In Schizosaccharomyces pombe, septum formation is coordinated with cytokinetic ring constriction but the mechanisms linking these events are unclear. In this study, we explored the role of the cytokinetic ring component Fic1, first identified by its interaction with the F-BAR protein Cdc15, in septum formation. We found that the fic1 phospho-ablating mutant, fic1-2A, is a gain-of-function allele that suppresses myo2-E1, the temperature-sensitive allele of the essential type-II myosin, myo2. This suppression is achieved by the promotion of septum formation and required Fic1's interaction with the F-BAR proteins Cdc15 and Imp2. Additionally, we found that Fic1 interacts with Cyk3 and that this interaction was likewise required for Fic1's role in septum formation. Fic1, Cdc15, Imp2, and Cyk3 are the orthologs of the Saccharomyces cerevisiae ingression progression complex, which stimulates the chitin synthase Chs2 to promote primary septum formation. However, our findings indicate that Fic1 promotes septum formation and cell abscission independently of the S. pombe Chs2 ortholog. Thus, while similar complexes exist in the two yeasts that each promote septation, they appear to have different downstream effectors.
Cytokinesis is the final process in the cell cycle which creates two independent daughter cells. Many eukaryotic organisms use an actin-myosin structure known as the cytokinetic ring (CR) to mark the plane of cell division and to drive membrane ingression (reviewed in Cheffings et al., 2016; Mangione and Gould, 2019). In organisms with cell walls, such as Schizosaccharomyces pombe and Saccharomyces cerevisiae, the CR alone is insufficient for cytokinesis (Jochova et al., 1991; Munoz et al., 2013; Proctor et al., 2012; Ramos et al., 2019; Schmidt et al., 2002). These organisms require the formation of a septum coupled to CR constriction to drive cell abscission (Cortés et al., 2007, 2015; Jochova et al., 1991; Proctor et al., 2012; Schmidt et al., 2002).
Yeast septa are trilaminar structures composed of a primary septum flanked by secondary septa (Humbel et al., 2001; Wloka and Bi, 2012). In S. pombe and S. cerevisiae, CR constriction promotes septation perpendicular to the cell cortex (Cortés et al., 2002, 2007; Johnson et al., 1973; Roncero et al., 2016; Schmidt et al., 2002). In S. cerevisiae the chitin synthase Chs2 polymerizes N-acetylglucosamine to form the primary septum (Sburlati and Cabib, 1986; Shaw et al., 1991; Silverman et al., 1988). In contrast, in S. pombe it is the glucan synthases Bgs1 and Ags1 that polymerize linear-β(1,3)glucans and α(1,3)glucans, respectively, to form the primary septum (Cortés et al., 2002, 2007, 2015, 2012).
S. cerevisiae Chs2 and septum formation are stimulated by a protein complex within the CR named the ingression progression complex (IPC), comprised of the ingression protein Inn1, the F-BAR protein Hof1, and Cyk3 (Devrekanli et al., 2012; Nishihama et al., 2009; Sanchez-Diaz et al., 2008). Analogous proteins exist in S. pombe. Specifically, S. pombe Fic1, Cdc15/Imp2, and Cyk3 are the orthologs of Inn1, Hof1, and Cyk3, respectively (Demeter and Sazer, 1998; Fankhauser et al., 1995; Pollard et al., 2012; Roberts-Galbraith et al., 2009). Fic1 was identified in a yeast-two hybrid screen using the SH3 domain of Cdc15 as bait and directly interacts with Cdc15 and Imp2 (Ren et al., 2015; Roberts-Galbraith et al., 2009). S. pombe Cyk3 was identified based on sequence similarity to S. cerevisiae Cyk3 and has been found to co-immunoprecipitate with Fic1 (Bohnert and Gould, 2012; Roberts-Galbraith et al., 2009). However, it is unknown if these S. pombe proteins cooperate to promote primary septum formation similarly to the IPC.
