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. 2022 Jan 21;33(2):ar17. doi: 10.1091/mbc.E21-04-0214

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis

Larissa V G Longo a, Evelyn G Goodyear a, Sha Zhang a, Elena Kudryashova b, Jian-Qiu Wu a,c,*
Editor: Rong Lid
PMCID: PMC9236143  PMID: 34910579

Abstract

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.

INTRODUCTION

Cytokinesis is the final step of the cell-division cycle, which partitions cellular contents from a mother cell into two daughter cells. Cytokinesis in fungi relies on successful coordination of assembly and constriction of an actomyosin contractile ring, plasma membrane deposition at the division site, and construction and remodeling of the division septum (Proctor et al., 2012; Thiyagarajan et al., 2015; Zhou et al., 2015; Meitinger and Palani, 2016; Pollard, 2019). Several cellular pathways involved in the regulation of these processes have been discovered, but the molecular mechanisms behind septum synthesis remain poorly understood.

In the fission yeast Schizosaccharomyces pombe and most fungi, the septum is composed mainly of glucans and has three layers with a primary septum sandwiched by secondary septa. Daughter cells separate when the primary septum is digested by glucanases and the secondary septa become the cell wall of new cell ends (Alonso-Nunez et al., 2005). Septum synthesis in S. pombe primarily depends on three glucan synthases: Bgs1, Bgs4, and Ags1. The primary septum is mainly composed of linear β-(1,3) glucan synthesized by Bgs1 (Cortes et al., 2002). Bgs4 synthesizes branched β-(1,3) glucan for secondary septum formation and the proper completion of primary septa (Muñoz et al., 2013). Ags1 is responsible for α-(1,3) glucan synthesis, which is found in the secondary septum and strengthens the primary septum (Cortes et al., 2012). These three glucan synthases are all essential transmembrane proteins that are delivered to the plasma membrane at the division site via the secretory pathway (Liu et al., 1999; Cortes et al., 2002, 2005, 2012; Muñoz et al., 2013).

Several regulators of the conserved glucan synthases have been identified in fungi, such as Rho GTPases (Drgonova et al., 1996; Kondoh et al., 1997; Beauvais et al., 2001) and the α-COP protein, which is involved in their intracellular translocation from endoplasmic reticulum to Golgi (Lee et al., 1999; Lee et al., 2002). In S. pombe, β-glucan synthases Bgs1 and Bgs4 are activated by Rho1 GTPase (Arellano et al., 1996), and Rho1 and Rho2 GTPases regulate the α-glucan synthase Ags1 through the protein kinase C Pck2 (Katayama et al., 1999; Calonge et al., 2000). We and others have previously identified Sbg1 as a novel regulator for the localization and stability of Bgs1 and found that Sbg1 is important for actomyosin ring constriction and septum synthesis (Davidson et al., 2016; Sethi et al., 2016). Bgs1 transport also depends on the F-BAR protein Cdc15 and clathrin light chain Clc1 (de Leon et al., 2013; Arasada and Pollard, 2014). Cdc15 helps deliver Bgs1 from the Golgi to the plasma membrane, where the Bgs1–Cdc15 interaction is important for contractile ring stability (Arasada and Pollard, 2014), while β-glucan synthase activity is diminished and Bgs1 localization is compromised without Clc1 (de Leon et al., 2013). Furthermore, the F-BAR protein Rga7 is critical for the delivery of Bgs4 to the cleavage site (Arasada and Pollard, 2015), and both rga7Δ and coiled-coil protein rng10Δ mutants reduce Bgs1, Bgs4, and Ags1 levels at the division site (Liu et al., 2016). However, much remains unknown about the coordination of these glucan synthases and the mechanisms of their delivery to and localization at the division site.

Smi1, also known as Knr4, is an intrinsically disordered protein (IDP) conserved in many fungi (Martin-Yken et al., 2016). In Saccharomyces cerevisiae, Smi1 acts as a hub that physically interacts with the key components of two pathways: Slt2 MAP kinase in the cell wall integrity pathway and the calcineurin phosphatase in the calcium–calcineurin pathway (Dagkessamanskaia et al., 2010a,b; Martin-Yken et al., 2016). The roles of Smi1 as a hub are also supported by its numerous interaction partners, including many synthetic lethal interactions (Goehring et al., 2003; Costanzo et al., 2010). Smi1 also regulates transcription of genes involved in cell cycle, cell wall synthesis, morphogenesis, and transcriptional responses to heat and cell wall stress in different fungi (Martin-Yken et al., 2002; Lagorce et al., 2003; Penacho et al., 2012). In the smi1 mutant, the SBF transcription factor is constitutively hyperactivated instead of peaking at the G1/S transition (Kim et al., 2010), and at least two cell cycle checkpoints are impaired: the morphogenesis checkpoint, which coordinates cell division with bud growth (Harrison et al., 2001; Mizunuma et al., 2001; Miyakawa and Mizunuma, 2007) and the mechanism controlling the daughter cell size (Dagkessamanskaia et al., 2010a,b). Smi1 homologues in other fungi exhibit similar functions and localization at polarized growth sites (Martin-Yken et al., 2003). However, it has not been reported that Smi1 directly regulates glucan synthases or glucanases in budding yeast and other fungi.

In this study, we investigated the role of the essential protein Smi1 in the regulation of septum formation and cell separation in fission yeast cytokinesis. We found that Smi1 and Bgs4 physically interact, colocalize in secretory vesicles, and travel together toward the division site. Localizations of glucan synthases, especially Bgs4, depend on Smi1. Collectively, we conclude that Smi1 is important for Bgs4 trafficking and localization to regulate late stages of cytokinesis in fission yeast.

