Introduction

Septins are a family of GTP-binding proteins that are highly conserved from yeast to mammalian cells, but absent in land plants (Longtine et al., 1996; Gladfelter et al., 2001; Cao et al., 2007; Nishihama et al., 2011; Onishi and Pringle, 2016; Marquardt et al., 2019; Woods and Gladfelter, 2021). They form hetero-oligomeric complexes that can assemble into different higher-order structures such as rings, gauzes, hourglasses, and carry out various functions (Frazier et al., 1998; Hsu et al., 1998; Kinoshita, 2003; Sheffield et al., 2003; Bertin et al., 2008; Garcia et al., 2011; Bridges et al., 2014). Septins can serve as scaffolds for protein recruitment at discrete cellular locations (Gladfelter et al., 2001; Versele and Thorner, 2005; Mostowy and Cossart, 2012; Finnigan et al., 2015; Meitinger and Palani, 2016; Perez et al., 2016; Marquardt et al., 2019). Septins are proposed to act as a diffusion barrier to ensure that cellular components are spatially segregated or compartmentalized (Dobbelaere and Barral, 2004; Caudron and Barral, 2009; Hu and Nelson, 2011; McMurray et al., 2011). They can also sense the membrane curvatures and/or deform the plasma membrane due to their lipid-binding properties (Bridges and Gladfelter, 2016; Cannon et al., 2017; Cannon et al., 2019; McMurray, 2019; Shi et al., 2023). The diverse roles of septins lead to their involvement in multiple processes including cytokinesis, mitosis, exocytosis, apoptosis, fungal or viral infections, neuronal spine morphogenesis, ciliogenesis, and spermiogenesis (Longtine et al., 1996; Kartmann and Roth, 2001; Mostowy and Cossart, 2012; Dolat et al., 2014; Momany and Talbot, 2017; Ageta-Ishihara and Kinoshita, 2021; Neubauer and Zieger, 2021; Woods and Gladfelter, 2021; Safavian et al., 2023).

One of the best-studied septin functions is their roles in cytokinesis in the budding yeast Saccharomyces cerevisiae (Hartwell, 1971; Haarer and Pringle, 1987; Ford and Pringle, 1991; Kim et al., 1991; DeMarini et al., 1997; Bi et al., 1998; Lippincott and Li, 1998; Longtine et al., 1998). The septin ring or hourglass structures at the presumptive bud site and bud neck are required for the recruitment and maintenance of various cytokinesis proteins (Bi et al., 1998; Lippincott and Li, 1998; Gladfelter et al., 2001; Longtine and Bi, 2003; Marquardt et al., 2019).

These roles occur through either direct interactions with proteins such as the F-BAR protein Hof1 (Vallen et al., 2000; Meitinger et al., 2013; Oh et al., 2013), or through septin-binding proteins such as Bni5, which links septins to the myosin-II heavy chain Myo1 (Lee et al., 2002; Fang et al., 2010; Finnigan et al., 2015). During cytokinesis, the septin double rings were proposed to function as a diffusion barrier for proteins such as the exocyst component Sec3 and chitin synthase II Chs2 at the division site (Dobbelaere and Barral, 2004). But other studies have challenged this view by showing that Chs2 localizes efficiently to the division site in the absence of septin rings (Wloka et al., 2011). Regardless of the debate, septins are known to play essential roles in budding yeast cytokinesis. However, no physical interactions between septins and the exocyst have been reported, even in the genome wide interactome studies (Michaelis et al., 2023). Moreover, budding yeast septins also serve as scaffolds for the localization of hundreds of proteins at the bud neck including signaling proteins, bud site selection proteins, and chitin synthases (Longtine et al., 1996; DeMarini et al., 1997; Gladfelter et al., 2001; Longtine and Bi, 2003; Marquardt et al., 2019).

Unlike in budding yeast, septins are not essential in the fission yeast Schizosaccharomyces pombe and their roles in cytokinesis remain obscure (Longtine et al., 1996; Berlin et al., 2003; Tasto et al., 2003; Wu et al., 2010; Zheng et al., 2024). Fission yeast has seven septins, Spn1 to Spn7, with Spn1 to Spn4 expressing in vegetative cells and functioning at the division site (Longtine et al., 1996; Berlin et al., 2003; Tasto et al., 2003; An et al., 2004; Petit et al., 2005; Onishi et al., 2010; Wu et al., 2010). None of the Spn1-Spn4 is essential, but loss of some or all of them causes a delay in cell separation, resulting in multi-septated phenotype (An et al., 2004). Spn1 and Spn4 are the more important components of the septin ring structure during cytokinesis (An et al., 2004). Septins accumulate to the division site shortly before the contractile ring constriction and form a single ring which quickly transitions into unconstricting double rings (Berlin et al., 2003; Tasto et al., 2003; Wu et al., 2003; Wu et al., 2010). It is only known that septin rings recruit the guanine nucleotide exchange factor Gef3 (Muñoz et al., 2014; Wang et al., 2015), the small GTPase Rho4 (Wang et al., 2015), and the glucanases Eng1 and Agn1 to the division site (Martin-Cuadrado et al., 2005). Thus, we know much less about fission yeast septins compared to those in budding yeast. It remains mysterious what the most conserved functions of septins are during evolution.

Previous studies have suggested that septins function in exocytosis (Hsu et al., 1998; Vega and Hsu, 2003; Martin-Cuadrado et al., 2005; Perez et al., 2015; Tokhtaeva et al., 2015). Fission yeast septins are proposed to work with the exocyst complex to regulate the secretion of glucanases at the appropriate location, but it was reported that septins and the exocyst are independent for localization (Martin-Cuadrado et al., 2005; Perez et al., 2015). The exocyst is a highly conserved, octameric complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) in exocytosis (TerBush et al., 1996; Wang et al., 2002; Hsu et al., 2004; Heider and Munson, 2012; Liu and Guo, 2012). It functions in late stages of exocytosis by promoting the tethering and fusion of post-Golgi secretory vesicles to the plasma membrane (TerBush et al., 1996; Hsu et al., 2004; Liu et al., 2018). Although studies suggest that septins regulate the exocyst complex and a possible involvement of septins in targeting secretory vesicles to the exocytic sites (Hsu et al., 1998; Vega and Hsu, 2003; Li et al., 2007; Gupta et al., 2015), no direct interactions between septins and the exocyst subunits have been reported in budding yeast and other organisms.