We previously found that Fic1 is phosphorylated on two sites by multiple kinases (Bohnert et al., 2020). Preventing phosphorylation at these sites produces defects in the establishment of normal cell polarity. Here, we pursued the observation that the fic1 phospho-ablating mutant, fic1-2A, also suppressed the myo2-E1 temperature-sensitive allele of the essential type-II myosin Myo2 (Balasubramanian et al., 1998; Kitayama et al., 1997). The inability of myo2-E1 cells to form a functional CR to guide septum formation prevents cytokinesis and leads to cell death (Balasubramanian et al., 1998). Time-lapse microscopy showed that fic1-2A suppressed myo2-E1 by promoting septum formation and daughter cell abscission and that cells lacking fic1 exhibited significant delays in septation. We determined that the ability of fic1-2A to suppress myo2-E1 required its interactions with Cyk3, Cdc15, and/or Imp2 but not Chs2. This work revealed that S. pombe's IPC analogs interact to promote septum formation through a mechanism that is functionally divergent from the IPC in S. cerevisiae.
Fic1 phospho-ablating mutant suppresses myo2-E1
To determine if Fic1's phosphorylation state impacts cytokinesis, we took a genetic approach and probed interactions between fic1 phosphomutants and deletions or temperature-sensitive alleles of genes involved in actin dynamics (cdc12), septum formation (sid2, bgs1, and bgs4), and CR constriction (cdc4 and myo2) (Fig. S1A). From this screen we observed one significant interaction: fic1's phospho-ablating mutant, fic1-2A, suppressed myo2-E1 (Fig. 1A,B and Fig. S1A). myo2-E1 is a temperature-sensitive allele of the essential type-II myosin, myo2, that inhibits Myo2's activity and produces non-constricting CRs at the restrictive temperature (Balasubramanian et al., 1998; Palani et al., 2017, 2018). Without CR constriction, cell wall accumulates at the division site but does not form a septum (Balasubramanian et al., 1998; Palani et al., 2017, 2018; Ramos et al., 2019). Interestingly, fic1Δ did not suppress myo2-E1 and no genetic interaction was observed between fic1-2D and myo2-E1 (Fig. 1B,C). This suggests fic1-2A is a gain-of-function allele, but whether this gain in function is due to alterations to Fic1's phosphorylation state is unclear. The individual phospho-ablating fic1 mutants only partially suppressed myo2-E1, and none of the fic1 phosphomutants were temperature-sensitive (Fig. S1B,C). We then pursued the underlying mechanisms behind myo2-E1's suppression to gain insight into the cytokinetic roles of Fic1, a CR protein of enigmatic function.
fic1-2A cells exhibit similar CR dynamics compared to wild-type cells
We postulated that myo2-E1 suppression by fic1-2A could be achieved by altering CR dynamics. fic1-2A could provide additional time for proper glucan synthase localization by prolonging CR maturation and/or constriction. Glucan synthases are trafficked to the site of cell division and localize diffusely on the cortex (Cortés et al., 2002; Hoya et al., 2017; Katayama et al., 1999; Mulvihill et al., 2006; Ramos et al., 2019). As the CR constricts the glucan synthases coalesce into a ring concentric with the CR, Bgs1 is activated, and primary septum formation begins (Ramos et al., 2019). By providing additional time for glucan synthase ring formation by prolonging CR maturation and/or constriction fic1-2A could effectively promote septum formation. Alternatively, fic1-2A could increase the rate of CR constriction, which could allow fic1-2A to suppress myo2-E1 by restoring the contractile function of the CR and septum formation.
To test these possibilities, we performed live-cell time-lapse imaging at 25°C and 36°C of wild-type, fic1Δ, fic1-2A, and fic1-2D cells containing a CR marker, rlc1-mNG, to monitor CR dynamics and a spindle pole body (SPB) marker, sid4-GFP, to monitor mitotic progression (Chang and Gould, 2000; Naqvi et al., 2000). The timing of anaphase B onset was similar between all strains at both temperatures (Fig. 1D,F), as was the timing of CR assembly and CR maturation (Fig. 1E,G). The timing of CR constriction was similar between wild-type, fic1-2A, and fic1-2D cells at both temperatures indicating that Fic1 phosphostate does not appreciably affect CR dynamics (Fig. 1E,G). However, fic1Δ took longer, an average of 25.1±0.6 and 44.2±2.1 min at 25°C and 36°C, respectively, whereas wild-type cells took 22.2±0.5 and 19.3±0.6 min at 25°C and 36°C, respectively (Fig. 1E,G). Delayed CR constriction in fic1Δ but not fic1-2A suggests that prolonging CR constriction is not how fic1-2A suppresses myo2-E1. Rather, because the rate of CR constriction is linked to the rate of septum deposition (Proctor et al., 2012; Ramos et al., 2019), the delay in CR constriction of fic1Δ suggests that Fic1 promotes septation and that the fic1-2A allele may enhance this function.