RESULTS

Identification of Smi1 as a β-glucan synthase Bgs4 binding partner

Identification of binding partners of glucan synthases, which are essential for cell wall and septum formation, is important for understanding the synthases’ functions. Recently, our lab and others characterized transmembrane protein Sbg1 as a regulator of the β-glucan synthase Bgs1 (Davidson et al., 2016; Sethi et al., 2016), which led us to search for Bgs4 binding partners. Purification of GFP-Bgs4 from an S. pombe extract using GFP-Trap (Rothbauer et al., 2008) followed by mass spectrometry identified Smi1 (SPBC30D10.17c) as a Bgs4-interacting protein (Supplemental Figure S1). Smi1 was more abundant in pull downs of cells expressing GFP-Bgs4 than in those from GFP-Bgs1 (Supplemental Figure S1A). Additionally, Smi1 coverage was higher from cells expressing GFP-Bgs4 than GFP-Bgs1 (Supplemental Figure S1B). Predictive modeling of Smi1 suggested that the N- (aa 1–130) and C- (aa 340–504) terminal regions of Smi1 are highly disordered, while its central portion (aa 131–339), which contains the Smi1/Knr4 domain (aa 153–301), is structured (Figure 1, A and B). The same pattern of disordered N- and C-termini and a structured central region was observed in the Smi1 homologue Knr4 in S. cerevisiae (Martin-Yken et al., 2016).

FIGURE 1:

FIGURE 1:

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The intrinsically disordered protein Smi1 is important for cell integrity during cell separation. (A) Computa­tional evaluation of intrinsic disorder propensity of Smi1 using PONDR-VSL1 score (http://www.pondr.com/). Values between 0 and 1 are scored considering amino acid frequencies, sequence complexity, ratio of net charge/hydrophobicity, and mean flexibility. Scores ≥0.5 indicate disorder. (B) Schematic of Smi1 domain organization, highlighting the three point mutations in smi1-1: D307G, E331G, and N426S. (C) DIC images of smi1-1 cells grown at 36°C for 4 h. (D) Quantification of cell lysis in WT and smi1-1 cells grown at 25°C or shifted to 36°C for 4 h. (E) Time-lapse images of cell-lysis scenarios during separation of smi1-1 cells at 36°C. None (top), one (middle), or both (bottom) daughter cells lysed are shown. Scale bars, 5 μm. See also Supplemental Movie S1. (F) Quantification of septation index in WT and smi1-1 cells grown at 25°C or shifted to 36°C for 4 h. ***p < 0.0001 compared with WT in t test.

Smi1 is important for septum integrity

Smi1 has been previously described as an essential gene in S. pombe genome-wide studies (Kim et al., 2010; Hayles et al., 2013). It was reported that Smi1 is involved in the entry and maintenance of the quiescent state (Sajiki et al., 2009). To investigate the roles of Smi1 in cycling cells, we generated a smi1-1 temperature-sensitive mutant (Figure 1B) by marker reconstitution mutagenesis (Tang et al., 2011). smi1-1 had three mutations, one in the disordered C-terminus and two in the central ordered region (Figure 1B). The major phenotype of smi1-1 was massive cell lysis, with ∼76% of cells lysed at the restrictive temperature (Figure 1, C and D). Time-lapse microscopy revealed that lysis occurred during cell separation, and either one or both daughter cells of smi1-1 lysed (Figure 1E; Supplemental Movie S1). smi1-1 cells also showed a significantly higher (38.5%) septation index than wild-type (WT) cells (11.5%) at 36°C (Figure 1F). The cell lysis and septation defects were confirmed by depleting Smi1 using P81nmt1-smi1 cells in medium containing thiamine (Supplemental Figure S2, A–D).

Movie S1.

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Cell division in smi1‐1 cells. Time‐lapse DIC images show different cell‐lysis scenarios during daughter‐cell separation of smi1‐1 at 36°C. Left, both daughter cells lyse; Right, only one of them lyses.

The smi1-1 lysis phenotype suggested a possible role of Smi1 in cell wall integrity during septation. Indeed, smi1-1 cells were sensitive to cell wall stresses including treatments with Calcofluor white and SDS, indicating a cell wall weakness (Figure 2A). This phenotype was rescued by sorbitol, which decreased the osmotic stress on the weakened cell wall (Figure 2A). Genetic evidence also supported that Smi1 works with glucan synthases for septum synthesis and cell wall integrity. smi1-1 was synthetic lethal with mutations in β-glucan synthases Bgs1 (bgs1-191 and bgs1-D277N [Liu et al., 1999; Dundon and Pollard, 2020]) and Bgs4 (cwg1-1 and cwg1-2 [Ribas et al., 1991; Muñoz et al., 2013]) at 30°C (Figure 2B and Table 1) and showed moderate genetic interaction with the α-glucan synthase Ags1 mutant mok1-664 (Katayama et al., 1999) (Table 1). In addition, smi1-1 had strong genetic interactions with mutations in the cell integrity pathway (protein kinase Cs pck1Δ and pck2-Δ1 [Toda et al., 1993; Viana et al., 2013] and arrestin art1Δ [Davidson et al., 2015]), in glucan synthase regulators rga7Δ and rng10Δ (Nakano et al., 2001; Kim et al., 2010; Liu et al., 2016, 2019), and in the septum initiation network (mob1-M17 [Salimova et al., 2000]) (Figure 2B and Table 1).

FIGURE 2:

FIGURE 2:

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Smi1 plays a role in cell wall integrity. (A) Sensitivity of smi1-1 cells to Calcofluor white and SDS. Cells were grown in YE5S medium (25°C), spotted in serial (five-fold) dilutions onto YE5S plates and YE5S plates containing the indicated treatments, and then incubated at 36°C for 48 h. (B) Synthetic genetic interactions between smi1-1 and mutations in genes involved in cell wall integrity. The cells were grown on YE5S plates at 30 or 36°C for 48 h.