Here we report septins regulate the exocyst localization and vesicle targeting in fission yeast via physical interactions. We find that the loss of septin rings alters the exocyst localization, with increased concentration to the center and reduced localization to the rim of the division plane. The initial recruitment of the exocyst is independent of septins, but the exocyst requires septin rings to maintain the rim localization during furrow ingression. Consistently, we found multivalent direct physical interactions between septins and the exocyst subunits. Loss of the exocyst ring leads to abnormal accumulation of secretory vesicles in septin mutants. As a result, the glucan synthase Bgs1 accumulates more to the center and the glucanase Eng1 is missing from the rim of the division plane, contributing to a thicker septum and a delayed cell separation in septin mutant cells. Our findings provide insights into the regulation of the exocyst localization and function on the plasma membrane by septins in other systems.

Results

Septin and the exocyst complex colocalize and are partially interdependent for localization at the division site

Both septins and the exocyst complex localize to the division site during cytokinesis (Longtine et al., 1996; Wang et al., 2002; An et al., 2004; Petit et al., 2005). To understand if and how they work together, we first examined the colocalization of the septin Spn1 and the exocyst subunit Sec3 in fission yeast. Spn1 is a key component in septin structures, and its loss leads to complete disruption of the septin rings from the division site (An et al., 2004). Sec3 is a spatial landmark for exocytosis in budding yeast (Finger et al., 1998; Boyd et al., 2004; Luo et al., 2014). The fission yeast Sec3 is an essential gene and crucial for exocyst localization (Kim et al., 2010; Bendezu et al., 2012; Jourdain et al., 2012). Spn1 and Sec3 colocalized at the division site as a single ring first, and later as double rings during septum formation (Figure 1, A and B). Sec3, but not Spn1, also concentrated at cell tips (Figure 1C). However, Sec3 arrived at the division site 13.4 ± 2.2 min after spindle pole body (SPB) separation, about 10 min earlier than Spn1 that arrived at 23.6 ± 1.8 min (Figure 1, C and D). Time lapse movies of Exo70-tdTomato and Spn1-mEGFP confirmed that the exocyst appeared at the division site earlier than septins (Video 1). This suggests that the colocalization required for their proper function occurs at a specific stage during cytokinesis rather than a general regulation throughout the cell cycle.

Figure 1.

Since septin and the exocyst colocalize at the division site and Sec3 arrives earlier, we tested whether septin localization depends on Sec3 and other exocyst subunits. In WT cells, Spn1 always formed ring structures at the rim of the division plane during septation (Figure 1E). In exocyst mutants exo70Δ and the temperature sensitive sec3-913 and sec8-1, Spn1 localization was comparable to WT at permissive temperature (Figure supplement S1A). At restrictive temperature, although Spn1 localized as ring at the division site before septation, a fraction of Spn1 abnormally spread onto the division plane following furrow ingression in sec3-913 and sec8-1 mutants (Figures 1E, red boxes; and Figure supplement S1B, middle focal plane). As exo70Δ cells have no severe defects (Wang et al., 2003), the exocyst complex may not be as compromised as in sec3-913 and sec8-1 mutants. Only minor mislocalization of Spn1 was observed in exo70Δ cells even at 36°C (Figure supplement S1B). This localization pattern in exocyst mutants suggested a possible correlation between septins and the furrow ingression. Indeed, some Spn1 followed the contractile ring marked with Rng8 (Wang et al., 2014) as it constricted, spread onto the new plasma membrane, and concentrated at the center of the division plane while maintaining its localization at the rim (Figure 1F). We also examined Spn1 levels at the division site in cells with no visible septum, forming septum, and closed septum. Spn1 levels were comparable or higher in exocyst mutants compared to WT at both 25°C and 36°C (Figures 1G and Figure supplement S1, C and D). FRAP analyses of Spn1 showed no difference in its dynamics in WT and sec3-913 cells at 36°C (Figure 1H and Figure supplement S1E). Collectively, despite some Spn1 mislocalizes to the center of the division plane in exocyst mutants, majority of Spn1 still localizes to the rim (Figures 1E and Figure supplement S1B). Thus, septins only partially depend on the exocyst for their localization.

Next, we examined the localization and levels of the exocyst complex (subunits Sec3, Exo70, and Sec8) in spn1Δ cells. In mitotic cells without a septum, the exocyst localized to the rim of the division plane in both WT and spn1Δ cells (Figures 2A and Figure supplement S1, F and G, yellow boxes). During septation, however, the exocyst spread across the division plane as a disk in spn1Δ cells while it remained at the rim in WT cells (Figures 2A and Figure supplement S1, F and G, red boxes). The levels of Sec3, Exo70, and Sec8 at the division site in spn1Δ cells were not significantly different from WT before septation (Figures 2B and Figure supplement S1H). During and after septum formation, the levels of all three exocyst components were significantly reduced at the division site (except Exo70 in cells with forming septum) and almost absent at the rim in spn1Δ cells (Figures 2A and Figure supplement S1H). These results were confirmed in cells expressed Sec8-GFP and myosin light chain Rlc1 as a contractile ring marker (Figure 2, D and E). However, the dynamics of Sec3 at the division site was not affected in spn1Δ cells (Figures 2C and Figure supplement S1I). The exocyst was much more dynamic than septins at the division site (Figures 1H and 2C), which was confirmed by high temporal resolution imaging of Exo70 (Video 2 and 3). Together, loss of septins results in exocyst mislocalization and its decreased levels, especially at the rim of the division plane during septum formation, suggesting that septins play an important role in regulating the exocyst localization at the division site.

Figure 2.

Collectively, our data suggest that septins and the exocyst complex are interdependent for localization at the division site during and after the contractile ring constriction, with the septin rings being more important for the exocyst localization. Thus, we conclude that the initial recruitment of the exocyst to the division site does not depend on septins, but its rim localization and maintenance during late cytokinesis require the septin rings.