fic1-2A myo2-E1 cells can complete cytokinesis
We next probed this possibility for Fic1 function that would be analogous to Inn1 in S. cerevisiae (Sanchez-Diaz et al., 2008). We performed time-lapse imaging at 36°C with wild-type, fic1-2A, myo2-E1, and fic1-2A myo2-E1 cells expressing the membrane marker LactC2-GFP, to monitor membrane ingression, and the SPB marker Sad1-GFP, to monitor mitotic progression (Curto et al., 2014; Hagan and Yanagida, 1995). The kinetics of septation were measured by timing membrane ingression and daughter cell abscission beginning from SPB separation at the onset of mitosis. The timing of anaphase B onset was similar between all genotypes (Fig. 2A,B). The initiation of membrane ingression was similar between wild-type and fic1-2A cells, averaging 14.9±0.4 and 16.2±0.3 min, respectively (Fig. 2A,C). However, both myo2-E1 and fic1-2A myo2-E1 cells exhibited delays in the initiation of membrane ingression compared to wild-type, averaging 28.4±1.0 and 28.8±1.9 min, respectively (Fig. 2A,C). Daughter cell separation was completed at similar times in the wild-type and fic1-2A cells, averaging 40.6±0.6 and 43.1±0.6 min, respectively (Fig. 2A,D). None of the myo2-E1 daughter cells separated, but 7 out of the 22 imaged fic1-2A myo2-E1 daughter cells took an average time of 117.9±20.2 min to separate (Fig. 2A,D). The ability of some fic1-2A myo2-E1 cells to complete membrane ingression and abscission is consistent with the idea that Fic1-2A enhances septum formation.
Fic1 directly interacts with Cyk3's SH3 domain
Because the involvement of Fic1 in promoting septation was reminiscent of the role of S. cerevisiae's IPC (Devrekanli et al., 2012; Nishihama et al., 2009; Sanchez-Diaz et al., 2008), we asked whether Fic1's interactions with Cdc15 and Imp2 were required for myo2-E1 suppression. Fic1 binds the SH3 domains of the F-BAR proteins Cdc15 and Imp2 (Roberts-Galbraith et al., 2009) through the P254,257 PxxP motif, and the fic1-P257A mutation, which disrupts Fic1's interactions with Cdc15 and Imp2 (Bohnert and Gould, 2012), prevented fic1-2A's suppression of myo2-E1 (Fig. 3A,B). These data suggest that Fic1's interaction with Cdc15 and Imp2 are required for fic1-2A's suppression of myo2-E1 and thus, Fic1's role in promoting septum formation.
We next asked if S. pombe Cyk3 was required for fic1-2A's suppression of myo2-E1. Indeed, cyk3Δ prevented fic1-2A from suppressing myo2-E1 (Fig. 3C). We then aimed to determine if Cyk3 bound Fic1 through an SH3-PxxP interface, similarly to Cyk3 and Inn1 in S. cerevisiae (Nishihama et al., 2009) (Fig. 3D). To test this, we generated recombinant Cyk3-SH3-GST and as a negative control, Cyk3-SH3-W43S-GST. Based on SH3 domain homology, the W43S substitution is predicted to disrupt Cyk3-SH3's ability to bind PxxP motifs (Saksela and Permi, 2012). Immobilized Cyk3-SH3-GST purified Fic1-FLAG3 from lysates of S. pombe arrested by cps1-191, a temperature-sensitive allele of bgs1, which allows CRs to form but prevents primary septum deposition (Liu et al., 1999), but Cyk3-W43S-SH3-GST did not (Fig. 3E). Finally, we found that Fic1-MBP directly bound Cyk3-SH3-GST indicating that Cyk3-SH3 directly binds Fic1 (Fig. 3F).