TABLE 1:

Genetic interactions between mutations in smi1 and genes involved in cytokinesis.

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Strong genetic interactions are highlighted in gray. +++, growth and morphology similar to WT; ++, some cell lysis, pink color on YE5S + PB plates; +, massive cell lysis, red color on YE5S + PB plates; –, inviable, unable to grow on YE5S + PB plates.

aStrain was woken up in YE5S + sorbitol plates, because it does not grow on YE5S plates.

We confirmed the defects in septa using electron microscopy. In cells with closed septa, the primary septum was uneven (Figure 3, red arrows) and thinner in smi1-1 cells than in WT (Figure 3A). The defects in the septum and cell wall adjacent to the division site were more obvious in separating smi1-1 cells (Figure 3B). Unilateral separation with a thinned adjacent cell wall was frequently observed in smi1-1 cells (Figure 3B, black arrows). Similar defects were seen when Smi1 was depleted (Supplemental Figure S2D), indicating that the septal defects were due to the lack of Smi1 function. Moreover, smi1-1 cells had lower Calcofluor intensity at the division site, which stains the primary septum (Figure 3C), confirming an impaired primary septum.

FIGURE 3:

FIGURE 3:

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Smi1 plays a role in septum and cell wall formation during septation. (A, B) Electron microscopy micrographs (left and middle) and quantifications of septum width (right) of septating WT and smi1-1 cells grown at 36°C for 4 h before (A) or during (B) daughter-cell separation. The red arrow marks defects in the primary septum, and the black arrows indicate the thinner adjacent cell wall (within 0.5 µm from the septum). Asymmetric separation in smi1-1 is also shown. Scale bars, 500 nm. (C) Micrographs and quantifications of defective primary septa in smi1-1 cells grown at 36°C for 3–4 h, as revealed by Calcofluor white staining. Scale bar, 5 μm. *p < 0.01, **p < 0.001, and ***p < 0.0001 compared with WT.

The glucan synthases are transmembrane proteins delivered to the plasma membrane via the secretory pathway (Liu et al., 1999; Cortes et al., 2002, 2005, 2012; Muñoz et al., 2013). To investigate the potential role of Smi1 in exocytosis, smi1-1 was crossed with different mutants in the exocytic pathway (Table 1), but only moderate genetic interactions with Munc13/UNC-13 mutant ync13-4 (Zhu et al., 2018) and the myosin-V mutant myo52Δ (Motegi et al., 2001; Win et al., 2001) were detected, suggesting that Smi1 is not a general player in the exocytic pathways. Interestingly, smi1-1 showed only mild genetic interactions with mutations in contractile ring components (Balasubramanian et al., 1998), suggesting that the ring machinery is not affected by the smi1 mutation (Table 1). Indeed, time-lapse microscopy revealed that the timing of ring formation, maturation, constriction, and disassembly was similar in smi1-1 and WT cells (Supplemental Figure S3). Collectively, the lysis phenotype, high septation index, genetic interactions, and electron microscopy data indicate that Smi1 is important for septum formation and/or cell septation.

Smi1 localizes to the division site and secretory vesicles

To better understand Smi1 function during cytokinesis, we tagged Smi1 at its C-terminus under its native promoter with tandem dimer Tomato (tdTomato) and monomeric enhanced GFP (mEGFP). Smi1 localized to cytoplasmic puncta and the division site (Figure 4A, arrows), forming a discrete ring in 87% (n = 300) of dividing cells in time-lapse movies (Figure 4B). With the spindle pole body (SPB) protein Sad1 as a cell cycle marker, we observed that Smi1 started to concentrate at the division site 18 ± 4 min (n = 15 cells) after SPB separation (Figure 4C, yellow arrow). To determine the identity of the Smi1 puncta, we compared Smi1 localization to known vesicle proteins at different stages of secretion (Figure 4, D and E). Smi1 puncta colocalized well with trans-Golgi marker Sec72 (Vjestica et al., 2008); ∼80% Smi1 puncta contained Sec72 (Figure 4E) while <40% Smi1 puncta colocalized with early Golgi proteins Anp1 and Sec24 (Supplemental Figure S4). Smi1 also colocalized well with the Rab11 family GTPase Ypt3, the TRAPP II complex subunit Trs120, and the v-SNARE Syb1 (Figure 4, D and E). The localization is consistent with Smi1’s roles in septum formation and/or cell septation.

FIGURE 4:

FIGURE 4:

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Localization of Smi1 to secretory vesicles and the division site. (A) Smi1 localizes to cytoplasmic puncta and the division site (arrows). (B) Three-dimensional projection of Smi1-tdTomato forming a ring structure at the division site in three different cells. (C) Time course of Smi1 localization with SPB protein Sad1 (its separation defined as time 0) as a cell cycle marker. (D, E) Micrographs (D) and quantifications (E) showing the colocalization of Smi1 with Sec72, Ypt3, Trs120, and Syb1 in vesicles in interphase and septating cells. N = number of vesicles in ∼10 cells for each protein. Scale bars, 5 μm.

