Abstract
The septin scaffold recruits and organizes the actomyosin ring (AMR) components, thus ensuring faithful cytokinesis. The septin-associated kinases - Elm1, Gin4, Hsl1, and Kcc4 are believed to stabilize the septins at the bud neck, but the underlying mechanisms are largely unknown. Here, we present a comprehensive, quantitative analysis of these four septin regulatory kinases and reveal major roles for Elm1 and Gin4 in septin stability. We find that Elm1 and Gin4 play an overlooked role in actomyosin ring organization and constriction. We report that Gin4 kinase directly interacts with F-BAR protein Hof1 via its C-terminal membrane-binding domain and may be involved in proper organization and anchoring of AMR component Hof1 at the bud neck, representing an unappreciated mode of regulation of cytokinesis by the septin kinase network. We also show that Gin4 controls septin organisation and AMR constriction in a kinase-independent manner similar to Elm1. We have also performed an extensive GFP-GBP-based tethering screen in Δelm1 and Δgin4 cells and found an important role for Hsl1 in maintaining septin organisation and cell shape in coordination with Elm1, Gin4, and Kcc4. Furthermore, our data indicate that Hsl1 acts downstream of Elm1, with its membrane-binding KA1 domain being critical for its function. Together, these findings reveal new insights into modes of cytokinesis regulation by kinases Gin4 and Elm1 and highlight a redundant role for Hsl1 in controlling septin organization and cytokinesis, revealing the in-built adaptability of the septin kinase network in S. cerevisiae.
Introduction
Septins are an evolutionarily conserved family of GTPases, although recent studies have revealed that some septin proteins have lost the ability to hydrolyze bound GTP during evolution1,2. These filament-forming proteins assemble into higher-order cytoskeletal structures, such as filaments, rings, bundles, gauzes, etc., on the membrane3–7. Septin structures act as scaffolds for assembling and organizing various proteins required for cytokinesis and cell division across organisms8,9. Septins act as a diffusion barrier at the cell division site in fungi and animals, contributing to contractile actomyosin ring (AMR) assembly, maturation, and constriction during cytokinesis10–13. However, studies have also highlighted that redundant mechanisms cooperatively regulate AMR components, suggesting that the septin diffusion barrier is not an essential requirement for normal cytokinesis9,14. The budding yeast Saccharomyces cerevisiae is an excellent model for studying the relationship between septins and cytokinesis. S. cerevisiae expresses five mitotic septins: Cdc3, Cdc12, Cdc10, Cdc11 and Shs115–19, which assemble into octameric complex to form filaments or septin rods that eventually organize into diverse higher-order architectures on the membrane3,20,21.
The presence of Cdc11 and Shs1 at the terminal positions of the octameric unit determines the type of higher-order septin structures in budding yeast, with Cdc11 promoting septin bundling and Shs1 facilitating the formation of septin rings22,23. The septin filaments span the mother bud neck as a stable “hourglass” structure, providing a scaffold for various proteins to assemble into an actomyosin ring7,9,10. The hourglass septin scaffold is remodeled into double rings upon mitotic exit and cytokinesis onset24–26. This allows AMR to anchor properly to the membrane, leading to normal constriction and centripetal septum deposition between the dividing cells10,27–30. The exact molecular events that regulate the hourglass-to-double-ring (HDR) transition are still not known, and the mitotic exit network (MEN) is believed to play a crucial role in initiating this transition25,26.
Septin-associated kinases regulate the dynamics of septins by associating with the septin hourglass structure from the G1 phase and leaving the bud neck at the time of the HDR transition31. Septin-associated kinases are one of many key candidates believed to drive the drastic septin rearrangement at the onset of cytokinesis32,33. Four major conserved septin-associated kinases – Elm133–36, Gin432,37–43, Hsl144–49, and Kcc450,51 are thought to have distinct and common regulatory effects on septin stability and organisation32,33,52–54. Despite their involvement in stabilizing septins, a comprehensive understanding of their crosstalk and coordinative effects on septin organisation and cytokinesis in S. cerevisiae is just beginning to be understood32.
In this study, through systematic analysis of these kinases on septin stability and actomyosin ring-mediated cytokinesis, we find a role for the Elm1 and Gin4 kinases in maintaining AMR organisation and constriction. The AMR components are disorganized upon loss of Elm1 and Gin4, and AMR displays asymmetric constriction, leading to abnormal cytokinesis defects. We show that the control of Gin4 over AMR organisation is independent of its kinase activity and may rely directly on its interaction with the septin-binding protein Hof1, representing a direct crosstalk between the septin kinase network and cytokinetic machinery. We find that artificial tethering of Hsl1 alone or in combination with Gin4/Kcc4 to the bud neck rescues morphological defects exhibited by Δelm1 cells. These results suggest an inherent redundancy in controlling septin architecture and cell polarity in budding yeast where Hsl1 kinase can compensate for the role of Elm1. Overall, our work unveils 1) a kinase-independent role of Gin4, 2) a novel physical association between the septin-associated kinase Gin4 and F-BAR domain containing AMR protein Hof1, involved in direct regulation of AMR organization and cytokinesis, 3) an previously uncharacterized role for Hsl1 kinase in regulating septin scaffold and AMR dynamics in the absence of major kinases Elm1 and Gin4 to ensure proper cell cycle progression and cytokinesis in S. cerevisiae.
