The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin Free

Septins are evolutionarily conserved GTPases and mainly localize to membrane structures within the cell (Bridges and Gladfelter, 2015; Fung et al., 2014; Marquardt et al., 2019; Mostowy and Cossart, 2012). On the cortical membrane, septins form high-order structures, including short filaments, rings, and gauzes, in a cell-cycle- and/or location-dependent manner (Bertin et al., 2010; Bridges and Gladfelter, 2015; Bridges et al., 2014; Marquardt et al., 2019; Sirajuddin et al., 2007). These high-order septin structures could function as scaffolds responsible for recruiting a wide range of proteins to regulate cytokinesis and/or as diffusion barriers to compartmentalize membranes (Caudron and Barral, 2009; Dobbelaere and Barral, 2004; Fung et al., 2014; Kozubowski et al., 2005; Marquardt et al., 2019; Trimble and Grinstein, 2015). In addition, septins play critical roles in membrane remodeling and in regulating cortical rigidity (Beber et al., 2019; Fung et al., 2014; Gilden and Krummel, 2010; Gilden et al., 2012; Mavrakis et al., 2014; Mostowy et al., 2011). Despite significant progress in understanding septin functions, how septins are localized to the cortical membrane remains elusive.

The membrane affinity of septins might be dictated by their intrinsic properties since an N-terminal polybasic region is present in most septins (Casamayor and Snyder, 2003; Fung et al., 2014; Zhang et al., 1999). Consistent with this idea, recombinant septins can bind silica beads decorated with phospholipids, dependent of septin concentration and bead size (Bridges et al., 2016). Moreover, in vitro lipid-binding assays have established that septins bind phosphoinositides (Bertin et al., 2010; Casamayor and Snyder, 2003; Onishi et al., 2010; Zhang et al., 1999). The fission yeast Schizosaccharomyces pombe has seven septins, i.e. Spn1–Spn7 (Onishi et al., 2010). Spn1–Spn4 function in vegetative cells, and Spn2 and Spn5–Spn7 mediate forespore membrane extension during sporulation (An et al., 2004; Onishi et al., 2010). Similarly, the N-terminal region of Spn1–Spn4 and Spn7 contains a cluster of basic amino acids, and in vitro phosphoinositide-binding assays with Spn2 and Spn7 showed that both have an affinity for phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate [PtdIns(4)P and PtdIns(5)P] (Onishi et al., 2010). Recent evidence suggests that septins are sensors of micron-scale membrane curvature and are often enriched at the membrane regions with high curvature, such as the yeast bud neck, the bases of membrane branches and the cell projection tips (Bridges et al., 2016; Cannon et al., 2019). Nevertheless, septins can also localize to the cortical regions with low curvature. For instance, during interphase, fission yeast septins localize as short filaments on the cell sides and form complex high-order structures through annealing and end polymerization (Bridges et al., 2014). How septins are localized to the cell cortex areas with low curvature remains poorly understood, and the function of the cortically localized septins is elusive.

In the budding yeast Saccharomyces cerevisiae, septins are recruited to the cortical region of the budding site by the Rho GTPase Cdc42, the master regulator of cell polarity, via the effector proteins of Cdc42, Gic1 and Gic2 (Caviston et al., 2003; Gladfelter et al., 2002; Iwase et al., 2006). The cortically localized septins subsequently recruit the Cdc42 GTPase-activating protein (GAP) to inhibit Cdc42 activity, and the inhibitory effect on Cdc42 is counteracted by polarized exocytosis (Okada et al., 2013). These feedback controls among Cdc42, septins and exocytosis then promote the formation of the septin ring at the bud neck (Okada et al., 2013). Therefore, the interplay between septins and Cdc42 GAP proteins play critical roles not only in septin ring formation but also in polarized cell growth. Interestingly, similar to septins, most RhoGAP proteins contain membrane-binding domains such as BAR, PH and a polybasic region (PBR) (Amin et al., 2016). Whether the interplay between RhoGAP proteins and septins is important for their localization to the cell cortex is an interesting question to pursue.

Nine RhoGAP proteins exist in the fission yeast (Villar-Tajadura et al., 2008). Among them, Rga3, Rga4 and Rga6 localize to the cell cortex and have GAP activity for Cdc42 (Das et al., 2007; Gallo Castro and Martin, 2018; Revilla-Guarinos et al., 2016; Tatebe et al., 2008). Functional characterization shows that Rga4 and Rga6 work in concert to regulate cell polarity by restricting active Cdc42 to the growing cell tip (Revilla-Guarinos et al., 2016).