Smi1 colocalizes with glucan synthases

To better understand the role of Smi1 in septum synthesis, we investigated the colocalization of Smi1 with glucan synthases (Figure 5). Whether the cells were in interphase or septating, Smi1 colocalized well in cytoplasmic puncta with glucan synthases Bgs4 (90–93%), Ags1 (82–87%), Bgs1 (65–76%), and the Bgs1 partner Sbg1 (76–79%), although Smi1 was less concentrated on the plasma membrane (Figure 5, A and B). A parallel analysis was performed to compare the colocalization between the glucan synthases Bgs1, Bgs4, and Ags1 (Supplemental Figure S5). No colocalization higher than 85% was seen in cytoplasmic puncta, suggesting that the high rate of colocalization between Bgs4 and Smi1 is specific. Thus, we analyzed GFP-Bgs4 and Smi1-tdTomato trafficking by total internal reflection fluorescence (TIRF) microscopy. We found that Smi1 and Bgs4 traveled in vesicles and arrived at the division site together (Figure 5C, n = 12; Supplemental Movie S2). These data support a role for Smi1 in glucan synthase delivery to the division site, especially for Bgs4.

FIGURE 5:

FIGURE 5:

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Smi1 mostly colocalizes with Bgs4, Ags1, Bgs1, and Sbg1 and travels together with Bgs4 in vesicles. Micrographs (A) and quantifications (B) showing colocalization of Smi1 with Bgs4, Ags1, Bgs1, and Sbg1 in vesicles in interphase and septating cells. (C) TIRF micrographs showing that Smi1 and Bgs4 move together in vesicles (an example marked by a red arrow) to the division site. Scale bars, 5 μm. See also Supplemental Movie S2.

Movie S2.

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Smi1 and Bgs4 travel together in vesicles to the division site. Video from total internal reflection fluorescence (TIRF) microscopy shows that Smi1 and Bgs4 travel together towards the division site in cytoplasmic puncta.

To further investigate the relationship between Smi1 and glucan synthases, we mislocalized Smi1 using the mitochondrial outer membrane protein Tom20 tagged with GFP-binding protein (GBP; Figure 6). Smi1-mEGFP mislocalized Bgs4 and Ags1 to mitochondria, where they colocalized in mitochondrial strands or clumps throughout the cell (Figure 6, A and B, yellow arrows). In contrast, Bgs1 and Sbg1 were not mislocalized by Smi1-mEGFP (Figure 6, C and D). The reverse experiments showed that only the mistargeted GFP-Bgs4, but not Ags1, Bgs1, or Sbg1, could mislocalize Smi1 to the mitochondria (Supplemental Figure S6). This could be due to a weaker interaction between Ags1 and Smi1 in comparison to that between Bgs4 and Smi1 or different configurations of proteins on the outer membrane of mitochondria. Consistently, we found that Smi1 coimmunoprecipitated with Bgs4 (Figure 6E). We confirmed that the mislocalization was not caused by signal bleed-through between channels (Supplemental Figure S7). Together, these results suggested that Smi1 interacts with Bgs4, which is important for Bgs4 localization.

FIGURE 6:

FIGURE 6:

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Mislocalized Smi1 can ectopically target Bgs4 and Ags1 to mitochondria. Micrographs of cells coexpressing Tom20-GBP, Smi1-mEGFP, and either RFP-Bgs4 (A), Ags1-Cherry (B), tdTomato-Bgs1 (C), or tdTomato-Sbg1 (D). Yellow arrows mark examples of colocalization on mitochondrial structures. Scale bars, 5 μm. (E) Smi1 coimmunoprecipitates with Bgs4. Solubilized proteins from the membrane fraction of the indicated strains were immunoprecipitated (IP) with anti-GFP antibodies. Proteins before (input) and after immunoprecipitation were transferred to the same membrane and blotted with monoclonal anti-GFP or anti-Myc antibody.

Smi1 helps to deliver glucan synthases and glucanases to the division site

To understand the importance of Smi1 for localization of glucan synthases, we measured their fluorescence intensities in the smi1-1 mutant (Figure 7). Bgs4 had lower global intensity in smi1-1 cells, while that of Ypt3, Ags1, Bgs1, and Sbg1 remained the same as in WT cells (Figure 7, A and B). This Bgs4 result was confirmed by Western blot showing that the smi1-1 strain had a lower GFP-Bgs4 level compared with WT control (Figure 7D). Together, these results suggest that Smi1 plays a role in Bgs4 stability or expression level.

FIGURE 7:

FIGURE 7:

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Localization of Ypt3, Bgs4, Ags1, Bgs1, and Sbg1 in WT and smi1-1 cells. (A–C) DIC and maximum-intensity projections of fluorescence images (A), global fluorescence intensity (B, n  =  70 cells each), and local intensity at the division site (C, n  =  15 cells each) of Ypt3, Bgs4, Ags1, Bgs1, and Sbg1 in WT and smi1-1 cells grown for 2 h at 36°C. Scale bar, 5 μm. For local fluorescence intensity, cells are grouped into three stages: before ring constriction (cells with compact ring before constriction), constricting ring (cells during ring constriction), and disk (cells with protein spreads to the whole division plane after ring constriction). *p < 0.01, **p < 0.001, and ***p < 0.0001 compared with the WT. (D) Western blot image (left) and quantification (right) of two independent experiments showing GFP-Bgs4 from whole cell lysates of GFP-bgs4 and smi1-1 GFP-bgs4 cells grown at 36°C for 2 h. α-TAT1 is a loading control. (E) Line scans of individual cell (left) and average (right; n = 10 cells) showing GFP-Bgs4 intensity in vesicles from WT (blue) and smi1-1 cells (red). Data in the right graph are mean ± SEM.