Results
Dynamics of Septin-associated kinases and their defects during the cell cycle
S. cerevisiae contains four major septin-regulatory kinases known to localize to the mother-bud neck – Elm1, Gin4, Hsl1, and Kcc4, contributing to septin stability and morphogenesis checkpoint during the cell cycle33,52. Three of these kinases, commonly referred to as Nim1-related kinases: Gin4, Hsl1, and Kcc4, consist of membrane-binding Kinase-Associated (KA1) domain at the C-terminus (Fig. 1A). The KA1 domain shows affinity to acidic phospholipids (phosphatidylserine) on the plasma membrane and is a requisite for bud neck targeting40,42,44,50. To understand their temporal kinetics and localized accumulation at the bud neck septin scaffold, we performed time-lapse imaging of GFP-tagged kinases in cells co-expressing the septin Cdc3-mCherry. All four kinases showed gradual recruitment to the septin hourglass scaffold starting from the G1 phase, reaching their peak intensity during M-phase (Fig. S1A-S1D, Movie-S1). Elm1 and Kcc4 were present at lower abundance at the bud neck (Fig. S1A, S1D) compared to the higher abundance of Gin4 and Hsl1, as observed from their fluorescence intensities (Fig. S1B, S1C). At the onset of cytokinesis, the septin hourglass scaffold undergoes remodelling into a double-ring structure, and we observed that all four kinases left the bud neck just before or during the HDR transition (Fig. S1E, S1F, Movie-S1). Interestingly, Elm1 and Kcc4 signal intensity started decreasing ∼4-6 mins before the HDR transition (Fig. S1E, S1F). In contrast, Gin4 and Hsl1 signal intensity started decreasing just before or during the HDR transition, suggesting them to be a possible trigger for septin remodelling (Fig. S1E, S1F). These observations suggest a defined temporal order of removal for these kinases from the bud neck, which may depend on the interdependency of their localizations on one another. It is also worth noting that since Gin4 and Hsl1 leave the hourglass septin scaffold very close to the HDR transition (Fig. S1E, S1F), they may act as molecular signals for the transition and a trigger for crosstalk of cytokinesis. Overall, the above data shows that the four septin kinases are majorly associated with the hourglass septin scaffold with similar recruitment but distinct removal kinetics, suggesting that they may play both overlapping and non-overlapping roles in septin hourglass stability and HDR transition.

Septin organization and actomyosin ring dynamics is defective in Δelm1 and Δgin4 cells.
(A) Domain-level architecture of Elm1, Gin4, Kcc4 and Hsl1. The KA1 domain represents the membrane-binding domain. (B) Representative time-lapse images of Cdc3-mCherry in kinase deletion strains (t0=spindle breakpoint). (C) Plot showing the kinetics of Cdc3-mCherry in indicated strains of (B) (n>25 cells/strain). (D) Representative time-lapse images of Inn1-mNG in wildtype (n=21), Δelm1 (n=13) and Δgin4 cells (n=23). (E) Quantification for Inn1-residence in indicated strains of (D). ****: p<0.0001, ns: p>0.05 (F) Plot of normalised signal intensity of Inn1-mNG in indicated strains of (D); a population of cells showing asymmetric constriction in Δelm1 and Δgin4 is plotted (t0=spindle breakpoint).
Septin-associated kinases Elm1 and Gin4 majorly regulate septin stability, HDR transition, and cell morphology
Septin rearrangement is believed to be partly governed by post-translational modifications like phosphorylation, ubiquitination, acetylation and sumoylation31,37,55–60. Previous studies have extensively highlighted the regulation of kinase-mediated modification on septin remodelling33,53,61,62. Septin-associated kinases are believed to play an essential regulatory role in stabilizing the septin hourglass structure and maintaining a timely transition to the double-ring structure upon mitotic exit in S. cerevisiae31–34,52. To understand the extent to which different septin kinases affect cell physiology and septin organisation, we characterized cell morphology, cell growth, and septin organisation in wildtype cells and cells harboring deletions for these septin-regulatory kinases – Elm1, Gin4, Hsl1, and Kcc4. We observed that cells exhibited elongated and clumped morphologies in the Δelm1 and Δgin4 backgrounds, respectively, at all temperatures tested (Fig. S2A, S2B). Δelm1 cells displayed a more severe defect, with nearly 100% of cells, whereas Δgin4 cells showed defects in over 70% of the population (Fig. S2A, S2B). Δhsl1 cells showed a fraction of cells with mild morphological defects, which increased with temperature, while no alterations in morphology were observed in Δkcc4 cells (Fig. S2A, S2B). Spot assay analysis showed a similar trend where Δelm1 and Δgin4 showed growth sensitivity at all temperatures tested, while Δhsl1 exhibited growth sensitivity at higher temperatures and Δkcc4 cells grew identical to wildtype (Fig. S2C).
The core septin Cdc3 fused to mCherry was used to visualize the septin structures during the cell cycle77. Cdc3-mCherry localized to the emerging bud site in wildtype, Δelm1, and Δgin4 cells but was also seen to be mislocalized to the bud tip in ∼100% Δelm1 and ∼ 70% Δgin4 cells (Fig. S2D, S2E, Movie-S2). In wildtype cells, septin formed an hourglass structure at the bud neck, which subsequently split into double rings coinciding with spindle breakage and mitotic exit (Fig. 1B). The septin hourglass architecture appeared abnormal in Δelm1 and Δgin4 cells (Fig. 1B, 1C) and also exhibited defective HDR transition (Fig. 1B, 1C, Movie-S2). Δelm1 cells showed a stronger defect in septin organisation than Δgin4 cells (Fig. 1B, 1C, S2D, S2E), suggesting a more prominent role for Elm1 in maintenance of septin filament architecture, consistent with previous literature33. Cells lacking the kinases Hsl1 and Kcc4 showed normal septin organisation and HDR transition similar to wildtype cells (Fig. 1B, 1C, S2D, S2E). However, approximately 15% of Δhsl1 cells displayed septin mislocalisation to the bud tip (Fig. S2D, S2E), indicating that Hsl1 and Kcc4 may also play minor or supporting roles in septin dynamics. These results demonstrate the requirement of Elm1 and Gin4 kinases for proper septin arrangement and dynamics at the cell division site.