To verify that at least one of Fic1's 11 PxxP motifs was necessary for interaction with Cyk3's SH3 domain, we generated recombinant Fic1-MBP with every PxxP motif mutated to AxxA, referred to as Fic1-11AxxA-MBP. As predicted, Fic1-11AxxA-MBP did not bind Cyk3-SH3-GST or Cyk3-W43S-SH3-GST (Fig. S2A). To identify which PxxP motif was required for the interaction, we generated Fic1-MBP fusion proteins with each individual AxxA mutation. Fic1-P174,177A-MBP and Fic1-P176,179A-MBP exhibited reduced binding to Cyk3-SH3-GST compared to Fic1-MBP (Fig. 3G,H). Because these are distinct from the PxxP motif involved in binding Cdc15 and Imp2, Fic1 might be able to bind Cyk3 and Cdc15 or Imp2 simultaneously (Bohnert and Gould, 2012) to form an analog of the IPC. Indeed, molecular modeling using ColabFold predicted that Fic1 could simultaneously bind the SH3 domains of Cdc15 and Cyk3 (Fig. 3I and Fig. S2B) (Jumper et al., 2021; Mirdita et al., 2022).
Because the Fic1-2A mutant eliminates phosphorylation on T178, we wondered whether disrupting the prolines required for Cyk3 binding around T178 might alter Fic1's phosphorylation status. We were especially cognizant of this possibility because T178 can be phosphorylated in vitro by CDK, a proline-directed kinase (Bohnert et al., 2020). To examine whether these proline mutations affected Fic1 phosphorylation in vivo, we analyzed the gel mobilities of Fic1-P174,177A and Fic1-P176,179A. Fic1-FLAG3 migrates as four bands. The top band represents dual phosphorylation at T178 and S241, the two intermediate bands are singly phosphorylated at T178 or S241, and the fastest migrating form is not phosphorylated (Bohnert et al., 2020). As predicted, Fic1-P176,179A formed only two bands, consistent with a loss of T178 phosphorylation (Fig. S2C) (Bohnert et al., 2020). Interestingly, Fic1-P174,177A displayed the wild-type pattern of phosphorylation suggesting that it could be used to selectively test the role of Cyk3 binding to Fic1 in the suppression of myo2-E1 (Fig. S2C). We found that fic1-2A-P174,177A did not suppress myo2-E1 (Fig. S2D) and similarly, inactivation of the Cyk3-SH3 domain by the cyk3-W43S allele disrupted fic1-2A's suppression of myo2-E1 (Fig. S3A). Taken together, these results suggest Cyk3 is required for Fic1's roles in septum formation.
Cyk3's SH3 domain and transglutaminase-like domain (TLD) are required for Fic1's roles in septum formation
In addition to its SH3 domain, Cyk3 has a central TLD within a larger cysteine protease-like domain (CPD), which has been implicated in Cyk3’s function but not thought to have enzymatic activity (Fig. 3D) (Pollard et al., 2012). To determine if Cyk3's TLD is required for fic1-2A's suppression of myo2-E1, we inactivated the TLD through the previously established H577A mutation (Pollard et al., 2012) and found that this mutation also disrupted fic1-2A's suppression of myo2-E1 (Fig. S3B). To ensure that cyk3-W43S and cyk3-H577A were not disrupting fic1-2A's suppression by destabilizing Cyk3 or by preventing Cyk3's localization to the CR, we measured the fluorescence intensity of GFP fusion proteins Cyk3-W43S and Cyk3-H577A. We found that both alleles had similar CR and whole-cell fluorescence as Cyk3-GFP (Fig. S3C-E), demonstrating these alleles were stably expressed and localized normally. Additionally, we found that both Cyk3-W43S and Cyk3-H577A co-immunoprecipitated with Cdc15 from lysates of cells arrested in prometaphase by the nda3-KM311 cold-sensitive beta-tubulin allele as Cyk3 does (Fig. S3F) (Bohnert and Gould, 2012; Roberts-Galbraith et al., 2010).