In this study, we identified Rga6 as a protein that interacts with the septin complex and demonstrated that the interaction between septins and Rga6 promotes the cortical localization of septins. On the cell cortex, Rga6 and septins collaborate to regulate cell growth by confining active Cdc42 to the cell end. Hence, this present work establishes a role of septins in regulating the targeted localization of active Cdc42 required for polarized cell growth.

In fission yeast cells, the building block of high-order septin structures is a heterooctamer containing two molecules of each Spn1, Spn2, Spn3 and Spn4 (An et al., 2004). Spn1 and Spn4 are essential for the formation of the heterooctamer, since high-order septin structures are absent in cells lacking either Spn1 or Spn4 (An et al., 2004; Zheng et al., 2018). Therefore, to identify the RhoGAP protein(s) that regulate the cortical localization of septins, we examined the localization of Spn1, which we tagged with tdTomato (Spn1–tdTomato), in wild-type (WT) and mutant cells lacking the individual RhoGAP protein (i.e. Rga2–Rga9).

Microscopy observation showed that Spn1–tdTomato localized as scattering dots/bars on the cell cortex in interphase and appeared to be enriched at the cortical sites adjacent to the growing cell tip marked by CRIB–GFP (labeling active GTP-bound active Cdc42; Tatebe et al., 2008) (Fig. 1A,B). This characteristic localization of Spn1 was found in WT and most of the RhoGAP deletion-mutant cells. Interestingly, the absence of rga6 or rga8 significantly impaired the characteristic localization of Spn1 on the cell cortex (Fig. 1A,B). To confirm this observation of the characteristic localization, we carefully measured the intensity of Spn1–tdTomato and CRIB–GFP along the cortex on one side of the cell that displayed CRIB–GFP signals (Fig. 1C). More than 50 cells were measured for each type of strain, and the maximum intensity value of CRIB–GFP (referred to as ‘Zero’ in Fig. 1C and which corresponds to the ‘0’ point on the x-axis on the plots shown in Fig. 1D) was used to align the data. As shown in Fig. 1D, in WT cells, the CRIB–GFP signal displayed the highest intensity at the zero point and decayed almost symmetrically on both sides of the zero point, while two apparent peak signals of Spn1–tdTomato were detected as flanking the CRIB–GFP signals. This result confirmed that Spn1–tdTomato is enriched on both sides of the crescent shape of CRIB–GFP. All types of the tested cells except rga6Δ and rga8Δ showed similar cortical localization profiles of Spn1–tdTomato and CRIB–GFP to the ones in WT cells (Fig. 1D,E). Intriguingly, no apparent average Spn1–tdTomato peak signals were found to flank CRIB–GFP in rga6Δ cells, suggesting that Rga6 is required for the enrichment of Spn1–tdTomato on both sides of the crescent shape of CRIB–GFP. In addition, the signals for Spn1–tdTomato and CRIB–GFP apparently overlapped at the cell end in rga8Δ cells, indicating that Rga8 is also required for proper localization of Spn1–tdTomato on the cell cortex. In this present work, we focus on addressing the role of Rga6 in regulating the localization and function of Spn1.

Next, we characterized the localization of Spn1–tdTomato in rga6+ and rga6 mutant cells. Time-lapse live-cell microscopic analysis showed that Spn1–tdTomato mainly localized on the side of a cell but not at the growing tip where Cdc42-GTP (marked by CRIB–GFP) resided (Fig. S1A, top panel). During cytokinesis, Spn1–tdTomato formed a ring and colocalized with CRIB–GFP at the middle of a cell, and dissolution of the septin ring was concomitant with the reappearance of CRIB–GFP at the old cell ends (Fig. S1A, bottom panel). Septins play a crucial role in cytokinesis in fission yeast (Berlin et al., 2003; Tasto et al., 2003; Wu et al., 2010). Therefore, we first tested whether the absence of Rga6 affects cytokinesis by altering the localization of septins. As shown in Fig. S1B,C, the signal intensity of Spn1–tdTomato was decreased significantly at the septin ring in rga6Δ cells, suggesting that the proper organization of the septin ring requires Rga6. Intriguingly, further measurements revealed that the duration time of cells from mitosis onset to formation of the septin ring or to cell separation was comparable in WT and rga6Δ cells (Fig. S1D). Hence, although Rga6 is required for the proper organization of the septin ring, Rga6 plays a minor role in regulating cell septation during cytokinesis.