Elm1 and Gin4 regulate actomyosin ring organisation and constriction
Next, we investigated the roles of the kinases Elm1 and Gin4 to understand their impact on AMR organisation and constriction. We used Inn1, a plasma membrane ingression protein that couples AMR constriction with primary septum (PS) formation, as a marker for AMR constriction63,64. Inn1-mNeonGreen (mNG) arrived at the bud neck just after spindle breakage and constricted symmetrically as part of the AMR with a residence time of ∼5 minutes and eventually disappeared from the bud neck in wildtype cells (Fig. 1D, 1E). In Δelm1 and Δgin4 cells, Inn1-mNG was recruited normally but displayed an abnormal constriction pattern and increased duration of constriction (Fig. 1D-1F, Movie-S3). On the other hand, in Δhsl1 and Δkcc4 cells, Inn1-mNG recruited normally and constricted, similar to wildtype (Fig. S3A-S3C, Movie-S3). Inn1 and Cyk3 function parallelly to activate a primary component of PS, Chitin Synthase II (Chs2)63,65–67. Thus, we monitored the constriction pattern and dynamics of Chs2-mNG and observed an increased residence time for Chs2 at the bud neck in Δgin4 cells (Fig. S3D-S3F). The C-terminal domain of Inn1 is necessary for its bud neck localisation and interacts with both F-BAR protein Hof1, which senses and induces membrane curvature63,68, and the Cyk3 protein63. The tripartite complex Hof1-Inn1-Cyk3, referred to as the Ingression Progression Complex (IPC), eventually activates Chs2 for functional PS formation63,64,69. Our observations suggest that loss of septin kinases Elm1 and Gin4 may result in an improper organisation and constriction dynamics of the AMR and possibly subsequent IPC complex formation. To test for AMR/IPC organization, we assessed the organization of the F-BAR protein Hof1, which is known to be associated with septin hourglass and transit to AMR during split ring trigger68–74. Hof1 deletion has been shown to exhibit asymmetric AMR constriction with improper chitin deposition during PS synthesis63. We then analyzed the residence time of Hof1-mNG at the bud-neck after the spindle breakpoint during cytokinesis. Hof1-mNG exhibited a residence time of 10-12 minutes, significantly increasing to >15 minutes in both Δelm1 and Δgin4 cells (Fig. 2A,2B, Movie-S4).In wildtype cells, Hof1-mNG appeared as a uniform ring at the bud neck, while in Δelm1 and Δgin4 cells, Hof1-mNG signal was present as a disconnected, broken ring in more than 60% of the cells (Fig. 2C-2E). These observations suggest that Elm1 and Gin4 regulate the proper spatial assembly of different AMR components to ensure robust cytokinesis. The effect on AMR dynamics observed in Δelm1 and Δgin4 cells may be an indirect effect caused by defects in septin organization. However, a previous study has shown that the disruption of the septin scaffold does not affect cytokinesis, suggesting that Elm1 and Gin4 could regulate AMR organization directly and constriction independently of their effects on septin organization9,14. Together, these results reveal an unappreciated role for Elm1 and Gin4 in AMR assembly, organization, and constriction, opening avenues for future investigation.

Gin4 regulates Hof1 organisation and dynamics by interacting with N-terminal membrane binding F-BAR domain.
(A) Representative time-lapse images of Hof1-mNG in wildtype, Δelm1 and Δgin4. (B) Quantification for Hof1-residence after spindle breakpoint in indicated strains of (A). **: p<0.01, ns: p>0.05 (C) Representative images showing Hof1-mNG organisation at the bud neck in indicated strains of (A). Scale bar-5µm. (D) Line-scan profile of (C). (E) Bar graph showing % phenotype for indicated strains of (A) (N=3 and n>200/strain). ****: p<0.0001, ns: p>0.05 corresponds to population of cells with disorganised Hof1 at the bud neck. (F) Representative images for Yeast Two-Hybrid assay depicting the interaction of full length Gin4, Gin4-KID and Gin4-KA1 with full length Hof1. (G) Images of Yeast Two-Hybrid showing the interaction between Gin4-KA1 and N-terminal F-BAR containing domain of Hof1. (H) Immunoblot representing the in vitro binding assay for the interaction between 6xHis-bdSUMO-Gin4KA1 and GST-Hof1N (1-350) fragments. (I) Quantification of (H). ****: p<0.0001 (J) Representative model for the possible mode of regulation between Gin4 and Hof1 at the plasma membrane.
Gin4 directly interacts with the F-BAR domain of AMR protein Hof1 to regulate proper cytokinesis
To understand how Elm1 and Gin4 kinase affect AMR dynamics, we performed a Yeast Two-Hybrid screen to find novel interacting partners at the bud neck . We found that the Gin4 fragments, KID (290-1142aa) and KA1 (1003-1142aa) but not the full-length protein, interacts with the full length Hof1 protein (Fig. 2F). Further, we found that the N-terminal Hof1 fragment (1-350aa) which contains its F-BAR domain and not the other fragments, interact with the Gin4-KA1 domain (Fig. 2G, S3I). In contrast neither full length Elm1 nor its C-terminal fragment (421-640aa) showed any interaction with Hof1 (Fig. 2F). To further validate the interaction between Gin4KA1 and Hof1N (1-350) domains, we purified 6His-bdSUMO-Gin4-KA1(1003-1142 aa) and GST-Hof1-N-terminus(1-350 aa) fragments from E. coli and performed an in vitro-binding assay. Strikingly, our results revealed that the N-terminal, membrane binding F-BAR containing domain of Hof1 (1-350 aa) interacts strongly with the C-terminal membrane-binding, KA1 domain of Gin4 (1003-1142 aa) (Fig. 2H, 2I). Overall, these results reveal a direct physical link between the Gin4 kinase and AMR component Hof1, suggesting a potentially novel and direct mode of regulation of cytokinesis by the septin kinase network in S. cerevisiae. The involvement of the membrane-binding interfaces of these proteins also suggests a possible coordinative mechanism where Gin4 may regulate Hof1 to control its proper assembly and anchoring to the membrane prior to cytokinesis (Fig. 2J). This is supported by the disorganized and irregular Hof1 ring structure observed in Δgin4 cells (Fig. 2C). Moreover, Δgin4 cells show asymmetric constriction (Fig. 2A) which has been reported earlier to be a characteristic phenotype of Δhof1 cells63. Thus, our data combined with these lines of evidence strongly suggest that Gin4 exerts direct control over AMR dynamics via Hof1.
Gin4 exerts its control over actomyosin ring components in a kinase-independent manner
Elm1 kinase activity is essential for its localisation to the bud neck but is dispensable for its role in septin stability33. Bni5 is a myosin-recruiting protein that stabilizes and crosslinks septin filaments by binding to the terminal septin Cdc1175–78. Elm1 regulates the septin hourglass structure and stability through its binding partner, Bni533. Therefore, we asked if Gin4 kinase activity is required for its functions in septin regulation and cytokinesis. To address this, we complemented Δgin4 cells with the kinase-dead gin4-K48A32,37,79 allele (henceforth referred to as Gin4KD), integrated into the leu2 locus. Interestingly, Gin4KD localized to the bud neck and complemented the abnormal septin organisation (Fig. 3A, 3B) and AMR constriction dynamics (Fig. 3C-3E) observed in Δgin4 cells. Additionally, Gin4KD restored Hof1-misorganisation defects when expressed in Δgin4 cells (Fig. S3G-S3H). Gin4 kinase stabilizes septins by phosphorylating its substrate, Shs143. Hof1 has been shown to exhibit synthetic lethality with Gin4, possibly by regulating septins in a parallel pathway71. We, therefore, complemented Gin4FL and Gin4KD in cells carrying Δgin4 Δhof1 double deletion (Fig. S3J). Interestingly, Gin4KD rescued the synthetic lethality, similar to Gin4FL, suggesting that the kinase activity of Gin4 is not required to restore viability in the absence of Hof1 (Fig. S3J). These findings provide strong evidence to support the idea that Gin4 kinase activity is majorly dispensable for its localisation and functions in septin organization and cytokinesis.