Fic1 and Cyk3 function independently of Chs2
S. cerevisiae Cyk3's TLD stimulates Chs2 (Foltman et al., 2016; Nishihama et al., 2009). While S. pombe's septum lacks chitin, S. pombe does have an orthologous protein to S. cerevisiae's Chs2 with the same name but lacking catalytic activity (Horisberger et al., 1978; Martin-Garcia et al., 2003; Matsuo et al., 2004; Sietsma and Wessels, 1990). S. pombe Chs2 possibly influences septum formation indirectly because, like fic1Δ and cyk3Δ cells, chs2Δ cells display delays in CR constriction (Martin-Garcia and Valdivieso, 2006). In the cases of fic1Δ and cyk3Δ, CR constriction delays correlate with a postponement in the onset of bipolar growth, also known as new end take-off (NETO), and a transition to invasive pseudohyphal growth (Bohnert and Gould, 2012). If Chs2 acts downstream of Fic1 and Cyk3 we would expect that chs2Δ cells to also exhibit NETO defects and invasive pseudophyphal growth. We analyzed bipolar growth establishment in chs2Δ, fic1Δ, cyk3Δ, and combination fic1Δ chs2Δ, cyk3Δ chs2Δ, and fic1Δ cyk3Δ chs2Δ mutants and found that interphase cells of each indicated genotype had an increase in cells growing from only one end (monopolar) compared to wild-type (Fig. 4A). However, chs2Δ cells did not exhibit polarity defects at the time of septation or invasive pseudohyphal growth (Fig. 4B,C). Further, deletion of chs2 did not disrupt fic1-2A's suppression of myo2-E1 and surprisingly, chs2Δ independently suppressed myo2-E1 (Fig. 4D). These data together indicate that Chs2 does not act downstream of Fic1 and Cyk3 in septation. In accord, ColabFold did not predict an interaction between Cyk3's CPD and Chs2 in S. pombe, and even the predicted interaction between Cyk3's CPD and Chs2 in S. cerevisiae was weak (Fig. 4E-H) (Jumper et al., 2021; Mirdita et al., 2022).
Fic1 is a CR component that supports proper CR constriction and disassembly, which influences growth polarity establishment in the subsequent cell cycle (Bohnert and Gould, 2012; Roberts-Galbraith et al., 2009). Fic1's role in cell polarity requires dynamic phosphorylation of two residues within its proline-rich C-terminus, a region that localizes Fic1 to the CR and supports it interactions with the SH3 domains of Cdc15 and Imp2 (Bohnert and Gould, 2012; Roberts-Galbraith et al., 2009). We presented evidence here to suggest that Fic1 promotes septum formation. This was revealed by the ability of the fic1-2A phospho-ablating allele to suppress myo2-E1. This suppression required Fic1's interactions with the SH3 domains of Cdc15 and/or Imp2 as well as a newly defined interaction with Cyk3's SH3 domains. These interactions were reminiscent of the IPC in S. cerevisiae, which promotes septum formation through Chs2 (Devrekanli et al., 2012; Nishihama et al., 2009; Sanchez-Diaz et al., 2008). While the interactions of orthologous proteins in S. pombe and S. cerevisiae were similar, we found that the Chs2 was not a downstream effector of the S. pombe protein network.
While these studies highlight Fic1's role in promoting septum formation, its downstream effectors remain elusive. Our finding that Chs2 is not the downstream effector aligns with previous findings demonstrating that the cell walls of vegetative S. pombe cells lack chitin (Horisberger et al., 1978; Sietsma and Wessels, 1990). Instead, glucan polymers comprise the septum, which makes the glucan synthases attractive potential downstream effectors to study (Cortés et al., 2002, 2007, 2015, 2012). We attempted to determine if Fic1's role in septation involved refining the localization of the glucan synthases at the site of cell division by performing time-lapse imaging of cells with GFP fusions of Bgs1, Bgs3, Bgs4, and Ags1 in wild-type, fic1-2A, myo2-E1, and fic1-2A myo2-E1 cells at myo2-E1's restrictive-temperature. Unfortunately, these fusion proteins disrupted fic1-2A's suppression of myo2-E1. It is likely that the glucan synthase fusion proteins are slightly hypomorphic and that without full function of these cell wall enzymes fic1-2A is unable to suppress myo2-E1.
While we have considered the idea that Fic1 and Cyk3 promote septum formation through the glucan synthases, an anonymous reviewer suggested that Fic1 and Cyk3 may promote septum formation by stimulating the exocyst complex given that there are two-stages of CR constriction, with the second more-rapid phase requiring exocyst function and the activation of the RhoGTPase Cdc42 by the RhoGEF Gef1 (Ramos et al., 2019). It is possible that fic1-2A promotes the initiation of the second stage of constriction, which could allow the first stage of constriction to be bypassed. It would therefore be interesting to probe for genetic interactions between the fic1 phosphomutants, Gef1, and the exocyst complex to determine if Fic1 is involved in either of these pathways.