Septins regulate the exocyst localization through direct physical interactions

Septins have been shown to play a role in the Rho GEF Gef3-Rho4 GTPase pathway to regulate the exocytosis of glucanases Eng1 and Agn1 for proper cell separation (Perez et al., 2015; Wang et al., 2015). Septins are essential for Gef3 localization to the division site (Wang et al., 2015). In spn1Δ cells, Gef3 localization on the plasma membrane is abolished, and Rho4 localization in cells with a closed septum was significantly reduced (Wang et al., 2015). Since Rho4 can interact with both the exocyst complex and septins (Perez et al., 2015), we tested whether the altered exocyst localization pattern that we observed in septin mutants was through Gef3 and Rho4. Although partially mislocalized to the center of the division plane, majority of Sec3 still localized as a ring at the rim of division plane in rho4Δ, gef3Δ, and rho4Δ gef3Δ cells (Figure supplement S2A). Moreover, Spn1 ring localization was not affected in rho4Δ gef3Δ cells (Figure supplement S2B). Thus, the different localization patterns of the exocyst in spn1Δ and rho4Δ gef3Δ cells suggest that septins can regulate exocyst localization independent of Gef3 and Rho4.

To test the hypothesis that septins regulate the localization of the exocyst directly, we examined the physical interactions between septins and the exocyst subunits. Sec3 and Exo70 are the most important subunits for the targeting of the octameric exocyst to the plasma membrane (Boyd et al., 2004; He et al., 2007; Bendezu et al., 2012; Luo et al., 2014; Yue et al., 2017; Liu et al., 2018; Synek et al., 2021), and Spn1 and Spn4 are essential for septin localization and functions (An et al., 2004). Therefore, we first tested the interactions between Spn1-Sec3, Spn1-Exo70, Spn4-Sec3, and Spn4-Exo70 using co-immunoprecipitation of cell extracts from fission yeast. Surprisingly, no physical interactions were detected among the four proteins.

Then we utilized AlphaFold2_advanced ColabFold algorithm (Jumper et al., 2021; Mirdita et al., 2022), whose highly accurate predictions of protein structures have revolutionized structural biology, to predict the physical interactions between all 32 combinations of the four septins and eight exocyst subunits. For the modeling, the complete sequences of each subunit of septins and exocyst complex were used except Sec8. Sec8 subunit was analyzed in two fragments with overlapping sequence due to the 1,400 amino acids input sequence limitation of AlphaFold2_advanced. Representative models generated are shown (Figures 3 and Figure supplement S3). Predicted interacting interface residues defined as amino acids of two possible binding partners with distance ≤4 Å were calculated from the rank 1 predicted model (Yin and Pierce, 2023). Moreover, the contacts between interface residues having pLDDT score >50 were calculated. Based on the above analyses, we predicted the following top six interactions between the septin and exocyst subunits: Spn2 and Sec15 (Figure 3), Sec15 and Spn1, Sec6 and Spn1, Spn2 and Sec5, Spn4 and Sec15, and Spn4 and Sec3 (Figure supplement S3).

Figure 3.

Next, we used reciprocal Co-IP assays of fission yeast extracts to confirm the predicted interactions between septin and exocyst subunits by AlphaFold2_advanced ColabFold. Out of the six predicted interactions, we found five of them were positive in Co-IP. We found that Spn2 physically interacted with Sec5 and Sec15, Spn1 with Sec15 and Sec6, and Spn4 with Sec15 (Figures 4 and Figure supplement S4). Sec15 interacts with three septins Spn1, Spn2, and Spn4, which was stronger than other combinations. We also utilized yeast two-hybrid assays to test whether these five-pair interactions are direct (Figure 4, E and F). X-gal overlay assay (insets) and quantification of β-galactosidase using ONPG confirmed that Sec15 directly interacted with Spn1, Spn2, and Spn4 (Figure 4E); and Sec6 interacted with Spn1 and its C-terminal fragment (Spn1[300-469]) that contains the coiled-coil motif (Figure 4F). The Spn2-Sec5 interaction could not be tested due to very high level of autoactivation of Sec5. Thus, we conclude that septins physically and directly interact with the exocyst complex in fission yeast via multivalent interactions.

Figure 4.

Septins are involved in concentrating Sec15 and Sec5 at the rim of the division site especially at the late-stage cytokinesis

We reasoned that septins localize the exocyst at the division site via their multivalent interactions with the exocyst subunits Sec15, Sec5, and Sec6. Weakened interactions between septins and the exocyst in the absence of a certain septin subunit could lead to mislocalization of the exocyst complex. Indeed, similar to the results presented in Figures 1, 2, and Figure supplement S2 with other exocyst subunits, the deletion of spn1 or spn4 led to mislocalization of Sec15 on the division plane in ∼75% of cells with a septum while Sec15 in ∼90% of WT cells localized as rings at the rim of the division plane in septating cells (Figure 5, A and B). Results from time-lapse microscopy of spn1Δ or spn4Δ cells were consistent with these findings. Sec15 was first recruited to division site as rings and then spread to whole division plane before signal disappearance, leading to some multiseptated cells (Videos 4-6).

Figure 5.

Although septin filaments have four subunits in vegetative cells (An et al., 2004), Spn2 is less important than Spn1 and Spn4 for septin functions and spn2Δ has a much weaker phenotype in septation than spn1Δ or spn4Δ (An et al., 2004; Wu et al., 2010; Zheng et al., 2018). Consistently, in spn2Δ cells, Sec15 and Sec5 localized normally at the division site before septation (Figure 5, A and C). Both Sec15 and Sec5 spread to the whole division plane in ∼50% of spn2Δ cells with obvious septa observed in the DIC channel (Figure 5, A-D). Unlike in spn1Δ or spn4Δ cells, a fraction of Sec15 and Sec5 can still localize to the rim in spn2Δ cells. Collectively, these data support that Spn1, Spn2, and Spn4 are important for restricting the exocyst to the rim of the division plane during cytokinesis through direct physical interactions.