Artificial tethering of Gin4-GFP to the bud neck, along with its related kinase Hsl1, restores functionality in Δelm1 cells in a kinase-independent manner.
(A) Representative images showing septin defects in Δgin4 and rescue by Gin4FL-GFP and Gin4KD-GFP expressed under pTEF promoter -scale bar-5µm. (B) Quantitation for septin mislocalisation shown in (A) (The n>30 cells/strain). (C) Representative time-lapse images of Inn1-mNG kinetics in Δgin4 and rescue by Gin4FL and Gin4KD (t0=spindle breakpoint). (D) Quantification for Inn1-residence in indicated strains of (C). ****: p<0.0001, ns: p>0.05 (E) Plot of normalised signal intensity of Inn1-mNG in indicated strains of (C); a population of cells showing asymmetric constriction in Δgin4-Empty vector is plotted (t0=spindle breakpoint; n>20 cells/strain). (F) A diagrammatic representation of GFP-GBP screen for artificial tethering of Protein of interest to the bud neck, along with its binding partners tagged with GBP. (G) Representative images showing artificial tethering of Gin4-GFP with Hsl1-GBP in Δelm1. Scale bar-5µm. (H) Bar graph showing % phenotype for indicated strains of (G) (N=3, n>350 cells/strain). **: p<0.01 (I) Representative images showing Gin4FL-GFP and Gin4KD-GFP tethering with Hsl1-GBP in Δelm1Δgin4. Scale bar-5µm. (J) Bar graph showing % phenotype for indicated strains of (I) (N=3, n>300 cells/strain). ****: p<0.0001, ns: p>0.05 corresponds to population of cells with round morphology. (K) Representative images showing artificial tethering of Gin4-GFP with Hsl1-Δka1-GBP in Δelm1. Scale bar-5µm. (L) Bar graph showing % phenotype for indicated strains of (K) (N=3, n>350 cells/strain). ****: p<0.0001, ns: p>0.05 corresponds to population of cells with round morphology.
Hsl1 kinase can partially compensate for Elm1 function upon forced tethering to the bud neck
Marquardt et al. (2024) have shown that Gin4 and Elm1 exhibit reciprocal regulation during different cell cycle stages, and their localisation to the bud neck is interdependent32,80. The study also suggests that Gin4 localisation to the bud neck is hampered during late cell cycle stages in Δelm1 cells32. We independently corroborated these results and observed that Gin4 was mislocalised to the bud cortex during bud emergence and exhibited defective localisation to the bud neck during the onset of cytokinesis in Δelm1 cells (Fig. S4A-S4D). Next, we asked if restoring Gin4 localisation to the bud neck could rescue elongated morphology in cells lacking Elm1 kinase. To test this hypothesis, we performed a GFP-GBP tethering screen to artificially restore Gin4 localisation at the bud neck in Δelm1 cells. Interestingly, we observed that tethering Gin4-GFP with the kinase Hsl1-GBP could restore normal morphology in ∼60% of Δelm1 cells (Fig. 3F-3H) while tethering with other binding partners like Shs143,81, Bni543 , and Anillin-like protein Bud482 did not show any rescue in the phenotype of Δelm1 cells despite restoration of bud neck localization (Fig. S4E, S4F). To test whether Gin4 kinase activity is required for this observed rescue, we tethered Gin4-GFP and Gin4KD-GFP via Hsl1-GBP in Δgin4 Δelm1 cells and observed that both constructs restored normal cell morphology (Fig. 3I, 3J). These results suggest that Gin4 tethering to the bud neck via Hsl1 may compensate for Elm1 function in a kinase-independent manner. Finally, we tested whether the membrane-binding Kinase-Associated (KA1) domain of Hsl1 is required for this function and found that Gin4-GFP tethering via Hsl1-Δka1-GBP did not rescue cellular morphology of Δelm1 cells (Fig. 3K, 3L). Together, these observations suggest that (i) restoring Gin4 localisation via Hsl1 is necessary to rescue the morphological defects exhibited by Δelm1 cells partially, (ii) Gin4 might be forming a complex with Hsl1, which becomes essential in the absence of Elm1 indicating that Elm1 might play a significant role in regulating Gin4-Hsl1 complex. (iii) proper membrane tethering of Hsl1 via its KA1 domain is essential for its function in Δelm1.
Intrigued by the rescue specifically observed after tethering Gin4-GFP with Hsl1-GBP, we extended our screen to its related kinases and tethered Hsl1-GFP and Kcc4-GFP via various bud-neck proteins in Δelm1 cells (Fig. 4A-4D, S5F, S5G). Our results revealed that Hsl1-GFP could restore normal cell morphology in ∼30% of Δelm1 cells when tethered with Shs1-GBP while tethering with septin kinases Gin4-GBP or Kcc4-GBP resulted in a significantly increased percentage of phenotype rescue (∼50%), which suggests possible coordination between these kinases (Fig. 4A, 4B). Strikingly, like Gin4-GFP, Kcc4-GFP tethering restored cell morphology similar to wildtype, only when tethered with Hsl1-GBP (Fig. 4C, 4D). However, Kcc4-GFP tethering via different bud neck proteins did not show any rescue of phenotype, but a minor rescue was observed upon tethering with Gin4-GBP, suggesting a coordination between Kcc4 and Gin4 (Fig. S5F, S5G). To support these results, we analyzed the localisation of Hsl1 and Kcc4 in Δelm1 and Δgin4 cells. As reported in a previous study33, Kcc4 exhibited mislocalisation to the bud cortex during the initial phases and exhibited defective localization at the onset of cytokinesis (Fig. S5B-S5E). Recruitment and localisation of Hsl1 to the bud neck are also found to be severely hampered in Δelm1 cells (Fig. 4E-4G, S5A), suggesting a role for Elm1 in maintaining Hsl1 localization at the bud neck. In contrast, recruitment and localisation of Hsl1 and Kcc4 were similar to wildtype in Δgin4 cells (Fig. 4E-4G, S5A-S5E). These data suggest that loss of Hsl1 at the bud neck may contribute majorly to the phenotype observed in Δelm1 cells and consistent with this idea, restoration of Hsl1 localization rescues the observed defects in our experiments. Our results reveal that Hsl1 regulates normal cell morphology, either through downstream signaling events caused by the presence of Elm1 in wildtype cells or by a parallel pathway in coordination with either Gin4 or Kcc4. Thus, Elm1 kinase may act upstream to control the localisation and activation of other regulatory kinases like Hsl1, Gin4 and Kcc4 at the septin scaffold in a precise, spatiotemporal manner in a network with multiple feedback regulatory links. Overall, the ability to restore normal function by simply rewiring the septin kinase network demonstrates the inherent redund-ancy and robustness of this network in S. cerevisiae.