In conclusion, our results suggest that mutating the two Fic1 phosphorylation sites to alanine enhances Fic1's normal role in septation to allow myo2-E1 suppression. Whether this is due to preventing Fic1 phosphorylation or changing Fic1 structure remains to be determined. However, Fic1's interactions with Cyk3, Cdc15, and/or Imp2 are required for this function and molecular modeling suggests Fic1 could simultaneously bind the SH3 domains of Cyk3 and Cdc15/Imp2 to form a complex similar to the S. cerevisiae IPC. Despite similar interactions to S. cerevisiae's IPC, these proteins promote septum formation independent of Chs2 and it will be interesting to determine how they influence this critical aspect of fission yeast cell division.
MATERIALS AND METHODS
S. pombe strains utilized in this study (Table S1) were cultured in yeast extract (YE) media (Moreno et al., 1991). Glutamate media was used for crosses (Moreno et al., 1991). fic1, cyk3, sid4, rlc1, and sad1 were tagged endogenously at the 3′ end of their open reading frames (ORFs) with FLAG3:kanR, GFP:kanR, mNG:hygR, V53:hygR, and/or mCherry:natR using pFA6 cassettes as previously described (Bahler et al., 1998). G418 (100 μg/ml; Sigma-Aldrich, St. Louis, MO, USA), Hygromycin B (125 µg/mL; Invitrogen, Waltham, MA, USA), and Nourseothricin (125 µg/mL; Gold Biotechnology St. Louis, MO, USA) in YE media was used for selecting kanR, hygR, and natR cells, respectively. mNG, a YFP derivative from the lancelet Branchiostoma lanceolatum, was selected for imaging experiments because of its superior brightness (Shaner et al., 2013; Willet et al., 2015). A lithium acetate transformation method (Keeney and Boeke, 1994) was used for introducing tagging sequences, and endogenous integration of tags were verified by whole-cell PCR and/or microscopy. Introduction of tagged loci into other genetic backgrounds was accomplished using standard S. pombe mating, sporulation, and tetrad-dissection techniques. Fusion proteins were expressed from their endogenous locus under control of their native promoter unless otherwise indicated. For serial-dilution growth assays, cells were cultured in liquid YE at 25°C in a shaking incubator, four 1:10 serial dilutions starting at 1.5×106 cells/ml were made, 2 µl of each dilution was spotted on YE agar, and cells were grown at the specified temperatures for 3-5 days. All spot assays were performed in triplicate and representative images are shown.
Mutants of fic1 were expressed from the endogenous loci. To make fic1 mutations, fic1+ gDNA with 500 bp 5′ and 3′ flanks was inserted between BamHI and PstI sites of pIRT2 (Bohnert and Gould, 2012) and site-directed mutagenesis was used to introduce the desired mutations, which were confirmed by DNA sequencing. fic1::ura+ was transformed with these pIRT2-fic1 constructs, and stable integrants resistant to 1.5 g/L 5-fluoroorotic acid (5-FOA) (Thermo Fisher Scientific, Hampton, NH, USA) were isolated. The correct insertion site and mutations were confirmed by whole-cell PCR and DNA sequencing.
cyk3 strains were made in a similar manner but pIRT2-cyk3 (W43S, H577A, W43S, H577A) mutant plasmids were constructed from cyk3 cDNA with 500 bp 5′ and 3′ flanks to allow these cyk3 alleles to be verified by whole-cell PCR once integrated.
Invasive growth assays
To assay pseudohyphal invasive growth, 5 μl containing a total of 105 cells were spotted on 2% YE agar and incubated at 29°C for 20 days. Colonies were subsequently placed under a steady stream of water and surface growth was wiped off using a paper towel, as described previously (Pohlmann and Fleig, 2010; Prevorovsky et al., 2009).
Live-cell imaging of S. pombe cells were acquired using one of the following: (1) a personal DeltaVision microscope system (Leica Microsystems, Wetzlar, Germany) that includes an Olympus IX71 microscope, 60× NA 1.42 PlanApo and 100× NA 1.40 UPlanSApo objectives, a pco.edge 4.2 sCMOS camera, and softWoRx imaging software or (2) a Zeiss Axio Observer inverted epifluorescence microscope with Zeiss 63X Oil (1.46 NA), a Axiocam 503 monochrome camera (Zeiss), and captured using Zeiss ZEN 3.0 (Blue edition) software. All cells were in log phase growth before temperature-sensitive shifts and/or live imaging. Time-lapse imaging was performed using an ONIX microfluidics perfusion system (CellASIC ONIX; EMD Millipore, Burlington, MA, USA). A suspension of 50 µl of 40×106 cells/ml YE was loaded into Y04C plates for 5 s at 8 psi. YE media was flowed through the chamber at 5 psi throughout imaging.