Septin mutants affect the sites of secretory vesicle tethering and cargo delivery at the division plane

Septin and exocyst mutations showed no or very mild synthetic genetic interactions (Tables 1 and 2), suggesting that septins and the exocyst complex function in the same pathway to regulate cytokinesis and septation. Surprisingly, they have different genetic interactions with the transport particle protein-II (TRAPP-II) mutants (Tables 1 and 2). The exocyst mutant sec8-1 is synthetic lethal with trs120-M1 and has severe synthetic cytokinesis defects with trs120-ts1 due to the overlapping function of the exocyst and TRAPP-II in exocytosis during fission yeast cytokinesis (Wang et al., 2016). However, spn1Δ trs120-M1 and spn1Δ trs120-ts1 double mutants were viable with no obvious synthetic interactions (Tables 1 and 2). Thus, septins and the exocyst also function in different genetic pathways in fission yeast.

Table 1.

Table 2:

The exocyst complex is the major tether of secretory vesicles at the plasma membrane (TerBush and Novick, 1995; TerBush et al., 1996; Wang et al., 2002; Luo et al., 2014). So, we tested whether exocyst mislocalization in septin mutants compromises the targeting of secretory vesicles and their cargos. We first performed electron microscopy to examine if secretory vesicles are accumulated at the division site in spn1Δ cells (Figure 6A). During septum formation, 7– and 2-fold more secretory vesicles accumulated at the division site in sec8-1 and spn1Δ cells, respectively, compared to WT (Figure 6, A and B). However, in cells with a closed septum, the number of secretory vesicles adjacent to the division site were not significantly different between WT and spn1Δ cells (Figure 6B). Consistently, secretory vesicle markers Rab11 GTPase Ypt3 and v-SNARE Syb1 accumulated more in the center of the division plane but diminished from the rim in spn1Δ cells (Figure 6, C and D). The accumulation of the secretory vesicles at the division plane and their mistargeting are consistent with exocyst mislocalization in spn1Δ cells.

Figure 6.

We next examined the distribution of two secretory vesicle cargos, β-glucan synthase Bgs1 and β-glucanase Eng1, which were delivered to the division site by the secretory vesicles during cytokinesis (Liu et al., 1999; Baladrón et al., 2002; Cortes et al., 2002; Martin-Cuadrado et al., 2003). More Bgs1 localized in the center of the division plane in spn1Δ cells compared to WT (Figure 7A). spn1Δ and sec8-1 cells also had a much thicker septum compared to WT cells (Figure 7B). Another cargo of secretory vesicles, Eng1, spread across the division plane as a disk with localization clearly missing at the rim in spn1Δ cells (Figure 7C). Lack of glucanase Eng1 at the rim could contribute to the delayed cell separation in spn1Δ cells since the junctions between septum and the cell wall cannot be efficiently digested, consistent with earlier studies (Baladrón et al., 2002; Martin-Cuadrado et al., 2003). Our studies on Bgs1 and Eng1 indicate an increase of vesicle tethering in the center and a loss at the rim of the division plane without septins.

Figure 7.

Collectively, our data indicate that septins play important roles in maintaining the proper localization of the exocyst complex on the plasma membrane. This regulation on the exocyst occurs specifically during late stages of cytokinesis in fission yeast. Loss of septins results in spreading of the exocyst across the division plane with significant reduction at the rim, which leads to the accumulation of secretory vesicles and mistargeting of downstream cargos.

Discussion

In this study, we reveal how septins and the exocyst complex physically interact to regulate exocytosis and ensure proper targeting of vesicle cargos to the plasma membrane.

Septins are important for proper membrane targeting of the exocyst complex to ensure successful cytokinesis

Septins are essential for cytokinesis and other cellular processes in budding yeast and many other organisms (Neufeld and Rubin, 1994; Longtine et al., 1996; Kinoshita et al., 1997; Gladfelter et al., 2001; Russell and Hall, 2005; Oh and Bi, 2011). However, the nature of their functions is only partially understood. It has been a mystery why the phenotypes of septin mutants are so mild in fission yeast ever since their discoveries in the early 1990s, yet their sequences and structures are evolutionarily conserved across species (An et al., 2004; Zheng et al., 2018; Zheng et al., 2024). In human, dysregulation of septins or exocyst complex leads to severe disorders including neurological diseases and cancers (Russell and Hall, 2005; Martin-Urdiroz et al., 2016; Halim et al., 2023; Werner and Yadav, 2023). Thus, it is critical to identify the functional links between septins and exocyst complex for better understanding and treatment of related diseases. In this study, we investigated the spatial regulation of the exocyst complex and roles of septins during cytokinesis in the fission yeast model system. Without septin rings, the exocyst complex, which specifies for the sites for vesicle fusion on the plasma membrane, cannot maintain its localization at the rim of the division plane. Instead, the exocyst complex follows actomyosin contractile ring constriction and spreads across the whole division plane. This localization dependence on septin rings occurs in a specific spatiotemporal manner at the division site during furrow ingression stage of cytokinesis. Although loss of septins does not affect the dynamics of the exocyst, the targeting sites of secretory vesicles and their cargos are altered, which contributes to a thicker septum and a delayed cell separation. Thus, fission yeast septins function in exocytosis through maintaining proper docking sites of the exocyst complex and secretory vesicles at the division site.

Fission yeast septins regulate the exocyst in specific temporal and spatial manners. They only regulate the localization of the exocyst during late stages of cytokinesis and are not responsible for its targeting to the cell tips during interphase or initial recruitment to the division site during early cytokinesis. Disruption of the contractile ring affects the localization of the exocyst to the division site (Wang et al., 2002; Dobbelaere and Barral, 2004). This suggests that the exocyst likely depends on the contractile ring for initial recruitment to the division site. However, this is not a universal mechanism. Although budding yeast Sec8 localization depends on the actomyosin ring, Sec3 localization is independent of actin cytoskeleton (Finger et al., 1998). The subcellular localization of the exocyst complex in rat brain cells is affected by microtubule-disrupting drugs, but not actin-disrupting drugs (Vega and Hsu, 2003). Thus, how the exocyst is initially recruited to the division site remains to be studied. Since fission yeast exocyst clearly depends on septins for proper localization during late stages of cytokinesis, its localization dependence must transition to septins from the contractile ring at some point before the onset of the contractile ring constriction. So, it will be of great interest to examine how this transition occurs. Although septins may act as either scaffolds or diffusion barriers for Sec3 in budding yeast, Sec3 localizes between the split septin rings during cytokinesis (Dobbelaere and Barral, 2004). However, in mammalian neurons, the exocyst subunits Sec6 and Sec8 colocalize with the septin SEPT7/CDC10 (Hsu et al., 1998). Thus, the colocalized septins and the exocyst in fission yeast may provide more insights in mammalian cells for understanding the molecular mechanisms of their interactions.