Restoring Hsl1 localisation to the bud neck can bypass the requirement of Elm1.
(A) Representative images showing artificial tethering of Hsl1-GFP via Shs1-GBP, Bud4-GBP, Kcc4-GBP, Bni5-GBP and Gin4-GBP in Δelm1. Scale bar-5µm. (B) Bar graph showing % phenotype for indicated strains of (A) (N=3 and n>350/strain). ***: p<0.001, ****: p<0.0001, ns: p>0.05 (C) Representative images showing artificial tethering of Kcc4-GFP with Hsl1-GBP in Δelm1. Scale bar-5µm. (D) Bar graph showing % phenotype for indicated strains of (C) (N=3, n>350 cells/strain). ****: p<0.0001. (E) Representative montages of the bud neck depicting the localisation of Hsl1 in Δelm1 and Δgin4 cells. (F) Plot of raw intensity of Hsl1-GFP in indicated strains of (E) (t0=spindle breakpoint). (G) Plot of normalised signal intensity of Hsl1-GFP in indicated strains of (E) (t0=spindle breakpoint).
Hsl1 and its membrane-binding ability are required for the rescue of Δgin4 cells by artificially tethered Elm1
Elm1 does not localise to the bud neck in the absence of Gin4 and can restore the morphological defects exhibited by Δgin4 cells upon artificial tethering to the bud neck septin scaffold32. We corroborated these findings independently and found that Elm1 is not recruited to the bud neck in Δgin4 cells (Fig. S6A-S6D). We find that Elm1-GFP tethering via Shs1-GBP, Bud4-GBP, and Hsl1-GBP could restore normal cell morphology in Δgin4 cells (Fig. S6E, S6F). Therefore, we asked whether presence of Hsl1 could be necessary for the observed rescue upon Elm1 tetehring in Δgin4 cells. To test this, we tethered Elm1-GFP to the bud neck via Shs1-GBP in Δgin4 and Δgin4 Δhsl1 cells. While cell morphology was restored to normal in Δgin4 cells, no rescue was observed in Δgin4 Δhsl1 cells, suggesting that Hsl1 is required downstream of Elm1-tethering in Δgin4 cells (Fig. 5A, 5B). A similar loss of rescue was also observed when Elm1-GFP was tethered to the bud neck in Δgin4 cells via Hsl1-Δka1-GBP, suggesting that Hsl1 acts downstream of Elm1 and its membrane-binding KA1 domain is required for its function (Fig. S6G, S6H).Next, we asked if Elm1-GFP tethering to the bud neck in Δgin4 cells could rescue other observed cellular defects. To test this, we first assessed the localisation of the type-II myosin Myo1, a core component of AMR, which localizes to the bud neck in a biphasic manner10,77.

A non-canonical role of Hsl1-Kinase in regulating septin organisation and AMR dynamics.
(A) Representative images showing septin defects in Δgin4 Δhsl1 with Elm1-GFP tethered to bud neck via Shs1-GBP. Scale bar-5µm. (B) Bar graph showing % phenotype for indicated strains of (A) (N=3, n>450 cells/strain). ****: p<0.0001, ns: p>0.05 corresponds to population of cells with round morphology. (C) Representative time-lapse montages for Inn1-3xmCherry in Δgin4 Δhsl1 with Elm1-GFP tethered to bud neck via Shs1-GBP. (D) Quantification for Inn1-residence in indicated strains of (C). *: p<0.05, ****: p<0.0001, ns: p>0.05
We find that Myo1-3xmCherry exhibited mislocalisation to the bud tip in Δgin4 cells (Fig. S6I). While Myo1-3xmCherry mislocalisation was restored upon Elm1-GFP tethering to the bud neck in Δgin4 cells, it exhibited misorganization upon deletion of Hsl1 (Fig. S6I). We also assessed Inn1-3xmCherry lifetime in these cells to measure AMR constriction time63,64. We find that Inn1-3xmCherry lifetime is similar to wildtype cells when Elm1-GFP is tethered via Shs1-GBP in Δgin4 cells but significantly increased when Elm1-GFP is tethered via Shs1-GBP in Δgin4 Δhsl1 cells (Fig. 5C, 5D). These results suggest that (i) Elm1 tethering to the bud neck restores normal Myo1 localisation and AMR constriction dynamics in the absence of Gin4, (ii) Hsl1 is required for Elm1 to exert its influence over AMR organisation and dynamics after bud neck tethering in Δgin4 cells, and (iii) Membrane-binding KA1 domain of Hsl1 is required for this activity. Taken together, our results suggest that Hsl1 may play redundant roles along with Elm1 and Gin4 in maintaining septin stability and cytokinesis (Fig. 6). From our observations, one significant role of Elm1 might be in properly recruiting Hsl1 to the bud neck, revealing that Hsl1 may act downstream of Elm1 in this pathway with a partial overlap in their function. The fact that Hsl1 is required for the rescue observed upon Elm1 tethering in Δgin4 cells also supports the idea that Hsl1 acts downstream of Elm1 in regulating septin stability and cytokinesis.

Representative model for the role of Hsl1 kinase in septin organization and actomyosin ring constriction downstream of Gin4 and Elm1.