Anaphase B onset was defined as the period from the separation of the SPBs to the initiation of SPB segregation towards opposite cell poles. CR assembly was defined as the period from the separation of the SPBs to the coalescence of the cytokinetic nodes into a clearly defined ring. CR maturation was defined as the period from the completion CR assembly to the initiation of CR contraction. CR constriction was defined as the period from CR contraction to the disappearance of the rlc1-mNG from the site of division.
Intensity measurements were made from non-deconvolved summed Z-projections of the images processed through ImageJ software (Schneider et al., 2012). For all intensity measurements, the background was subtracted by selecting a region of interest (ROI) in the same image in an area free of cells. The background raw intensity was divided by the area of the background, which was multiplied by the area of the measured object. This number was then subtracted from the intensity measurement of that object. Max intensity Z projections are shown in representative images.
To visualize birth scars by Calcofluor staining, cells were washed in PBS and then resuspended in PBS containing 5 µg/ml Calcofluor and allowed to incubate on ice for 30 min. Cells were then washed three times in PBS and images were acquired. Using the proximity of birth scars to cell ends, growth/morphology was categorized as one of the following: monopolar (i.e. growth on one end), bipolar (i.e. growth on both ends), monopolar and septated, or bipolar and septated. All cells stained with Calcofluor were grown to log phase at 25°C.
Cells were lysed by bead disruption in NP-40 buffer in denaturing conditions as previously described (Gould et al., 1991). Immunoblot analysis of cell lysates and immunoprecipitates was performed using anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO, USA), anti-PSTAIR Cdc2 (Sigma-Aldrich, St., Louis, MO, USA), anti-GFP (Roche, Indianapolis, IN, USA), or anti-GFP (VUIC9H4) antibodies or serums raised against GST-Cdc15 (amino acids 1–405; VU326; Cocalico Biologicals, Stevens, PA, USA) as previously described (Bohnert et al., 2009). Cyk3-SH3-GST (aa1-66), Fic1-MBP, GST-Cdc15-SH3 (aa867-927), and GST-Imp2-SH3 (aa608-670) recombinant proteins and their variants were purified from E. coli using standard biochemistry techniques. In vitro binding assays were performed in 20 mM Tris (pH 7.4) and 150 mM NaCl and allowed to be incubated at 4°C for 1 h while nutating. The beads were washed three times with reaction buffer before performing SDS-PAGE and Coomassie staining. Affinity purifications using bead bound Cyk3-SH3-GST recombinant proteins and S. pombe lysate were performed by lysing cells in Cyk3 lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.5% NP-40, 0.1% SDS, 1 mM DTT, 1 mM PMSF, 1.3 mM Benzamidine, and cOmplete protease inhibitors (Roche, Indianapolis, IN, USA). S. pombe lysates and Cyk3-SH3-GST recombinant proteins were incubated at 4°C for 1 h while nutating. The bead bound recombinant proteins were washed three times in the lysate buffer before SDS-PAGE and immunoblotting or Coomassie staining.
We are grateful to Yolanda Sánchez for providing strains. We thank Alaina Willet, Sierra Cullati, Kazutoshi Akizuki, and Jun-Song Chen for critical reading of the manuscript.
Conceptualization: A.M.R., K.A.B., K.L.G.; Validation: A.M.R., K.A.B., K.L.G.; Formal analysis: A.M.R., K.A.B.; Investigation: A.M.R., K.A.B.; Resources: K.L.G.; Data curation: A.M.R., K.A.B.; Writing - original draft: A.M.R., K.L.G.; Writing - review & editing: A.M.R., K.A.B., K.L.G.; Visualization: A.M.R., K.A.B.; Supervision: K.L.G.; Project administration: K.L.G.; Funding acquisition: K.L.G.
A.M.R. was supported by a National Institutes of Health grant (T32 GM008554). This work was supported by National Institute of General Medical Sciences grant (R35 GM131799) to K.L.G. Deposited in PMC for immediate release.
All relevant data can be found within the article and its supplementary information.
The authors declare no competing or financial interests.