The local intensity of Bgs1, Bgs4, and Sbg1, but not Ags1, at the division site was significantly lower in smi1-1 than WT cells in all cytokinesis stages. The Bgs4 level at the division site decreased the most, >80%, which is higher than the decrease in global intensity (Figure 7, A–D). The Bgs4 level at the cell tip of interphase cells was also dramatically decreased, and the cytoplasmic puncta looked more diffuse in smi1-1 (Figure 7A). Line scan analyses of cytoplasmic puncta showed that Bgs4 intensity indeed decreased in smi1-1 cells in comparison to WT (Figure 7E). This decrease in local intensity was specific to the glucan synthases studied, as the levels of the Rab11 GTPase Ypt3, a general regulator of vesicle trafficking at the plasma membrane (Cheng et al., 2002), and contractile ring component Myo2 at the division site were not significantly altered in smi1-1 cells (Figure 7, A and C, Supplemental Figure S3A, and unpublished data). In addition, localization and intensity of Ypt3 and Syb1 in cytoplasmic puncta were similar in P81nmt1-smi1 and WT cells grown under repression condition (Supplemental Figure S8).

Previously, our group showed that Sbg1 is involved in Bgs1 localization and trafficking to the division site (Davidson et al., 2016). To compare the importance of Smi1 and Sbg1 in the localization of glucan synthases, we measured the local intensity of Bgs1, Bgs4, and Ags1 at the division site in the temperature-sensitive mutant sbg1-3 (Sethi et al., 2016). Bgs1, but not Bgs4 or Ags1, intensity at the division site was reduced during the late stage of septation (Supplemental Figure S9). Together, these results confirmed that Sbg1 is specific to Bgs1, while Smi1 regulates the levels of both Bgs4 and Bgs1 at the division site, with a more important role for Bgs4 (Figure 7, A–C).

To investigate whether cell lysis in the smi1-1 mutant is due to improper digestion of the septum, we also measured the levels of glucanases at the division site. The endo-(1,3)-β-glucanase Eng1 and the (1,3)-α-glucanase Agn1 are the two most studied glucanases involved in septum degradation during cell separation (Martin-Cuadrado et al., 2003; Dekker et al., 2004). By contrast, glucanases Bgl2 and Exg1 are less studied (Duenas-Santero et al., 2010). We analyzed the fluorescence intensity of Eng1, Agn1, Bgl2, and Exg1 at the division site in WT and smi1-1 cells (Figure 8). Agn1 and Bgl2 intensities were significantly lower in both early and late stages of septation (Figure 8, A–C), while Eng1 intensity was not significantly reduced. Interestingly, the intensity of Exg1 increased in smi1-1, especially during early septation. However, the increased level of Exg1 was not the cause for cell lysis in smi1-1 cells because the double mutant smi1-1 exg1Δ had a similar percentage of cell lysis as the smi1-1 single mutant at the restrictive temperature (Supplemental Figure S10). Together, these results suggest that Smi1 has functions in maintaining the proper levels of both glucan synthases and glucanases at the division site.

FIGURE 8:

FIGURE 8:

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Localization and intensity of glucanases Eng1, Agn1, Bgl2, and Exg1 in smi1-1 cells. (A–C) DIC and fluorescence confocal images (A) and quantifications of fluorescence intensity of Eng1, Agn1, Bgl2, and Exg1 in cells without (B) or with (C) visible septum under DIC. WT and smi1-1 cells were grown for 2 h at 36°C. Scale bar, 5 μm. *p < 0.01, **p < 0.001 compared with the WT.

DISCUSSION

In this study, we characterized Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 plays an essential role in maintaining septum integrity during the late stages of cytokinesis by regulating levels of glucan synthases (especially Bgs4) and glucanases at the division site to ensure proper cell division.

Roles of Smi1 in regulating septum integrity during cytokinesis

Except for its reported involvement in the entry and maintenance of quiescence (Sajiki et al., 2009), the roles of Smi1 in fission yeast have not been studied. Our results provide several lines of evidence to suggest that Smi1 is important for septum integrity during cytokinesis. We showed that smi1-1 cells had defective septa (Figure 3), which lead to massive cell lysis at restrictive temperature (Figure 1 and Supplemental Movie S1). Additionally, smi1-1 was sensitive to cell wall stresses (Figure 2A) and was synthetic lethal with mutations in β-glucan synthases and the cell integrity pathway (Figure 2B and Table 1). Smi1 colocalized well with glucan synthases, especially Bgs4 (Figure 5 and Supplemental Movie S2), and Smi1 and Bgs4 physically interacted (Figure 6 and Supplemental Figure S6). Finally, the local intensities of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1, Bgl2, and Exg1 were significantly altered at the division site in smi1-1 (Figures 7 and 8). Taken together, our data indicate that Smi1 plays important roles in septum integrity during the late stages of cytokinesis by regulating the proper delivery and localization of glucan synthases and glucanases to the division site.

Several proteins have been identified as regulators of glucan synthases or glucanases (see Introduction). However, few proteins are known to regulate the localization of both classes of enzymes. Recently, Cdc42 GTPase was shown to promote recruitment of Bgs1 to the division site and be critical for proper localization of Eng1 and Agn1 (Onwubiko et al., 2020). Here we showed that Smi1 is important for division-site localization of both glucan synthases and glucanases, suggesting an interesting dual role of Smi1 in septum integrity.