Discussion
Septins are considered as the fourth cytoskeletal elements due to their extensive structural and functional diversity83. They play critical role in various fundamental processes such as cytokinesis, ciliogenesis, spermatogenesis and phagocytosis etc17,84–86. While their involvement in cytokinesis and cell division has been well-characterized, recent advancements have provided deeper insights into their regulation3–5,7,20. Septin architecture and remodeling have emerged as dynamic areas of investigation, as these processes are spatiotemporally controlled by septin-interacting proteins and post-translational modifications (PTMs)31,39,55–57,59,60. Despite significant progress in the understanding of septin organisation, the mechanisms regulating septins and their modulation by associated kinases in yeast and higher eukaryotes remain incomplete. In Saccharomyces cerevisiae, four septin-associated kinases-Elm1, Gin4, Hsl1, and Kcc4 have been reported to majorly regulate septin-dependent processes33,52,87. Previous studies suggest that Elm1 acts as the most upstream kinase, regulating the Nim1-related kinases (Gin4, Hsl1, and Kcc4) via phosphorylation 80,87,88. Interestingly, Elm1 also exhibits kinase-independent roles in septin regulation, potentially through direct interaction with Bni533. Gin4 primarily phosphorylates Shs143, while Hsl1 regulates Swe1 and morphogenesis checkpoint48. However, the precise mechanisms through which Nim1-related kinases influence septin architecture—whether directly or via interactions with binding partners—remain poorly understood and requires further investigation.
Septin remodeling marks the onset of actomyosin ring (AMR) constriction and cytokinesis9,10,26. However, the molecular players regulating the crosstalk between these processes remain largely unknown. Septins are severely mislocalized to the bud cortex in Δelm1 and Δgin4 cells, suggesting their role in organizing septins at the bud neck consistent with previous reports32,33,52. The defective dynamics of AMR component proteins, Inn1 and Hof1, in the absence of Elm1 and Gin4 kinases further indicate that these kinases regulate intermediate signaling events during cytokinesis and highlight an underappreciated role in maintaining AMR organization and dynamics (Fig. 1D-1E, 2A-2E).
A key question arising from these observations is whether the crosstalk between the septin kinase network and AMR dynamics is direct and septin-independent or indirect and septin-dependent. Previous work has shown that AMR constriction is unperturbed in septin mutants which affect septin organization9, suggesting that the deregulation of cytokinesis observed in our study may be a direct effect of the septin kinases, Elm1 and Gin4, on AMR organization and constriction. To support this idea, we also find that Gin4 kinase interacts with the F-BAR protein Hof1 through Yeast-Two Hybrid and direct in vitro binding assays (Fig. 2F-2I). The fact that the membrane-binding domains of Gin4 and Hof1 are involved in this interaction (Fig. 2G-2H) suggests that Gin4 activation at the membrane as previously suggested42,50 may directly involve or affect Hof1 organization and maturation of the AMR. This also suggests a potential mechanism of tethering of Hof1 to the membrane prior to cytokinesis (Fig. 2J). Interestingly, previous work has showed that Gin4 and Hof1 are synthetic lethal and that Hof1 exhibits mislocalization in Δgin4 cells71. These results also point towards a coordinative mechanism where septin-associated kinase Gin4 directly crosstalk with an AMR component Hof1 to regulate AMR organization and constriction. To our knowledge, this is the first evidence that physically links the septin kinase network to AMR proteins and highlights a novel regulatory input on AMR assembly in S. cerevisiae. Future work is required to delineate the molecular mechanisms underlying this regulation.
Our study shows that the kinase activity of Gin4 is not required for its role in septin organization, AMR constriction, and even for maintaining viability in the absence of Hof1 (Fig. 3A-3E, S3J). This highlights an important aspect of Gin4 function in cells and is similar to kinase-independent function reported for Elm133. This poses the question: What is the role of the kinase activity of Elm1 and Gin4? Our study and a previous study32,52 have now established that there are in-built redundancies within the septin kinases (Elm1, Gin4, Hsl1, Kcc4) in terms of function but their recruitment to the bud neck is also interdependent on each other. The kinase activities may play an important role in regulating the signaling that coordinates the precise arrival and spatiotemporal activation of these kinases at the bud neck. These results also raise important questions about post-translational regulatory mechanisms of septin filament organization as a number of studies have shown that these kinases directly phosphorylate septins43. Thus, the role of post-translational regulation on septins will be better understood with future work in this direction.
Although these kinases exhibit distinct functions, they also display interdependence on each other32,80. Recent studies propose a new perspective, revealing reciprocal regulation between Gin4 and Elm1 through direct binding and phosphorylation at different stages of the cell cycle32. An unresolved question over the years has been the difference in phenotype observed in Δelm1 vs Δgin4. Δelm1 cells shows a greater percentage of abnormality in cell shape and septin organization as compared to Δgin4 cells even though Elm1 is also known to be absent from the bud neck in Δgin4 cells32,52. Our work offers a possible explanation for this by analysis of the temporal kinetics of Hsl1 and Kcc4 in Δelm1 and Δgin4 cells, which are perturbed only in Δelm1 and not in Δgin4 cells (Fig. 4E-4G, S5A-S5E), possibly explaining why cells lacking Elm1 exhibit more pronounced defects. These findings suggest that the localization of Hsl1 and Kcc4 to the bud neck is crucial for normal function and may contribute to the less prominent phenotype observed due to loss of Gin4, emphasizing and strengthening the redundant roles of Hsl1 and Kcc4 in septin organization.
We also performed an extensive GFP-GBP screens in Δelm1 cells and found that restoring the localization of Hsl1 with Gin4 and Kcc4 can bypass the requirement for Elm1 during the cell cycle (Fig. 3G, 3H, 4C and 4D) highlighting a functional redundancy among the Nim1-related kinases in achieving successful cytokinesis. Our results also suggest that Hsl1 plays a non-canonical role in regulating septin organization and actomyosin ring dynamics downstream of Elm1 and Gin4, providing novel insights into Hsl1’s role in cytokinesis (Fig. 5A-5D, S6I). Therefore, we speculate synergy exists between Elm1 and Gin4 to regulate Hsl1 in a downstream signaling pathway that can regulate septin organization and cytokinesis. The requirement of Hsl1’s kinase activity in the absence of Elm1 or Gin4 could offer further understanding of the molecular mechanisms underlying Hsl1’s regulation in future studies. Strikingly, the inability to rescue this defect upon deletion of the membrane-binding KA1 domain of Hsl1 (Fig. 3K, 3L, S6G, S6H) highlights the importance of membrane-binding for the interplay and activation of these kinases. How membrane binding facilitates Hsl1 function remains to be explored. The underlying molecular mechanisms and actual cause-effect relationships of how Hsl1 and other kinases regulate septin organisation at the filament-level remain to be understood and approaches such as Platinum-replica electron microscopy (PREM) and invitro reconstitution assays will be key to generate such insights in future studies21,33,68. It promises to reveal critical insights into the modes of septin regulation through the coordinated action and signaling inputs from multi-septin kinase modules across eukaryotes.