For the convenience of identifying interphase cells, we next expressed Sid4–GFP, a protein localizing to the spindle pole (Johnson and Gould, 2011), in the rga6+ and rga6 mutant cells. First, the intensity of Spn1-tdTomato fluorescence along the plasma membrane in rga6+ and rga6Δ cells was measured by line-scan analysis (Fig. 2A,B). The measurement results showed that Spn1–tdTomato signals along the plasma membrane were generally weaker in rga6Δ cells than in WT cells (Fig. 2B), and the diminished signals were not due to the altered expression of Spn1–tdTomato in rga6Δ cells (Fig. S2A,B). This observation was confirmed by quantification of the average Spn1–tdTomato signal intensity along the plasma membrane (Fig. 2E). We then assessed the effect of Rga6 overexpression (i.e. Rga6 expressed from the nmt41 promoter in medium without thiamine) on the cortical localization of Spn1. Microscopic analysis showed that the localization of Spn1–tdTomato to the cell cortex was remarkably enhanced in Rga6-overexpressing cells (Fig. 2C,D,F), and the enhanced Spn1 signals were not due to the altered expression of Spn1–tdTomato in Rga6–GFP-overexpressing cells (Fig. S1C,D). We further expressed Rga6–GFP from the nmt41 promoter, a promoter capable of being repressed by thiamine, in media lacking thiamine to allow for variable expression of Rga6–GFP (Fig. 2G). In the cells carrying the nmt41 promoter, the average intensity of Rga6–GFP and Spn1–tdTomato signals along the cell cortex was measured, and correlation analysis showed that Rga6–GFP and Spn1–tdTomato signals are significantly linearly related (Fig. 2G; Fig. S2E). These results suggest that proper localization of septins to the cell cortex depends on Rga6.

The above relationship between Spn1 and Rga6 on the cortical localization prompted us to examine colocalization of the two proteins and to test their interaction. Microscopic observation of WT cells expressing Spn1–tdTomato and Rga6–3GFP showed that Spn1–tdTomato and Rga6–3GFP only partially colocalized on the cell cortex (Fig. 3A). Nonetheless, we noticed that Spn1–tdTomato was enriched at the sites adjacent to the cell tip and Rga6–3GFP was also present at the Spn1–tdTomato-enriched sites (Fig. 3A). Consistent with the published data (Revilla-Guarinos et al., 2016), Rga6 decorated the cell sides more strongly than the cell tips during interphase. Co-immunoprecipitation assays were then performed to test the interaction between Spn1 and Rga6, Spn2 (positive control), or Bub1 (the spindle assembly checkpoint protein, serving as a negative control). As shown in Fig. 3B, Spn1–tdTomato co-precipitated Spn2–3GFP and Rga6–3GFP, but not Bub1–GFP, suggesting that Spn1 interacts with Rga6 (Fig. 3B). We further tested the interaction between the septin complex and Rga6 by GST pulldown assays. Specifically, Spn1, Spn2–GST, Spn3 and Spn4 were coexpressed in Escherichia coli, and the septin complex composed of Spn1–Spn4 (hereafter referred to as septins) was purified with glutathione resins. We then performed pulldown experiments with GST–septins, GST and cell lysates containing Rga6–GFP. As shown in Fig. 3C, GST–septins, but not GST, was able to co-precipitate Rga6–GFP. Similarly, GST–septins was able to co-precipitate the recombinant protein His–Rga6–GFP, but not His–GFP, and GST was not able to co-precipitate both His–Rga6–GFP and His–GFP (Fig. 3D). Together, these biochemical data suggest that Rga6 physically interacts with the septin complex.

We further asked whether Spn1 plays a role in localizing Rga6 to the cell cortex. Microscopy analysis showed that the localization of Rga6–3GFP on the cell cortex was comparable in WT and spn1Δ cells (Fig. 3E), suggesting that the cortical localization of Rga6 is independent of Spn1. Hence, proper localization of septins to the cell cortex requires Rga6, but not vice versa.

Rga6p contains an N-terminal serine-rich (SR) region [amino acids (aa) 187–253], a GAP domain (aa 329–547) and a C-terminal PBR domain (aa 700–733) (Fig. 4A) (Revilla-Guarinos et al., 2016). We attempted to identify the Rga6 domains/region(s) that interact with Spn1. To this end, co-immunoprecipitation was performed to test the interaction between Spn1 and full-length or the deletion-truncation Rga6 mutants (Fig. 4A,B). As shown in Fig. 4B, Spn1–tdTomato was able to be co-precipitated by full-length Rga6–GFP and Rga6(ΔN1)–GFP, but not Rga6(ΔN2)–GFP, Rga6(ΔN3)–GFP, Rga6(ΔC)–GFP and MBP–GFP (Fig. 4B). Note that septins physically interact with Rga6 in vitro (Fig. 3D). Together, these results indicate that the N-terminal SR-containing and C-terminal PBR regions are required for the interaction of Rga6 with Spn1.