Most of our results indicate that Smi1 mainly regulates Bgs4 and the smi1-1 phenotype more closely resembles that of Bgs4 than that of Bgs1 mutants. After culture at restrictive temperature, Bgs1 mutants display very slow or complete failure in actomyosin ring constriction (Liu et al., 1999; Dundon and Pollard, 2020), as well as multiseptation and ring sliding due to less efficient anchoring (Arasada and Pollard, 2014; Cortes et al., 2015). None of these characteristics was observed in the smi1-1 mutant, and ring kinetics was not affected in smi1-1 (Supplemental Figure S3). In contrast, the lysis phenotype of smi1-1 is very similar to that of Bgs4 mutants. Bgs4 is required for the proper synthesis of the cell wall surrounding the primary septum and compensates for excessive cell wall degradation during cell separation (Cortes et al., 2005; Muñoz et al., 2013). Bgs4 depletion leads to the absence of the lateral cell wall at the start of cell separation (Cortés et al., 2016) and results in excessive cell wall degradation (Cortes et al., 2005; Muñoz et al., 2013), which was observed in smi1-1 cells under electron microscopy (Figure 3). Despite all these similarities, cells lacking Bgs4 show phenotypic changes not seen in smi1-1, such as ring sliding, obliquely positioned rings and septa, and misdirected septum synthesis. Moreover, Bgs4 is essential for secondary septum formation and for correct primary septum completion (Muñoz et al., 2013), which is not consistent with the smi1 mutant phenotype. It is possible that the remaining Bgs4 at the division site in smi1-1 is sufficient to maintain some of the Bgs4 functions.

We and others recently characterized Sbg1 as a regulator of glucan synthase Bgs1 (Davidson et al., 2016; Sethi et al., 2016). However, Smi1 differs from Sbg1 in how it regulates glucan synthases. First, Smi1 is an IDP (Figure 1, A and B), while Sbg1 is a transmembrane protein (Davidson et al., 2016), which allows Sbg1 to localize as a stable ring at the division site during septum formation until abscission (Davidson et al., 2016) and to link the plasma membrane, the actomyosin ring, and the division septum assembly machinery (Sethi et al., 2016). In contrast, Smi1 accumulates transiently at the division site and localizes mostly in cytoplasmic vesicles (Figure 4, A–C) and is not involved in ring stability or kinetics (Supplemental Figure S3). Second, Sbg1 and Smi1 have different specificities. Sbg1 plays a specific role in Bgs1 localization and does not affect other glucan synthases (Davidson et al., 2016), while Smi1 mainly regulates Bgs4 but also affects the localization of Bgs1 and Sbg1, as well as glucanases Agn1, Bgl2, and Exg1, thus playing a broader role in septum synthesis (Figures 7 and 8). The mechanism used by Smi1 to traffic Bgs4 to the division site still needs to be determined in future studies.

Smi1 proteins have diverse functions beyond cytokinesis

Our findings that Smi1 regulates septum integrity and the localization of Bgs4 and other proteins highlight some novel roles for Smi1. It has not previously been reported that Smi1 regulates glucan synthases or glucanases in budding yeast and other fungi. In S. cerevisiae, Smi1 acts as a hub that physically interacts with the key components of two pathways: Rho GTPase-protein kinase C-MAP kinase in the cell wall integrity pathway and the calcineurin phosphatase in the calcium–calcineurin pathway (Dagkessamanskaia et al., 2010a,b; Martin-Yken et al., 2016). It will be interesting to investigate in the future whether Smi1 regulates septum integrity through these pathways. Smi1 also regulates transcription of genes involved in cell cycle, cell wall synthesis, morphogenesis, and heat and cell wall stress in a variety of fungi (Martin-Yken et al., 2002; Lagorce et al., 2003; Penacho et al., 2012). It will be interesting to determine whether Smi1 has these functions in fission yeast besides its role in cytokinesis, especially whether Smi1 also regulates Bgs4 transcription.

Smi1 has homologues throughout the fungal kingdom, but not in higher eukaryotes including humans, making the essential Smi1/Knr4 family proteins attractive targets for new antifungal drugs. In the human pathogen Candida albicans, Smi1 expression is induced in the pathogenic hyphal cells (Harcus et al., 2004), and the smi1Δ/smi1Δ mutant shows reduced cell wall β-glucan synthesis and biofilm formation and less biofilm-associated fluconazole resistance (Nett et al., 2011). Moreover, the functional interaction between S. cerevisiae Smi1 and calcineurin supports the potential importance of studying Smi1 as an antifungal target, because calcineurin is implicated in the morphogenic switches responsible for virulence in pathogenic fungi (Martin-Yken et al., 2003).

In conclusion, we found that Smi1 has at least two novel roles in cytokinesis: involvement in the cell wall integrity pathway and in recruiting glucan synthases and glucanases to the division site for successful septation. It will be interesting to determine whether these roles are also conserved in other fungi.

MATERIALS AND METHODS

Strain construction and genetic methods

All strains used in this study are listed in Supplemental Table S1. To delete or tag genes at their endogenous locations, we used PCR-based gene targeting by homologous recombination (Bähler et al., 1998). All tagged proteins were expressed under their native promotors, except where specified. To deplete Smi1, we used the P81nmt1 promoter, which is repressed by thiamine (Maundrell, 1990; Basi et al., 1993). All smi1 strains were tagged at their C-terminus, and their functionalities were confirmed by growing cells on YE5S and YE5S + phloxin B (PB) plates at 25°, 30°, and 36°C.

The temperature-sensitive smi1-1 mutant was created using marker reconstitution mutagenesis (Tang et al., 2011; Lee et al., 2014). Briefly, smi1 was cloned into a pHis5C plasmid, and this construct was used as a template in an error-prone PCR using a mutagenic cocktail (8 mM dTTP, 8 mM dCTP, 48 mM MgCl2, and 5 mM MnCl2). The PCR product was then transformed into strain JW9102 (smi1-3′UTR-his5∆C-kanMX6 his5∆ ade6-210 leu1-32 ura4), and positive transformants were selected for histidine prototroph and temperature sensitivity at 36°C. Finally, we sequenced the mutants to identify the mutations in the smi1 open reading frame (ORF). smi1-1 showed three mutations: D307G, E331G, and N426S.