In summary, our study provides a comprehensive quantitative analysis of the cellular functions of septin-regulatory kinases in septin organization and cytokinesis. Notably, we identified Elm1 and Gin4 as key players in septin regulation, uncovering their previously uncharacterized roles in actomyosin ring organization and constriction. These findings pave the way for future investigations into the intricate molecular mechanisms and cross-regulatory interactions within the septin-kinase network and with actomyosin ring proteins during the cell cycle in Saccharomyces cerevisiae and other related organisms where septins and related proteins play a role in cell division.
Materials and methods
Strain construction
All the yeast strains in this study were constructed using the S. cerevisiae wildtype strains ESM356 and YPH499 derived from S288C genetic background. The strain number and the genotype are listed in supplementary table S1. The C-terminal epitope tagging, and endogenous gene deletions were carried out based on the PCR-based integration strategy89,90. All the strains were cultured at 23°C with 180rpm. Synthetic complete (S.C.) media lacking tryptophan, uracil, leucine and histidine were used to select auxotrophic recombinants. Antibiotic selections for endogenous gene manipulation were done on YPD agar plates supplemented with 200µg/ml G418 (Sisco Research Laboratories Pvt. Ltd.; catalogue no. 58327), 100µg/ml of hygromycin-B (Sisco Research Laboratories Pvt. Ltd.; catalogue no. 67317) and 100µg/ml of nourseothricin (Jena Biosciences; catalogue no. AB-102). To visualize the septin dynamics, Cdc3-mCherry, YIP211-cdc3-mCherry77 and YIP204-cdc3-mCherry91 plasmids were cut with BglII (catalogue no. R0144S; New England Biolabs) and was integrated into CDC3 locus. Cell cycle dynamics were visualized using a yeast expression vector with mRuby2-Tub1 under His3 promoter (pHIS3p:mRuby2-Tub1+3"UTR::Ura3)92 and pAFS125-GFP-TUB193, which were cut with ApaI (Catalogue no. R0114S; New England Biolabs) and StuI (catalogue no. R0187S; New England Biolabs) respectively and were integrated into ura3 loci. Yeast transformation for epitope tagging and endogenous gene deletions were performed using a lithium acetate (LiOAc)-based protocol described94.
Oligonucleotides and Plasmids
All the oligonucleotides used for endogenous gene tagging and deletion in this study are listed in the supplementary table S2. The plasmids used and generated for this study are listed in the supplementary table S3. For a generation of clones used in Fig. 3A, 3B the Gin4FL was amplified from the endogenous locus and was integrated into pRS305 under the pTEF promoter. The ligation reaction was performed using NEB Hifi-Builder (New England Biolabs; cat.no: E2621L). Catalytically dead Gin4KD(K48A) was constructed based on the site-directed mutagenesis strategy using the oligos (P855, P856, P857 & P1024). The kinase-dead mutant was confirmed correct by sequencing. To construct Gin4FL and Gin4KD used in Fig. 3C-3E, S3G, S3H and S3J the Gin4 gene fragment was amplified from genomic loci with the endogenous promoter using PCR and was ligated into a pRS305 integration vector cut with BamHI-HF (Catalogue no. R3136S; New England Biolabs).
Live-cell imaging
The cells were inoculated in S.C. media and incubated overnight at a 23°C. The secondary culture was diluted to log-phase (Optical density 0.4 to 0.6) from the overnight saturated culture. The 6% Concanavalin A type 6 (Sigma-Aldrich; catalogue no. C2010) was coated onto a confocal dish (ibidi GmbH-catalogue no. 81218-200; Cellvis-catalogue no. D35C4-20-1.5-N) and was incubated at 23°C incubator. The secondary culture cells were pelleted at 3200 rpm for 3 minutes. The cells were washed in S.C. media to remove non-adherent cells and subjected to live-cell imaging.
The laser point scanning confocal microscope Olympus FV-3000, equipped with high-sensitivity GaAsP photomultiplier tube (PMT) detectors and solid-state lasers (488 nm and 561 nm), was used to acquire time-lapse imaging for Fig. S1A-S1E. The images were acquired at a one-minute time interval with 0.5µm step size and seven z-slices for 2-3 hours. The raw images were three-dimensional (3D) deconvolved in Olympus CellSens Dimension (3.1) software. Maximum-intensity projection images were used for representation.
The Olympus IX83 widefield microscope equipped with a CoolLED PE-4000 LED light source and Prime-BSI ScMOS camera was used for acquiring images represented in Fig. 1B, 1D, 2A, 3C, S2D, S3A and S3D. All the images were acquired in a 100x oil-immersion objective with 1.45 N.A., 1µm step-size and three z-slices at a 2-minute time interval for 60-150 minutes. The raw images were processed for 3D deconvolution using Olympus CellSens Dimension (3.1) software. Maximum-intensity projection images were used for representation.
The images for Hof1 mis-organization and artificial tethering experiments in Fig. 2C, 3G, 3K, 4A, 4C, S3G, S4E, S5F, S6E and S6G were acquired using the Oxford Andor Dragonfly 502 spinning disc confocal system equipped with a fully motorized Dmi8 inverted setup. The raw images were captured in 100x oil immersion objective with 0.5µm step-size, 11 z-slices in Andor Sona scMOS camera and deconvolved using Andor Fusion software. The 3D-deconvolved and max-projected images were used for representation.
Olympus IX-83 Laser scanning spinning disc Yokogawa-W1 microscope was used to capture Fig. 3A, 3I, 5A and S6I images. The raw images were acquired in a 100x oil-immersion objective with 1.45 N.A., using 1µm step-size and five z-slices. The time-lapse imaging for Fig. 4E,5C, S4A, S4B, S5A-S4C, S6A, S6B was done at a one-minute time interval in 60x objective with 1.42 N.A. The images and time-lapse montages were 3D-deconvolved in Olympus CellSens Dimension (3.1) software. 3D-Deconvolved and maximum intensity projection images were used for representation.