The expression of Rga6 from its own promoter appeared to be very weak (Fig. S4A). Therefore, full-length (FL) Rga6 and its truncation variants tagged with GFP were expressed from the ase1 promoter for microscopy observation (Fig. 4C). Expression of the GFP-tagged proteins was confirmed by western blotting analysis (Fig. 4D), and the expression levels of Spn1–tdTomato appeared to be slightly increased in all rga6Δ cells ectopically expressing Rga6–GFP variants but comparable between these Rga6–GFP variant-expressing cells (Fig. 4D). Interestingly, Rga6(ΔN2)–GFP and Rga6(ΔN3)–GFP signals on the cell cortex were very weak, and Rga6(ΔC)–GFP did not localize to the cell cortex. By contrast, cortical signals for Rga6(FL)–GFP and Rga6(ΔN1)–GFP were apparent. Quantification of the cortical localization of Spn1–tdTomato showed that the average fluorescent intensity of Spn1–tdTomato along the cell cortex was fully rescued by ectopic expression of full-length Rga6–GFP and, to a lesser degree, by Rga6(ΔN1)–GFP in rga6Δ cells (Fig. 4E). Rga6(ΔN2)–GFP, Rga6(ΔN3)–GFP, and Rga6(ΔC)–GFP did not appear to significantly rescue the localization of Spn1–tdTomato in rga6Δ cells (Fig. 4E). Collectively, these results suggest that the presence of Rga6 on the cell cortex plays a crucial role in regulating the cortical localization of septins.

We next sought to test the affinity of the recombinant proteins Rga6 and septins for the membrane by in vitro liposome reconstitution assays. Using phosphatidylcholines (PCs) and phosphatidylinositol (PI), we followed the previously developed method (Bridges et al., 2016) to generate silica microspheres coated with small unilamellar liposome vesicles. For visualization of the septin complex, a GST-S tag on Spn2 in the septin complex, as shown in Fig. 3C,D, was replaced with a tdTomato-S-tag. The septin complex containing the Spn2–tdTomato-S tag (hereafter referred to as Spn2–tdTomato) was then purified from E. coli and was subjected to further purification by size-exclusion chromatography (Fig. 5A). Similarly, control recombinant proteins His–tdTomato and His–GFP were purified from E. coli and were subjected to further purification by size-exclusion chromatography (Fig. 5A). Rga6–GFP–His was expressed in insect cells. Using these recombinant proteins, we first examined the localization of each recombinant protein alone by spinning-disk microscopy. As shown in Fig. 5B,C, Rga6–GFP–His (0.152 µM) and Spn2–tdTomato (0.06 µM) alone, but not His–tdTomato (0.06 µM), were able to decorate liposome-coated microspheres. At the concentration of 1.3 µM, His-GFP decorated liposome-coated microspheres only weakly (Fig. 5C). Intriguingly, the presence of Rga6–GFP–His, but not His–GFP, greatly enhanced the association of Spn2–tdTomato–His with liposome-coated microspheres (Fig. 5B–D). By contrast, the presence of Rga6–GFP–His did not significantly enhance the association of His–tdTomato with liposome-coated microspheres (Fig. 5C). Interestingly, quantification of the intensity of the Rga6–GFP–His signals on liposome-coated microspheres showed that Rga6–GFP–His signals on the microspheres were diminished slightly by the presence of Spn2–tdTomato, but not His–tdTomato (Fig. 5E). Thus, these results support a model whereby Rga6 enhances the affinity of septins for the plasma membrane.

It has been demonstrated that Rga6 is a GAP for Cdc42 (Revilla-Guarinos et al., 2016). Therefore, it is conceivable that Spn1 and Rga6 may work in concert to regulate cell polarity. To test this hypothesis, we examined the localization of Spn1 and active Cdc42 (marked by CRIB–GFP) simultaneously in rga6+, rga6Δ and Rga6-overexpressing (Rga6-OE) cells by spinning-disk live-cell microscopy. As shown in Fig. 6A, in rga6+ cells, Spn1 was excluded from the growing cell tip, where active Cdc42 resided, but was enriched at the adjacent regions to the growing cell tip. This was confirmed by line-scan analysis of the fluorescent intensity of Spn1–tdTomato and CRIB–GFP along the CRIB–GFP-positive cell ends (Fig. 6B). By contrast, in rga6Δ cells, no obvious regional enrichment of Spn1–tdTomato was detected on the cell cortex while active Cdc42 appeared to spread over a wider area at the growing cell tip (Fig. 6A,B). In contrast to rga6Δ cells, Rga6-OE cells displayed very dense Spn1–tdTomato on the cell cortex except at the growing cell tip and active Cdc42 appeared to be restricted to a much smaller area (Fig. 6A,B).