Examples of localization dependence between septins and the exocyst have been reported in other systems. The most prominent cases come from fungal pathogens (Eisermann et al., 2023). Magnaporthe oryzae infects plants through a specialized infection cell called appressorium, which breaches through the cuticle of the leaf to allow entry into plant tissues (Dagdas et al., 2012; Gupta et al., 2015; Zhang et al., 2021). The exocyst assembles in appressorium at the point of plant infection in a septin-dependent manner. Septin deletion causes mislocalization of the key component for the exocyst assembly, Sec6, at the appressorium pore (Gupta et al., 2015). Similarly, the root-infecting phytopathogenic fungus Verticillium dahliae also assembles the exocyst at the penetration peg of the hyphopodium in a septin-dependent manner (Zhou et al., 2017). The absence of septin VdSep5 impairs the delivery of secretory proteins to the penetration interface (Zhou et al., 2017). Another example is Candida albicans septins, which localize at the hyphal tips where tip growth occurs with active exocytosis in this human opportunistic pathogen (Li et al., 2007). Deletion of septin CDC10 or CDC11 causes mislocalization of the exocyst marked by Sec3 (Li et al., 2007). Thus, one of the conserved roles of septins is to regulate the proper membrane targeting of the exocyst complex to the plasma membrane and to ensure spatiotemporal fidelity of vesicle tethering and fusion. However, how septins and the exocyst physically interact had not been systematically investigated. Our current study will provide insights on how fungal pathogens infect their hosts.

The exocyst complex docks on septins on the plasma membrane through multivalent physical interactions

Despite the relationships between septins and exocyst mentioned above, whether and how they physically interact with each other remain obscure. In budding yeast, the exocyst subunits have been shown to interact physically with a number of proteins, including Sec15 with Rab GTPase Sec4 and type V myosin Myo2; and Sec6 with v-SNARE protein Snc2, t-SNARE protein Sec9, and Sec1/Munc18 family protein Sec1 (Guo et al., 1999; Sivaram et al., 2005; Jin et al., 2011; Shen et al., 2013; Lepore et al., 2016). Evidence indicates that the septin dynamics is essential for exocytosis, but no direct interaction has been detected yet between septins and the exocyst (Tokhtaeva et al., 2015). Recently, Michaelis and colleges have mapped S. cerevisiae protein interactome and found no interactions between septins and exocyst in pull down experiments (Michaelis et al., 2023).

However, several interactions between septins and the exocyst have been identified by Co-IPs to support the role of septins in the regulation of the exocyst localization in other cell types (Hsu et al., 1998; Beites et al., 1999; Vega and Hsu, 2003; Li et al., 2007; Gupta et al., 2015). In rat brain lysates, the septins SEPT2, 4, 6 and 7 have been shown to associate with exocyst complex containing Sec6/8 (Hsu et al., 1998). Sec6 is found to be colocalized with SEPT7 on synapse assembly sites in isolated neurons where active membrane remodeling is required (Hsu et al., 1998). From rat brain cells, exocyst subunits Sec8 and Exo70 along with tubulin co-immunoprecipitated with septin Nedd5 (Vega and Hsu, 2003). During hyphal development in C. albicans, association of Sec3 and Sec5 was detected by co-immunoprecipitation with Cdc3 (Li et al., 2007). In M. oryzae, mislocalization of Sec6 was reported with deletion of septin Sep3. This was supported by pull down and mass spectrometry data where Sep4 and Sep5 were pulled down by Exo84 while Sep3 by Sec6 (Gupta et al., 2015). Here we have presented the most comprehensive studies on explaining importance of septins to regulate exocytosis by direct physical interactions with the exocyst complex in fission yeast.

In our study, we systematically investigated all the potential pairwise interactions between septin and exocyst subunits using AlphFold2 predictions. We experimentally confirmed five out of the six predicted interactions by co-IPs: Spn1-Sec15, Spn1-Sec6, Spn2-Sec15, Spn2-Sec5, and Spn4-Sec15 and validated four of them by yeast two-hybrid assays (except Spn2-Sec5 due to high levels of Sec15 autoactivation). These multivalent interactions are able to ensure that the exocyst tethers secretory vesicles on the plasma membrane with high temporal and spatial fidelity even individual interaction may not be very strong. Interfaces of exocyst subunits Sec15, Sec6, and Sec5 are known to be available in the exocyst complex to interact with many proteins as mentioned above in budding yeast and in other systems for different cellular functions (Sjölinder et al., 2002; Fukai et al., 2003; Zhang et al., 2004; Feng et al., 2012; Du et al., 2015; Guo et al., 2016; Yang et al., 2022). The interactions between septins and the exocyst that we identified in fission yeast will provide important insights into the mechanisms of exocyst regulations by septins. During evolution, fission yeast may have lost many but these most conserved aspects of septin functions including septin-exocyst interactions.

It is known that the octameric exocyst complex consist of two subcomplexes (Heider et al., 2016; Ahmed et al., 2018; Lepore et al., 2018; Mei and Guo, 2018; Mei et al., 2018; Ganesan et al., 2020). Subcomplex 1 consist of Sec3, Sec5, Sec6, and Sec8 while subcomplex 2 consist of Sec10, Sec15, Exo70, and Exo84. In our study, we found that septins can interact with both of the exocyst subcomplexes with multivalent interactions by Alphafold predictions, reciprocal Co-IPs and yeast two-hybrid assays. Future studies are needed to map out the residues involved in the interactions. The predicted interacting residues from AlphaFold are too numerous and have to be refined. Preliminary examinations suggested that many of the predicted conserved residues on Sec15 and Sec5 are accessible for interactions with other proteins based on the structure of budding yeast exocyst (Lepore et al., 2018; Mei et al., 2018). In addition, tests are needed to figure out if posttranslational modifications are necessary for the interactions between septins and the exocyst. Because the colocalization of septins and the exocyst required for their proper function occurs at a specific stage during cytokinesis rather than a general regulation throughout the cell cycle, septin filament formation and posttranslational modifications of the involved proteins may be required, which make it challenging to tease out the interactions in vitro (Dobbelaere et al., 2003; Hernández-Rodríguez and Momany, 2012; Ren and Guo, 2012; Tay et al., 2019; Sharma and Menon, 2023; Werner and Yadav, 2023). Moreover, we cannot rule out that RhoA GTPase and PI(4,5)P2 are involved in septin-exocyst interactions as both has been reported to interact with septins and/or the exocyst in other cell types (Guo et al., 2001; He et al., 2007; Bertin et al., 2010; Bendezu and Martin, 2011; Perez et al., 2015; Carim and Hickson, 2023; Safavian et al., 2023).