To mistarget Smi1, Bgs1, Bgs4, Ags1, or Sbg1 to mitochondria, strains expressing mEGFP/GFP-tagged proteins were crossed to a strain expressing the mitochondrial outer membrane protein Tom20 tagged with GBP (Yamamoto et al., 2011).

Affinity purification and mass spectrometry

We purified the GFP fusion protein from S. pombe extracts to identify the Bgs4 binding partners. We expressed the GFP control under the 41nmt1 promoter and GFP-Bgs4 and GFP-Bgs1 under their native promoters. Cells were grown in YE5S medium at 25°C for 48 h. Lyophilized cells stored at –80°C were thawed and ground with pestle and mortar to a homogeneous fine powder at room temperature and mixed with cold immunoprecipitation buffer (50 mM HEPES, pH 7.5, 1% NP-40, 200 mM NaCl, 1 mM EDTA, 20 mM β-glycerophosphate, 0.1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF], and one protease inhibitor tablet/50 ml buffer [Roche, Mannheim, Germany]). Cell extract was then centrifuged twice at 4°C (21,000 rpm for 30 min and 21,000 rpm for 10 min), and the protein concentration was measured via Bradford assay (Bio-Rad Laboratories, Hercules, CA). Then, cell extract was diluted with dilution/wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) and incubated with prewashed GFP-Trap magnetic agarose beads (Chromotek, Planegg-Martinsried, Germany) at 4°C for 2 h. Beads were washed five times with dilution/wash buffer and eluted with SDS-sample buffer (Laemmli) by boiling the beads for 5 min at 95°C. For mass spectrometry analysis, protein samples were run through a stacking SDS–PAGE gel (Liu et al., 2010) and excised as a one-sample band, which was sent for mass spectrometry analysis at the Mass Spectrometry and Proteomics Facility at The Ohio State University, Columbus, OH.

For mass spectrometry, gel pieces were digested overnight at room temperature with 10 µg/ml trypsin (Promega, Madison, WI) in 50 mM ammonium bicarbonate. The peptides were extracted with 50% acetonitrile/5% formic acid. Peptides were dried in a vacufuge and resuspended in 20 μl of 50 mM acetic acid. Capillary-liquid chromatography-nanospray tandem mass spectrometry (capillary-LC/MS/MS) was performed on a Thermo Scientific Orbitrap Fusion equipped with an EASY-Spray Source operated in positive ion mode. Samples were separated on an easy spray nano column (Pepmap RSLC; C18, 3 µ, 100 A, 75 µm × 150 mm; Thermo Scientific) using a two-dimensional RSLC high-performance liquid chromatography system from Thermo Scientific. Mass spectrometry data were analyzed using Mascot Daemon by Matrix Science version 2.5.1 (Boston, MA) and searched against the most recent SwissProt or NCBI databases. The mass accuracy of the precursor ions was set to 10 ppm, and fragment mass tolerance was set to 0.5 Da. Considered variable modifications were oxidation (Met), deamidation (N and Q), and carbamidomethylation (Cys). Four missed cleavages for the enzyme were permitted. A decoy database was used to determine the false discovery rate (FDR), and peptides were filtered at FDR of 1%. Proteins identified with at least two unique peptides were considered for a reliable identification.

Cellular methods and pharmacological treatments

To stain the primary septum, we incubated cells with 10 μg/ml Calcofluor for 10 min in the dark before imaging. To compare the sensitivities of the WT and smi1-1 cells to various stresses, cells were grown exponentially in YE5S medium at 25°C, serially (5×) diluted, and spotted onto YE5S plates and YE5S plates containing 100 µg/ml Calcofluor white, 0.0075% SDS, or 0.0075% SDS + 1 M sorbitol, and incubated at 36°C for 48 h.

Microscopy and image analyses

Strains stored at –80°C were streaked onto YE5S plates and grown for 2 days at 25°C. Fresh cells were inoculated into YE5S liquid media and grown exponentially for ∼2 days at 25°C. When indicated, cells were transferred to YE5S + thiamine medium to repress the nmt1 promoters. For microscopy, cells were collected by centrifugation at 3000 rpm for 30 s, washed once with EMM5S to reduce autofluorescence and once with EMM5S plus 5 μM n-propyl-gallate (to reduce phototoxicity and photobleaching during imaging). Cells were then imaged on a thin EMM5S with 20% gelatin pad with 5 μM n-propyl-gallate. For imaging at 36°C, the cells were cultivated in liquid culture at 25°C and then shifted to 36°C for the indicated time. Cells were then collected (3000 rpm, 30 s), placed on a prewarmed coverglass-bottom dish (0420041500C; Bioptechs, Butler, PA), and covered with prewarmed YE5S agar before being imaged at 36°C in a temperature-controlled chamber (Stage Top Incubator INUB-PPZI2-F1 with UNIV2-D35 dish holder; Tokai Hit, Shizuoka-ken, Japan).

For fluorescence images and time-lapse movies, microscopy was performed as previously described. Briefly, we used a 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 microscope. Nikon Plan-Apo oil objective lenses of 100×/1.4 or 100×/1.45 numerical aperture (NA) were used. When only differential interference contrast (DIC) images were taken, we used a Nikon Eclipse Ti inverted microscopy equipped with a DS-QI1 Nikon cooled digital camera (Nikon, Melville, NY).

TIRF microscopy was used to track the movement of GFP-Bgs4 and Smi1-tdTomato cytoplasmic puncta simultaneously. Images were collected using a Nikon Eclipse Ti-E microscope equipped with a Nikon TIRF illuminator, Nikon perfect focus system, a Nikon Plan Apo 100×/1.45 NA oil objective, and an iXon Ultra 897 EMCCD camera (Andor Technology) controlled with NIS Elements software (Nikon, Melville, NY).