We further measured the length of CRIB–GFP at the growing cell tips in rga6+, rga6Δ, spn1Δ, rga6Δspn1Δ, Rga6-OE and spn1ΔRga6-OE cells (Fig. 6C). Consistent with the imaging data shown in Fig. 6A, the CRIB–GFP length was slightly but significantly longer in rga6Δ cells than in rga6+ cells, but significantly shorter in Rga6-OE cells than in rga6+ cells (Fig. 6D). Moreover, the CRIB–GFP length was comparable in rga6Δ, spn1Δ and rga6Δspn1Δ cells, suggesting that Rga6 affects Cdc42-GTP, likely via the cortical septin complex (Fig. 6D). Similar results were obtained when cell width of the indicated cells above was measured (Fig. 6E). Considering the canonical role of septins in diffusion restriction (Trimble and Grinstein, 2015), we interpreted these data as evidence supporting a model whereby the Rga6-dependent localization of septins to the cell cortex functions to confine active Cdc42 to the growing cell end. Consistent with this, further measurements showed that the absence of Spn1 was able to widen the CRIB–GFP area at the cell tips in Rga6-OE cells, and that the absence of Rga6 or Spn1 caused a comparable widened length of CRIB–GFP (Fig. 6C,D). Taken together, we conclude that Rga6 promotes the accumulation of septins on the cortical sites near the growing cell tip to confine active Cdc42 for regulating cell growth.

Cortical localization of septins enables the assembly of high-order septin structures required for directing a wide range of crucial cellular activities, including membrane compartmentalization, membrane remodeling and cytokinesis (Bridges and Gladfelter, 2015; Fung et al., 2014; Marquardt et al., 2019). How the cortical localization of the septin complex is regulated in space and time is, therefore, a fundamental question that needs to be addressed. In this present study, we identified the RhoGAP Rga6 as a protein that interacts with the septin complex (Fig. 3) and demonstrated that Rga6 is required for the proper localization of septins to the cell cortex (Fig. 2). Our data support a model whereby Rga6 promotes the localization of septins to the cell cortex, particularly to the region near the growing cell tip, so that the GAP activity of Rga6 and the diffusion-barrier function of septins may be integrated to regulate polarized localization of active Cdc42 (Fig. 7).

It has been postulated that the polybasic stretch at the N-terminus enables septins to bind negatively charged lipids on the membranes (Casamayor and Snyder, 2003; Fung et al., 2014; Zhang et al., 1999). Particularly, in vitro biochemical work established that septins have an affinity for phosphoinositides (Bertin et al., 2010; Casamayor and Snyder, 2003; Onishi et al., 2010; Zhang et al., 1999), and membrane association appears to promote assembly of high-order septin structures (Bertin et al., 2010). Therefore, the cortical localization of septins is likely due to their intrinsic membrane-binding ability. In this regard, septins should decorate the entire cell cortex homogenously. However, within the fission yeast cells, septin enrichment is often found near the growing cell tip (Figs 1, 2A and 6A). One possible explanation is that the cortical septin localization is also regulated by assisting proteins. In this present study, we found that the presence of Rga6, a RhoGAP protein for Cdc42, greatly enhances the affinity of septins for liposome membranes (Fig. 5). The enhancement effect is also true within the cell because the absence of Rga6 and Rga6 overexpression compromises and enhances the cortical localization of Spn1, respectively (Fig. 2). Moreover, Rga6 interacts with the septin complex and partially colocalizes with the septin complex on the cell cortex (Fig. 3). Therefore, our work establishes that, in addition to the intrinsic membrane-binding ability, septins depend on the RhoGAP Rga6 for localizing to the plasma membrane.

Rga6 is a multi-domain protein containing a polybasic region at the extreme C-terminus, and the polybasic region is essential for localizing Rga6 to the cell cortex (Revilla-Guarinos et al., 2016; also see Fig. 4C). Although Rga6 is required for the proper localization of septins to the cell cortex, the absence of Spn1 does not appear to affect the localization of Rga6 (Fig. 3E). Therefore, we favor the model whereby Rga6 localizes to the cell cortex through its C-terminal polybasic region and subsequently recruits septins to the cell cortex. It is likely that multiple regions/domains in Rga6 are responsible for interacting with septins because deletion of the N-terminal regions in Rga6 compromises the interaction between the Rga6 mutants and Spn1 and the localization of septins to the cell cortex (Fig. 4B,C). It has been established that membrane interaction can promote septin assembly (Bertin et al., 2010) and septins generally form low-order structures within the cytoplasm (Bridges et al., 2014; Fung et al., 2014). Therefore, it is conceivable that the involvement of Rga6 in localizing septins to the cell cortex can locally concentrate low-order septin complexes from the cytoplasm and subsequently promote formation of high-order septin structures on the cortex.