In summary, we found that septins are important for exocyst targeting to the division site during late stages of cytokinesis through multivalent interactions between septins and the exocyst subunits. The proper exocyst localization at the rim of the division plane is critical for timely and successful cytokinesis. Our results will provide insights into future studies of the interactions and functions of septins and the exocyst complex in other cell types.

Materials and methods

Strains and molecular methods

Fission yeast strains used in this study are listed in Supplemental Table S1. Strains were constructed using PCR-based gene targeting and standard genetic methods (Moreno et al., 1991; Bähler et al., 1998). Tagged genes were expressed under endogenous promoters and integrated at their native chromosomal loci except where noted. The glucan synthase gene bgs1 is integrated at the leu1 loci under endogenous promoter, with the endogenous copy deleted (Cortes et al., 2002).

The functionalities of the tagged proteins (Spn1, Sec3, Exo70, Spn2, Sec5, Sec6, and Sec15) were tested by growing the strains at 25°C and 36°C on YE5S media or crossing to mutants. The growth and morphology of majority of the tagged strains were comparable to WT. However, Spn1 fused to red fluorescence tag (tdTomato and mCherry) localized abnormally to the plasma membrane on the cell sides at 25°C, indicating that these tagged proteins were not fully functional. tdTomato tagged Spn4 localized abnormally to the plasma membrane on the cell sides in addition to the division site. Although Spn4-mScarlet-I localized specifically to the division site, it is not fully functional with a small percentage of elongated and multiseptated cells at 25°C. These nonfunctional strains were not used.

Microscopy

Cells were normally grown at the exponential phase in YE5S liquid medium at 25°C for 40-48 h before microscopy or temperature shift. Confocal microscopy was performed as previously described (Wang et al., 2014; Davidson et al., 2015; Davidson et al., 2016; Zhu et al., 2018). Briefly, cells were collected from liquid culture by centrifuging at 3,000 rpm for 30 s at room temperature and washed with EMM5S twice to reduce autofluorescence. A final concentration of 50 nM n-propyl-gallate (n-PG) from a 10x stock (in EMM5S) was added in the second wash to protect cells from free radicals during imaging. Live cells were imaged on a thin layer of EMM5S with 20% gelatin and 50 nM n-PG at ∼23°C. To image cells at 36°C, concentrated cells were grown in coverglass-bottom dish and covered with EMM5S agar (Davidson et al., 2016).

We imaged cells using two confocal microscopy systems with 100x/1.4 or 100x/1.45 numerical aperture (NA) Plan-Apo objective lenses (Nikon, Melville, NY). Most fluorescence images were taken using a PerkinElmer spinning disk confocal system (UltraVIEW Vox CSUX1 system; PerkinElmer, Waltham, MA) with 440-, 488-, 515-, and 561-nm solid-state lasers and back thinned electron-multiplying charge-coupled device (EMCCD) cameras (C9100-13 or C9100-23B; Hamamatsu Photonics, Bridgewater, NJ) on a Nikon Ti-E inverted microscope. For better spatial resolution, Figure 1A was imaged using another spinning disk confocal microscope (UltraVIEW ERS; PerkinElmer) with 568-nm solid-state laser and 488-nm argon ion lasers and a cooled charge-coupled device camera (ORCA-AG; Hamamatsu Photonics) on a Nikon Eclipse TE2000-U microscope. We used TIRF microscopy controlled by NIS Elements software to examine the dynamic localization of the exocyst subunit Exo70 and the septin Spn1 at the division site for some movies. A Nikon Eclipse Ti-E microscope equipped with a TIRF illuminator, Plan Apo 100x/1.45NA oil objective, and an Andor iXon Ultra 897 EMCCD was used.

Image analysis

We analyzed images using ImageJ (National Institutes of Health, Bethesda, MD) and Volocity (PerkinElmer). Fluorescence images are maximum-intensity projections from z-sections spaced at 0.5 μm except where noted. Images of 3D projections (end-on views) and deconvolution (Figure 7C, Eng1) were generated from images with z-sections spaced at 0.05 μm. For quantification of fluorescence intensity at the division site, we summed the intensity from all z-sections using sum projection. A rectangular ROI1 was drawn to include majority of division site signal for intensity measurement. Then the intensity in a second ROI2 approximately twice the area of ROI1 (including ROI1) was measured and used to subtract cytoplasmic background as described previously (Coffman et al., 2011; Davidson et al., 2015; Davidson et al., 2016).

For comparing the colocalization at the rim of the division plane (Figure 1B), a line along the cell long-axis was drawn across the division plane at the same position for both Spn1 and Sec3 channels using maximum intensity projection images. Then the width of the line was adjusted to cover all signals at the division site, generating an ROI of 1.5 μm x 3.5 μm (x-y) (see Figure 1B). The mean intensity of all pixels in y-axis was measured along x-axis and plotted.

Line scans (Figures 6D, 7A, and 7C) across the division plane were made in the middle focal plane of the fluorescence images. A line along the cell short-axis was drawn across the division plane to cover the whole cell diameter. The width of the line was 3 pixels to reduce signal variations caused by measurements on a single focal plane. Mean intensity (average of 3 pixels) was measured across cell diameter. For Syb1 (Figure 6D), cells at the end of ring constriction (indicated by an Rlc1 dot at the center of the division plane) were measured; and line scans were aligned by referencing the peak intensity of Rlc1 signal. For Bgs1 (Figure 7A), cells with full septa were measured; and line scans were aligned by the peak intensity of Bgs1 signal. For Eng1 (Figure 7C), cells with full septa were measured; and line scans for WT cells were aligned by the middle of the two peaks; and the ones for spn1Δ cells were aligned by referencing the middle of septa in DIC images.

