interaction of a protein with Cdc42-GDP is unusual, since in most reported cases Cdc42, like other small G proteins, binds to its effectors in its active, that is GTP-bound state. However, also other proteins such as Msb3 and Msb4, which are GAP proteins, that are, like Gic1, involved in cell polarization, were shown to directly interact with Cdc42-GDP (Tcheperegine et al., 2005).

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Figure 11

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To identify the Cdc42 binding site on Cdc10, we calculated homology models of Cdc3, Cdc10, Cdc11 and Cdc12 using the SEPT2 structure (PDB 2QA5) as a reference and mapped regions of conservation between the four structures on their surfaces. Examining the Cdc10-Cdc10 interface, which is an N/C interface, we found that the N-terminal region is the least conserved, and would therefore present an ideal site for specific Cdc42-GDP binding (Figure 12A–B). We deleted the N-terminal 29 residues of Cdc10 and produced complexes whose Cdc10 was replaced by Cdc10(30-322). It was previously shown that deletion of the N-terminal residues of Cdc10 results in non-polymerizing septin complexes forming tetramers and inside-out octamers (Bertin et al., 2010). We analyzed the particles by EM and SPA, which showed that most of the complexes were indeed tetramers or octamers, and that the polymerization of the complexes was clearly impaired, as we did not observe filaments at low-salt conditions (Figure 13A–C). We then incubated the oligomers with high concentrations of Cdc42-GDP and looked for additional densities corresponding to Cdc42-GDP by EM and SPA. However, no additional protein was bound to the tetramers or octamers (Figure 13D–F). Gel filtration experiments corroborated this result and showed that indeed Cdc42-GDP does not bind to Cdc10(30-322) (Figure 13G). This indicates that the N-terminal region of Cdc10 is essential for the binding of Cdc42-GDP to septin filaments. Interestingly, the same region on Cdc10 was shown to be important for the interaction of septin filaments with PIP2 (Bertin et al., 2010).

Figure 12

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Figure 13

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From our previous experiments, we know that in septin-Gic1 complexes Cdc42-GDP binds neither to septins nor to Gic1 (Figure 8). Since both Gic1 and Cdc42-GDP bind to Cdc10 and very probably compete for the same binding site, Gic1 must have a higher affinity for Cdc10 than Cdc42-GDP. In order to prove this, we added Gic1 to septin-Cdc42-GDP complexes (Figure 14A–B). As expected, we found long railroad track-like ordered filaments indicating that Gic1 replaced Cdc42-GDP to interact with Cdc10, resulting in septin polymerization.

Figure 14

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We also observed septin-Gic1 filament complexes when we mixed Gic1 with octamers under conditions that normally inhibit polymerization, such as high-salt buffer (Figure 14C–D). This was surprising, since Gic1 binds and crosslinks Cdc10 proteins while high salt weakens the interaction at the N/C-interface between two Cdc11 proteins. Thus, the stabilizing effect of Gic1 must be stronger than the weakening effect of high salt. We confirmed this observation by performing the inverse experiment, that is septin-Gic1 complexes were dialyzed against a high-salt buffer. As expected, septin-Gic1 complexes were stable and did not dissociate (Figure 14E–F). Even septin complexes completely lacking Cdc11, which do not polymerize at low-salt conditions, form filaments when Gic1 is added (Figure 2G). Taken together, we conclude that Gic1 not only scaffolds septin filaments, but also increases their stability.

We then asked whether Gic1 would also stabilize septin filaments whose assembly is weakened at the Cdc10-Cdc10 instead of the Cdc11-Cdc11 interface. As shown above, complexes whose Cdc10 is replaced by Cdc10(30-322) do not polymerize at all and form tetramers or inside-out octamers (Figure 13A–C). Interestingly, Gic1 did not induce filament formation of such impaired septins (Figure 13H), but still bound to Cdc10(30-322) as indicated by an additional density attached to the septin oligomers and by size-exclusion chromatography (Figure 13I–L).

Our data (see Figure 15 for overview) provide important insights into the interaction of Gic1, Cdc42 and septins. Although our analysis focuses on only three proteins that are important for septin recruitment, ring formation and disassembly, which are complex processes involving many proteins (reviewed in Park and Bi, 2007), we think that the new information provided by our study is coherent enough to suggest the following mechanism for the interplay between Gic1, Cdc42 and septins during budding or the cell cycle.

Figure 15

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Because Cdc42-GDP binds to Cdc10 in septin complexes and prevents their polymerization, we propose that Cdc42-GDP rather than Gic1 recruits septin octamers (with Cdc10 at the caps) to the bud site (Figure 16A). At the bud site Cdc42 interacts with its GEF Cdc24, which catalyzes the exchange of its nucleotide (Figure 16B). As a result, Cdc42 dissociates from the septin complexes, which in turn polymerize. Besides activating many other proteins involved in septin recruitment and ring formation Cdc42-GTP recruits its effector Gic1 to the bud site (Figure 16C). Gic1, which has a high affinity for Cdc10, binds, scaffolds and stabilizes septin filaments (Figure 16D). Gic1-Cdc42-GTP-septin cables generated in this manner form a ring at the bud neck. Since our data show that the process of Gic1-septin cable formation does not necessarily require Cdc42-GTP, we propose that upon GAP-supported GTP hydrolysis, Cdc42-GDP, which has a much lower affinity to Gic1 than Cdc42-GTP, dissociates from the Gic1-septin complex (Figure 16E) and recruits more septin octamers to the bud site (Figure 16F).

Figure 16

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At later stages of the cell cycle the local Cdc42-GTP concentration increases at the bud neck, probably caused by an up-regulation of Cdc24 and/or identical spatial localization (Butty et al., 2002; Goryachev and Pokhilko, 2008). As a result, Cdc42-GTP binds to Gic1-septin complexes and competes with Cdc10 for Gic1, which finally results in the disassembly of the complex and thereby septin ring rearrangement (Figure 16G). Gic1 will then be either degraded or relocated. After GTP hydrolysis, Cdc42-GDP in the absence of Gic1 specifically binds to septin filaments and dissociates them (Figure 16H). Thus, septins can be reassembled and reused for the next cycle, as was observed by McMurray and Thorner when tracking labeled septins through several cell divisions (McMurray and Thorner, 2009). The model elegantly explains the cycles of GTP loading and hydrolysis by Cdc42 that were observed by Gladfelter et al. during budding (Gladfelter et al., 2002). In conclusion, Gic1 and Cdc42 in combination with many other factors, such as the nucleotide state of septins, specific interaction with lipids and protein posttranslational modifications regulate septin recruitment, ring formation and disassembly. Most importantly, Cdc42 does not only act as a regulator, but seems to be also involved in septin recruitment.

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Author details

1. Yashar Sadian

   Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

   Contribution

   YS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Christos Gatsogiannis

   Competing interests

   The authors declare that no competing interests exist.

2. Christos Gatsogiannis

   Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

   Contribution

   CG, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Yashar Sadian

   Competing interests

   The authors declare that no competing interests exist.

3. Csilla Patasi

   Department of Molecular Microbiology, Institute of Molecular Biology SAS, Bratislava, Slovak Republic

   Contribution

   CP, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Oliver Hofnagel

   Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

   Contribution

   OH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. Roger S Goody

   Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

   Contribution

   RSG, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Marian Farkašovský

   Department of Molecular Microbiology, Institute of Molecular Biology SAS, Bratislava, Slovak Republic

   Contribution

   MF, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Stefan Raunser

   Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany

   Contribution

   SR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   raunser@mpi-dortmund.mpg.de

   Competing interests

   The authors declare that no competing interests exist.

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