Septins are generally enriched on the cortical region adjacent to the growing cell tip (Figs 1A, 2A and 6A). Our findings also revealed that the expression levels of Rga6 affect both the cortical localization of septins and the tip localization of active Cdc42 (Fig. 6A,B). Specifically, the absence of Rga6 compromises the cortical enrichment of Spn1 and enables active Cdc42 to spread over a wider area at the growing cell end, whereas Rga6 overexpression enhances the cortical localization of Spn1 and restricts active Cdc42 to a smaller area at the cell tip (Fig. 6). Importantly, the restriction effect of Rga6 overexpression on Cdc42 at the cell tip depends on Spn1 (Fig. 6C,D). Considering the canonical functions of septin filaments as a diffusion barrier and Rga6 as a Cdc42 GAP, respectively, we favor a model whereby the confinement of active Cdc42 to the growing cell tip may be regulated by a collaborative effect of septins and Rga6 (i.e. septin-dependent compartmentalization and Rga6-dependent Cdc42 GAP activity). It is also possible that Rga6 promotes the localization of septins to the cortical region where both proteins function to regulate cell polarity independently. Given the fact that septin cytoskeleton interacts with catenin complex to organize a functional domain to separate apical from basal membranes in polarized epithelial cells (Wang et al., 2021), it would be of great interest to determine the spatiotemporal assembly dynamics of septin and catenin complex in polarized epithelial cells.

The tight relationship between septins and Cdc42 has been established previously in Saccharomyces cerevisiae (Caviston et al., 2003; Gladfelter et al., 2002; Iwase et al., 2006). Instead of Cdc42 GAP proteins, the Cdc42 effectors Gic1 and Gic2 play an important role in recruiting septins to the budding site (Iwase et al., 2006). Interestingly, at the budding site, septins also interact with Cdc42 GAP proteins and recruit the GAP proteins to inactivate Cdc42 (Okada et al., 2013). Therefore, the interaction between Cdc42 GAP proteins and septins may have been conserved through evolution but evolves to function differently in an organism-dependent manner. In human cells, similar to Rga6 in this case, many of the RhoGAPs, particularly the Cdc42 GAP proteins, contain membrane-binding domains (Amin et al., 2016). Given the conservative nature of septins, it would be interesting to explore whether the RhoGAPs containing membrane-binding domains also similarly regulate the cortical localization and functions of septins in higher eukaryotic cells using spectral imaging analyses (Liu et al., 2020).

Yeast strains were created either by the random spore digestion method or by tetra-dissection analysis. Gene deletion and tagging were performed by the PCR-based method using the pFA6a series of plasmids, and yeast transformation was carried out by the lithium acetate method (Forsburg and Rhind, 2006). The strains used in this study are listed in Table S1. For live-cell imaging, the strains were cultured in Edinburgh minimal medium supplemented with adenine, leucine, uracil, histidine, and lysine (0.225 g/l each) (FORMEDIUM). For biochemistry, cells were cultured in yeast extract (YE) medium containing the five supplements indicated above.

Plasmids used in this study are listed in Table S2. The restriction enzymes used for molecular cloning were purchased from NEB, and cloning was carried out by the conventional digestion/ligation method or by ClonExpress II One Step Cloning (Vazyme).

For generating the septin constructs, the four septin genes were first amplified by PCR with a yeast cDNA library. For generating the pETDuet plasmid carrying Spn1 and Spn2–GST-S-tag (pCF.3623), spn1 was digested with BamHI and NotI, and spn2 was digested with BglII and XhoI. Both digested products were then cleaned with a PCR purification column (TIANGEN), and ligation was then performed using the T4 DNA ligase (NEB). The GST tag was amplified by PCR and was inserted into the pETDuet plasmid, downstream of spn2, by the recombination method with a ClonExpress II One Step Cloning kit (Vazyme).

For generating pACYCDuet carrying Spn3 and Spn4 (pCF.3301), spn3 was digested with PstI and NotI and spn4 was digested with BglII and XhoI. Ligation was then performed using the T4 DNA ligase (NEB).

For generating pFastBac-Rga6-GFP-His (pCF.3805), rga6 was amplified from pCF.3080 and digested with BssHII and NotI for further ligation.

For generating Rga6 full-length and deletion-truncation mutant plasmids, the inserts obtained by PCR were digested with BglII and NotI and ligated into a pJK148 vector with a T4 DNA ligase (NEB).