FRAP analysis

FRAP was performed using the photokinesis unit on the UltraVIEW Vox confocal system at either ∼23°C or 36°C (Coffman et al., 2009; Laporte et al., 2011; Zhu et al., 2013). Half of the division site signals at the middle focal plane were photobleached to <50% of the original fluorescence intensity. Five pre-bleach images and 150 post-bleach images for spn1Δ cells, or 70 post-bleach images for sec3-913 cells, were collected at every 0.33 s or 10 s, respectively. For image analysis, the background and photobleaching during image acquisition were corrected using empty space and unbleached cells within the same image. The pre-bleach intensity was normalized to 100%, and the first post-bleach intensity was normalized to 0% (Laporte et al., 2011; Zhu et al., 2018). Intensities of three consecutive post-bleach time points were rolling averaged to reduce noise (Vavylonis et al., 2008). Data were plotted and fitted using the exponential decay equation y = m₁ + m₂ exp(-m₃x), where m₃ is the off-rate. The half-time for recovery was calculate by t_1/2 = ln2/m₃.

Predictions of septin-exocyst interactions using AlphaFold analysis

The development of computer algorithms to predict three-dimensional protein structures from amino acid sequence involves two complementary ways that concentrate on either the physical interactions or the evolutionary history (Jumper et al., 2021). AlphaFold utilizes cutting-edge neural network topologies and training techniques to predict the 3D coordinates of a primary amino acid sequence (Jumper et al., 2021). We made the AlphaFold models of interactions between different septin and exocyst subunits using Google Colab Platform and AlphaFold2_advanced option that does not need templates at: https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/beta/AlphaFold2_advanced.ipynb#scrollTo=ITcPnLkLuDDE. Sequences of each subunit were searched against genetic databases with msa_method = mmseqs2, pair_mode = unpaired. The default mode of sampling options was used; num_models = 5, ptm option, num_ensemble = 1, max_cycles = 3, num_samples = 1. Total 5 models were ranked according to their Predicted Local-Distance Difference Test (pLDDT) score between 0 to 100, from low to high confidence level. Septin and exocyst subunits were input in 1:1 ratio. For each of the 32 pairs of septin and exocyst subunits, the protein sequences were entered in both orders (for example, Spn1:Sec3 and Sec3:Spn1). We found that the order of input sequence has effect on prediction results. So, we predicted all septin-exocyst combinations in both input sequence orders (septin first exocyst later and exocyst first septin later). We then selected the top septin-exocyst combinations that showed interactions in both input orders. The structure figures were drawn with PyMOL version 2.0 (Schrodinger, Inc.).

Co-IP and Western blotting

We carried out Co-IP and Western blotting as previously described (Laporte et al., 2011; Lee and Wu, 2012; Ye et al., 2012). Briefly, mEGFP, GFP, mYFP, or 13Myc tagged septin or exocyst subunits were expressed under native promotors in fission yeast. Cells were grown in YE5S liquid medium at 25°C for ∼48 h before harvesting and lyophilization. Lyophilized cells (200 mg) were ground into a homogeneous fine powder using pestles and mortars. IP buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 50 mM NaF, 20 mM glycerophosphate, and 0.1 mM Na₃VO₄, 1 mM PMSF, and protease inhibitor [Roche] 1 tablet/30 ml buffer) was added according to the ratio of 10 µl: 1 mg lyophilized cell powder. 60 µl Dynabeads protein G beads (Invitrogen) were incubated with 5 µg polyclonal GFP antibody (Novus Bio) for 1 h at room temperature. After three washes with PBS and one wash with 1 ml IP buffer, the beads were incubated with cell lysate for 2 h at 4°C. After 5 washes at 4°C with 1 ml IP buffer each time, proteins were eluted by boiling with 80 µl sample buffer. The protein samples were separated with SDS-PAGE gel and detected with monoclonal anti-GFP antibody (1:1,000 dilution; 11814460001; Roche, Mannheim, Germany), monoclonal anti-Myc antibody (1:500 dilution, 9E10, Santa Cruz Biotechnology, Dallas, TX) and anti-tubulin TAT1 antibody (1:10,000 dilution)(Woods et al., 1989). Secondary antibody anti-mouse immunoglobulin G (1:5,000 dilution; A4416, Sigma-Aldrich) was detected using SuperSignal Maximum Sensitivity Substrate (Thermo Fisher Scientific) on iBright CL1500 imager (Thermo Fisher Scientific).

Electron microscopy

Electron microscopy was performed at the Boulder Electron Microscopy Services at the University of Colorado, Boulder (Boulder, CO) as previously described (Lee et al., 2014; Wang et al., 2016). Briefly, yeast cells were grown at 25°C for ∼41 h and then shifted to 36°C for 4 h before harvesting using Millipore filters. Samples were prepared using high-pressure freezing with a Wohlwend Compact 02 Freezer in the presence of 2% osmium tetroxide and 0.1% uranyl acetate in acetone. Thin sections with a thickness of 70 nm were cut and embedded in Epon-Araldite epoxy resin, which were post stained with uranyl acetate and lead citrate. Imaging of EM samples was done using a Philips CM100 transmission electron microscope (FEI, Hillsboro, OR).

Yeast two hybrid assays

Yeast two hybrid assays were performed as described previously using X-gal overlay and β-D-galactosidase activity quantifications (Amberg et al., 2006; Paiano et al., 2019). DNA or cDNA (for genes with introns) sequences of Spn1, Spn1(aa 300-469), Spn2, Spn4, Sec15, Sec5, and Sec6 were cloned into pVP16 or pGBT9 vectors having VP16 transcription activation domain (AD) or GAL4 transcription factor DNA-binding domain (BD), respectively. Constructed plasmids were confirmed by restriction digestions and Sanger sequencing. Pairs of plasmids were then co-transformed into S. cerevisiae strain MAV203 (11281-011; Invitrogen) and plated on synthetic drop-out medium lacking leucine and tryptophan (SD-L-W) for selection. For X-gal overlay assay, grown colonies were re-streaked on YPD (yeast extract-peptone-dextrose) plates to grow overnight. We used 10-12 ml chloroform per plate to permeabilize cells for 10 min and then dried for additional 10 min. 0.5% agarose was prepared in 25 ml PBS (pH 7.5) and 500 µl X-gal (20 mg/ml stock in DMSO) was added after cooling. After mixing thoroughly, agarose containing X-gal was overlaid onto the colonies and incubated at 30°C. Plates were checked every 30 min for development of blue color.