Microscopy images were analyzed using Volocity (PerkinElmer) and ImageJ (National Institutes of Health, Bethesda, MD) (Schneider et al., 2012). Fluorescence images shown in the figures are maximum-intensity projections of image stacks with 0.4–0.6 µm spacing except where indicated. For intensity quantification, only cells within the central ∼75% of the imaging field were used to reduce interference from uneven illumination. To measure the global protein level in a whole cell using the fluorescence intensity, the polygon region of interest (ROI) tool in ImageJ was applied to trace cell boundaries. Then, the fluorescence intensity in WT cells with no fluorescent tag was used to deduct the background. For quantification of fluorescence intensity at the division site, a rectangular ROI1 (∼4 µm2) was drawn at the division site to measure the mean intensity. Another rectangular ROI2 approximately twice the size of ROI1 elongated along the cell long axis was drawn to calculate cytoplasmic background as described (Wu and Pollard, 2005; Zhu et al., 2013; Coffman and Wu, 2014). Tracking of vesicles moving toward the division site was performed manually using ImageJ. For vesicle colocalization analysis, the plot profile in both channels was analyzed using ImageJ, and the distance between the peak of profiles, which was considered the centroid of a vesicle, was calculated. If the distance was less than the vesicle radius, we considered the vesicles as colocalized. Data in the figures represent mean values ± SD unless otherwise described. Graphs were made in KaleidaGraph software, and the p values were calculated using a two-tailed Student's t test.

Electron microscopy

We used electron microscopy to investigate cell wall and septum morphology in WT (JW81), smi1-1, and 81nmt1-smi1 strains. Cells were grown in YE5S + thiamine medium exponentially for 48 h at 25˚C and then fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 M sodium phosphate, pH 7.4, and 0.1 M sucrose) for 1 h. The fixed cells were submitted to the Campus Microscopy and Imaging Facility at The Ohio State University, where the samples were further fixed with 1% osmium tetroxide, embedded in agarose block, dehydrated in alcohol, and embedded in Epon8 epoxy resin as described before (Hayat, 1986). Thin sections of 70–90 nm were cut with a Leica EM UC6 ultramicrotome. Sections were stained with uranyl acetate and lead citrate and imaged using a FEI Tecnai G2 Spirit transmission electron microscope at 80 kV (Watanabe et al., 1988; Hayat, 2000).

Coimmunoprecipitation and Western blotting

We carried out coimmunoprecipitation and Western blotting similarly to previously described methods (Cortes et al., 2012; Lee and Wu, 2012; Gerien et al., 2020). Lyophilized cells (200 mg) of each strain were ground into fine powder. For whole cell protein extracts, cell powder was dissolved with lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM NaCl, 0.5% Tween 20, 100 µM PMSF, and protease inhibitor [1 tablet/50 ml; Roche]), and proteins were separated by SDS–PAGE before Western blotting. For protein extracts from membrane enriched fractions, cell powder was dissolved with 2 ml lysis buffer without detergent (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM NaCl, 100 μM PMSF, and cocktail protease inhibitor). Then the cell lysates were centrifuged for two rounds at 4500 × g for 1 min and 16,000 × g for 1 h at 4°C, respectively. The pellet containing the membrane fraction from the second centrifugation was resuspended in 600 μl of immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 200 mM NaCl, 0.5% Tween 20, 100 μM PMSF, and cocktail protease inhibitor). Next, the membrane suspension was centrifuged (21,000 × g, 30 min, 4°C) and the supernatant was collected. The solubilized membrane proteins were incubated with 30 µl of protein G magnetic beads (Invitrogen) bound with polycolonal anti-GFP antibody (Novus Biologicals) at 4°C for 2 h. The beads were washed with 1 ml immunoprecipitation buffer three times and then resuspended in sample buffer.

Proteins were separated by 4–20% Tris-glycine gradient SDS–PAGE (Mini-PROTEAN; Bio-Rad) and transferred to Immobilon-P membrane (Millipore). The membrane was blotted to detect GFP- or 13Myc-tagged proteins with the corresponding antibodies (monoclonal anti-GFP; 1:1000; [Roche]; and monoclonal anti-Myc; 1:2000; [Roche]) and the enhanced chemiluminescence (ECL) detection kit (SuperSignal; Thermo Scientific). α-TAT1 (1:20,000 dilution) was used as a loading control (Woods et al., 1989).

Supplementary Material

Click here for additional data file. (21MB, pdf)
Click here for additional data file. (209.4KB, pdf)

Acknowledgments

We thank Mohan Balasubramanian, Kathy Gould, Sophie Martin, Thomas Pollard, and Takashi Toda for strains; the Stephen Osmani and Dmitri Kudryashov laboratories for equipment; the Campus Microscopy and Imaging Facility, Mass Spectrometry and Proteomics Facility, and Samantha Nusbaum and Tiffany Ko at The Ohio State University for technical support; and current and past members of the Wu laboratory for helpful discussions and suggestions. The Fusion Orbitrap instrument was supported by National Institutes of Health (NIH) grant S10 OD018056. The work was supported by the National Institute of General Medical Sciences of the NIH (grant R01 GM118746 to J.-Q.W.) and Pelotonia (Postdoctoral fellowships to L.V.G.L and S. Z and an Undergraduate fellowship to E.G.G). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or Pelotonia.

Abbreviations used:

GBP

GFP-binding protein

IDP

intrinsically disordered protein

PB

phloxin B

SPB

spindle pole body

TIRF

total internal reflection fluorescence

WT

wild type.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E21-04-0214) on December 15, 2021.

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