Live-cell imaging was performed with a PerkinElmer Ultraview spinning disk confocal microscope equipped with a Nikon Apochromat TIRF 100×1.49 NA objective and a Hamamatasu C9100-23B EMCCD camera, following the method as described previously (Tran et al., 2004). Briefly, cells in the exponential phase were collected and sandwiched between an EMM (with the five supplements indicated above) agar pad and a coverslip. Images were acquired with Volocity (PerkinElmer) at room temperature. Unless otherwise specified, image stacks containing 11 planes were taken with a 0.5 μm step size and the time-lapse images were taken every 2 min (500 ms exposure for GFP and tdTomato).

For the colocalization of Rga6–3GFP and Spn1–tdTomato, imaging was performed with a ZEISS LSM980 (Airyscan) confocal microscope equipped with a ZEISS flat field achromatic 100×1.4 NA objective and a laser scanner (ZEISS).

Images were analyzed and measurements were performed using MetaMorph 7.7 (Molecular Devices) and ImageJ 1.52 (NIH). Graphs were generated and statistical analysis was performed using KaleidaGraph 4.5 (Synergy).

For measurement of the length of CRIB–GFP, line-scan intensity analysis was performed to obtain a plot of signal intensity against time for each cell. The plot was generally a bell-shape curve and the intensity values on the two tails of the bell-shape curve were small and fluctuated only slightly, we then followed the decreasing values on both sides of the bell-shaped curve and determined the first values that reached the baseline of the two tails. The baseline values on the left and right sides of the bell-shaped curve were used for calculation of the length of the CRIB–GFP. Cell width was measured directly at the middle of a cell.

For producing the septin complex with a GST tag and GST proteins, BL21(DE3) Escherichia coli cells were transformed with the duet septin expression plasmids, selected with ampicillin and chloramphenicol, or the plasmid pGEX-6p-1, and induced to express with 1 mM IPTG at an optical density at 600 nm (OD₆₀₀) of 0.8–1.0. After 24 h of growth at 22°C, cells were harvested by centrifugation at 5000 g for 15 min. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, and 300 mM NaCl) and crushed for 2 min with a high-pressure crusher (700-800 pressure). The cell lysate was then centrifuged at 4°C for 40 min at 16,200 g. The supernatant was then collected and incubated with 500 μl glutathione–Sepharose resins (GE Healthcare) for 90 min at 4°C. The resins were washed with GST wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 0.1% Triton X-100) (10X column volume) and were kept in storage buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1% Triton X-100 and 20% glycerol) at −20°C.

For producing Rga6–GFP–His, Sf9 insect cells (Thermo Fisher Scientific) were transfected with the bacmid pFast-Rga6-GFP-His. After three rounds of virus infection, cells were harvested by centrifugation at 5000 g for 10 min. Cell pellets were lysed in lysis buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, and 30 mM imidazole) and crushed for 2 min with a high-pressure crusher (200–300 MPa), and the cell extract was centrifuged for 20 min at 16,200 g at 4°C. The supernatant was then incubated with 2 ml Ni-NTA Superflow resins (QIAGEN) for 30 min. After the resins were washed with lysis buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, and 30 mM imidazole; 10× column volume), bound proteins were eluted with elution buffer (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, and 300 mM imidazole).

For producing recombinant proteins, Spn2–tdTomato–His, His–tdTomato or His–GFP proteins, BL21(DE3) E. coli cells carrying the corresponding plasmids were induced to express with 1 mM IPTG at an OD₆₀₀ of 0.8–1.0. A similar procedure to that above for purifying GST recombinant proteins was then used for protein purification. The recombinant proteins were concentrated to 2 ml and were further purified by size-exclusion chromatography (SEC) with a HiLoadTM 16/600 superdexTM 200 pg column (GE Healthcare) in SEC buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl₂ and 1 mM DTT).

Co-immunoprecipitation (co-IP) assays were performed with Dynabeads protein G beads (Thermo Fisher Scientific) and strains expressing the indicated GFP and tdTomato proteins in Tris-buffered saline (TBS) containing 0.1% Triton X-100. Cell lysates were prepared by grinding in liquid nitrogen with a mortar grinder RM 200 (Retsch), and dissolved in TBS lysis buffer containing 0.1% Triton X-100 and cocktail protease inhibitors. Dynabead–Protein G beads bound with the GFP antibody (made in house; GenScript; 5 μg) were incubated with the cell lysates for 1 h at 4°C, followed by washing with 1× TBS buffer containing 0.1% Triton X-100 five times and with 1× TBS once. Immunoprecipitated proteins were then analyzed by western blotting with antibodies against GFP (600-101-215; Rockland; 1:2000 dilution) and tdTomato (made in house; GenScript; 1:2000 dilution).