Interactions were then quantified by β-D-galactosidase activity using the o-nitrophenyl– β-D-galactopyranoside (ONPG) assay (48712-M; Sigma Aldrich) according to the published methods (Amberg et al., 2006; Paiano et al., 2019). For interactions between Sec15 with Spn1, Spn2, and Spn4, the Amberg et al. method was used (Amberg et al., 2006). Briefly, cells were grown in SD-L-W liquid medium at 30°C overnight. 40 ml culture with OD₅₉₅ >1 was collected and washed with 1 ml distilled water. Then cells were broken in 110 µl breaking buffer (100 mM Tris-Cl, pH 7.5, 1 mM DTT, and 20% glycerol) using glass beads on bead beater. 10 µl of the lysate was diluted with 90 µl distilled water and spun down to remove cell debris, and the supernatant was used to estimate protein concentration by Bradford assay. To the remaining 100 µl of lysate, 0.9 ml Z-buffer (100 mM sodium phosphate, pH 7.5, 10 mM KCl, and 2 mM MgSO₄) and 0.2 ml ONPG (8 mg/1 ml Z buffer) were added and incubated at 28°C until pale yellow color develops in at least one of the samples. All the reactions were stopped by adding 0.4 ml 1 M Na₂CO₃. Debris were removed by centrifuging at 15,700 g for 10 min and OD₄₂₀ was measured using 1 ml of supernatant. Time elapsed from adding ONPG to adding stop solution was recorded and activity of β-galactosidase was calculated using the formula: β-galactosidase activity (nmol/min/mg) = OD₄₂₀ × 1.7/ [0.0045 × protein (mg/ml) × extract volume (ml) × time (min)]

For the interaction between Spn1 and Sec6, the Painano et al. method was used (Paiano et ^al., 201⁹). Briefly, cultures were diluted to OD₅₉₅ = 0.30 and incubated for 2 hrs at 30°C. For each sample, cells from 9 ml culture were collected and washed with 1 ml Z buffer and then resuspended in 0.1 ml Z buffer. Cells were broken by three freeze-thaw cycles in liquid nitrogen. 0.7 ml Z buffer with β-mercaptoethanol (27 µl β-mercaptoethanol in 9.973 ml Z buffer) and 160 µl ONPG was added to the cell lysates and incubated at 30°C until a yellow color develop in at least one of the samples. Reactions were stopped by adding 0.4 ml 1 M Na₂CO₃. Debris were removed by centrifuging at 15,700 g for 10 min and OD₄₂₀ was measured using 1 ml of supernatant. Time elapsed from adding ONPG to adding stop solution was recorded and β-galactosidase activity were calculated using following formula: β-galactosidase Units = 1000 × OD₄₂₀/ [T × V × OD₅₉₅], where T is the elapsed time (minutes), V is the volume (ml) of culture used, and OD595 is the optical density of yeast culture.

Statistical analysis

Data in graphs are mean ± 1 SD except where noted. The p-values in statistical analyses were calculated using the two-tailed Student’s t tests except Figure 4. The P values in Figure 4 (E and F) was calculated using one way ANOVA with Tukey’s post hoc test for quantification of yeast two hybrid analysis. Data is shown in Mean ± SD except where noted.

Supplemental material

Fig S1 shows localization, levels, and dynamics of septin and exocyst subunits. Fig S2 shows localization of Sec3 and Spn1 in gef3, rho4, and gef3 rho4 mutants. Fig S3 presents Alphafold predicted models of septin and exocyst subunit interactions. Fig S4 shows that septin subunits interact with exocyst subunits in reciprocal Co-IP. Video 1 shows accumulation of Spn1-mEGFP to the division site with the exocyst Exo70-tdTomato. Video 2 and 3 represents the dynamic localization of Exo70-tdTomato at the division site. Video 4 shows the localization of Sec15-mEGFP as a ring in WT cells. Video 5 and 6 shows the mislocalization of Sec15-mEGFP from a ring to disc on division plane with deletion of spn1 and spn4 respectively during late-stage of cytokinesis.

Additional information

Funding

Pelotonia Graduate Fellowship to Yajun Liu, and the National Institute of General Medical Sciences of NIH grant GM118746 to Jian-Qiu Wu.

Author Contributions

Performed the experiments, methodology, validation, and formal data analysis: D. Singh, Y. Liu, Y.-H. Zhu, S. Zhang, S. Naegele. Visualization, figure and table preparations: D. Singh and Y. Liu. Writing and editing manuscript: Y. Liu, D. Singh, and J-Q Wu. Supervision, conceptualization, investigation, review: J.Q. Wu. Funding acquisition: Y. Liu and J-Q Wu.

Competing Interest

The authors declare no competing interest.

Abbreviations used in this paper

• Co-IP: Co-immunoprecipitation

• EMCCD: Electron-multiplying charge-coupled device

• FPS: Frame per second

• FRAP: Fluorescence recovery after photobleaching

• NA: Numerical aperture

• PB: Phloxin B

• pLDDT: Predicted Local-Distance Difference Test

• ROI: Region of interest

• SPB: Spindle pole body

• TIRF: Total internal reflection fluorescence

• TRAPP-II: Transport particle protein-II

• WT: Wild type

Acknowledgements

We thank Pilar Pérez for strains; Eileen O’Toole and Garry Morgan at University of Colorado, Boulder for help with electron microscopy; Anita Hopper, Steve Osmani, Dmitri Kudryashov, Elena Kudryashova, Damien Wilburn, and Emily Vais for equipment and technical support; and members of the Wu laboratory for helpful discussion and suggestions.

Supplementary figures

Figure S1.

Figure S2.

Figure S3.

Figure S4.