For analysis of protein expression levels, protein extracts were prepared by the trichloroacetic acid (TCA) lysis method. Briefly, exponentially growing cells were collected and washed once with 1 ml distilled deionized water (ddwater). The collected cells were resuspended with 50 μl 20% TCA and vortexed. About 200 μl glass beads was added, and cells were disrupted using Retsch MM400 (Retsch). Vortex was performed after 50 μl 20% TCA was added, and vortex was performed again after 400 μl 5% TCA was added. After removing the glass beads, samples were centrifuged at 16,200 g for 10 min at 4°C to remove the supernatant. Finally, the precipitate was resuspended with 100 μl 1× SDS sample buffer and 25 μl 1.5 M Tris-HCl pH 8.0, followed by boiling at 100°C for 5 min. The proteins were analyzed by western blotting with antibodies against GFP (600-101-215; Rockland; 1:2000 dilution), tdTomato (made in-house; GenScript; 2000-fold dilution) and tubulin (63-160; Bio Academia; 1:10,000 dilution).

For GST pulldown assays with only recombinant proteins, recombinant proteins Rga6–GFP–His and GFP–His were incubated with 20 μl glutathione resin bound with GST-fused proteins or with empty glutathione resins in 200 μl TBS buffer (50 mM Tris-HCl pH 7.5 and 150 mM NaCl) supplemented with 0.1% Triton X-100 and 40 mM imidazole. After a 90 min incubation at 4°C, the resins were washed five times with TBS plus 0.1% Triton X-100 and once with TBS. The resins were boiled in SDS-PAGE sample buffer for 5 min, and protein samples were analyzed by SDS-PAGE and immunoblotting with antibodies against GFP (600-101-215; Rockland; 1:2000 dilution) and GST (WH080027/AE006; Abclonal; 1:2000 dilution).

For testing the interaction of recombinant GST-fused proteins with Rga6–GFP, cell lysates containing Rga6–GFP was prepared and used for incubation with the indicated recombinant GST-fused proteins. After incubation of 90 min at 4°C, the GST resins were washed five times with TBS plus 0.1% Triton X-100 and once with TBS. The resins were boiled in SDS-PAGE sample buffer for 5 min, and protein samples were analyzed by SDS-PAGE and immunoblot with antibodies against GFP (600-101-215; Rockland; 1:2000 dilution) and GST (WH080027/AE006; Abclonal; 1:2000 dilution).

For testing each component of the septin complex, western blotting assays were performed with antibodies against His (WH080027/AE003; Abclonal; 2000-fold dilution), GST (WH080027/AE006; Abclonal; 1:2000 dilution), and S tag (A00625; Genescripts; 1:2000 dilution).

We followed the method reported previously (Bridges et al., 2016) to perform liposome reconstitution. For preparing small unilamellar vesicles (SUVs), we used a lipid composition of 75% phosphatidylcholines (PCs) and 25% phosphatidylinositol (PI) (both Avanti Lipids). Briefly, lipids were mixed in chloroform solvent (75 µl PC plus 25 µl PI, both at 10 mg/ml), dried under a steam of nitrogen for 5 min, and dissolved with 1 ml liposome buffer (20 mM Tris-HCl pH 8.0, 300 mM KCl, and 1 mM MgCl₂) to hydrate 30 min at 37°C, followed by bath sonication for 5 min to allow formation of SUVs. SUVs were then adsorbed on silica microsphere beads (3.17 μm in a mean diameter) (Bangs Laboratories) by mixing 75 µl SUVs with 0.002 g silica microspheres and rotating the mixture gently for 1 h at room temperature. After incubation, excess SUVs were removed by centrifugation of the liposome-coated beads for 1 min at 900 g, followed by three washes with wash buffer (50 mM Tris-HCl pH 8.0, and 100 mM KCl). To test the affinity of proteins for the liposome-coated microspheres, 25 µl protein samples were added to 75 µl microsphere solution (100 mM KCl, 50 mM Tris-HCl pH 8.0, 0.1% BSA and 1 mM DTT), and the mixture was incubated for 60–90 min at room temperature. After the incubation, the protein-containing mixture was then mixed by pipetting and 10 µl of the mixture was added to a glass profusion chamber for imaging with a spinning-disk confocal microscope.

We thank Dr Erfei Bi (UPENN) for suggestions and members in the Fu laboratory for insightful discussion. We would like to thank NBRP (Japan) for providing strains.

The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.259228.