Protein accumulation kinetics quantification and analysis
The live-cell imaging data acquired was opened in Fiji (ImageJ2) image analysis software95 using the protocol established96. In brief, a region of interest (ROI) was drawn around the bud neck of the sum-intensity projected image. The extracted fluorescent intensity values were corrected using background values. All the individual fluorescent intensity values obtained in an Excel sheet at each time point were normalized using the minimum and maximum values according to the formula below:
The data points ranging between the -20 and +20 time points were separately extracted for the ‘n’ number of cells indicated with each graph, respectively. Mean, and standard deviation was calculated. A line plot was generated using Origin-Pro (2015, Sr2, 69.2.272; Origin Lab, USA) image analysis software to represent the temporal accumulation kinetics of the protein of interest. The small bud is considered as a T0 for quantifications involving bud-emergence. The complete drop in Cdc3-mCherry intensity is considered as HDR transition in Figure S1E. The Spindle breakpoint is considered as T0 or reference time-point for measuring Hof1, Inn1 or Chs2 temporal dynamics during cytokinesis.
Quantitative analysis of septin and Hof1 misorganization was done manually by counting the number of cells exhibiting septin localisation at the bud cortex during bud emergence and the defective/discontinuous Hof1 ring at the bud neck.
Time-lapse imaging of AMR components was done to analyze the residence time. The time was calculated as the difference between the first-time point where the signal initially appears and the final time point where the signal diminishes completely from the bud neck.
Yeast Two Hybrid Assay
Genes or gene fragments of interest were amplified and cloned into NotI restriction enzyme (NEB; Catalogue No. R3189S) cut pMM5S and pMM6S plasmids consisting of DNA Binding Domain of LexA (LexDBD) and Activation Domain of Gal4 (Gal4AD) respectively. The clones were transformed into the yeast strains SGY37 (MATa) and YPH500 (MATalpha). The transformants were selected on S.C. media lacking Histidine and Leucine, respectively. Mating was performed on YPD plates and was incubated at 30°C for 2 days followed by replica plating on SC-His-Leu double selection plates. The plates were incubated for 2 days at 30°C, subsequently overlayed with freshly prepared X-Gal mix (500 mM sodium phosphate buffer (pH7.0), 10% SDS, 1 M KCl, 1 M MgCl2, 0.04% X-gal and 0.4% low melting agarose) for detection of β-galactosidase activity97,98. Interaction of LexADBD-Protein of interest A with Gal4AD-Protein of interest B resulted in the expression of the lacZ gene encoding the β-galactosidase, which converted X-gal to a product with blue colour99. The plates were scanned and documented after 14-16 hours of incubation with overlay mix.
Protein expression, purification and In vitro-Binding Assay
The N-terminal fragment of Hof1 containing the F-BAR domain (1-350 aa) and KA1 fragment of Gin4 (1003-1142 aa) were PCR amplified from the S. cerevisiae genomic DNA and was cloned into pGEX-5X1 cut with EcoRI restriction enzyme (Catalogue No. R3101S) and pET28a-6His-bdSUMO vector cut with NdeI restriction enzyme (Catalogue No. R0111S) respectively using NEB Hifi Builder. Both the plasmids were transformed into BL21-DE3 E.coli competent cells and was selected on Luria Bertani (LB) plate containing Kanamycin (50mg/ml) for pGEX-5X1-Hof1N (1-350) and Ampicillin (100mg/ml) for pET28a-6His-bdSUMO-Gin4KA1. The LB broth with Kanamycin or Ampicillin was used to grow primary culture overnight at 37°C with constant shaking at 200 rpm. The primary culture was diluted to 0.1 O.D. in 500ml LB broth with Kanamycin or Ampicillin. The protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) once the O.D. reached log phase (0.4-0.5). The culture was incubated at 30°C with shaking of 200 rpm. for 3-4 hrs. The cells were pelleted down 4°C, 7000 rpm for 20 min. Media was aspirated and the pellets were washed with cold phosphate buffered saline (PBS) containing 1mM phenymethylsulfonyl fluoride (PMSF) and was stored at -80°C.
Purification of 6His-bdSUMO-Gin4KA1
Pellets were kept on ice for 10 min. and was subjected to lysis with the Lysis Buffer (50 mM Tris pH-8, 200 mM NaCl, 10% glycerol). The lysed pellets were clarified at 4°C and 15,000xg. The clarified lysate was mixed with Ni-NTA agarose resin (cat. no. 786-940, G-Biosciences), washed with wash buffer (50 mM Tris pH-8, 200 mM NaCl, 10 mM Imidazole) and was incubated at 4°C for 2 hrs. The lysate with Ni-NTA resins were then transferred to poly-prep chromatography columns (cat. no. 731-1550, BIO-RAD laboratories Inc.), and was subjected to gradient elution with increasing concentration of imidazole. The eluted protein fractions were buffer exchanged with storage buffer (50 mM Tris-HCl pH 7.4, 2000 mM NaCl, 1 mM DTT and 10% glycerol) using PD Midi Trap G-10 Sephadex columns (GE Healthcare, cat. No. GE28-9180-11). The purified proteins were flash frozen in liquid Nitrogen and was stored at -80°C. The protein concentration was estimated in spectrophotometer at 280nm and was analyzed along the known concentration of Bovine Serum Albumin (BSA) standards on Sodium Dodecyl-Poly Acrylamide Gel electrophoresis (SDS-PAGE).
Purification of GST-Hof1N (1-350)
Pellets were kept on ice for 10 min. and was subjected to lysis with the Lysis Buffer (PBS, 0.5 mM EDTA, 1 mM DTT, 1 mg/ ml lysozyme, and complete mini-EDTA-free protease inhibitor cocktail tablets). The clarified lysate was incubated with 1% Triton-X-100 for 20 min. on ice and centrifuged at 4°C, 22,000xg for 30 min. The lysate was then transferred to a tube containing glutathione sepharose-4B resin (cat. no. GE17-0756-01, GE) washed with wash buffer (5x) (PBS, 0.5 mM EDTA and 1 mM DTT) and was incubated in 4°C for 2-3 hrs. The lysate with glutathione Sepharose resins were then transferred to poly-prep chromatography columns after incubation, washed with wash buffer and was stored in 4°C.
In vitro Binding assay
GST or GST– Hof1N (1-350) (∼3 µg) bound to glutathione beads were mixed with ∼1 µg 6His–bdSUMO-Gin4KA1 and incubated in binding buffer (PBS, 0.2% NP-40) for 1hour at 4°C. The beads were washed 3-5 times with wash buffer (PBS, 1% NP-40) and were analyzed by western blot using Anti-HIS (H-3) mouse monoclonal IgG1 (Santa Cruz/#sc-8036/Lot. No. C0421), Anti-GST (B-14) mouse monoclonal IgG1 (Santa Cruz/#sc-138/Lot. No. K1020) and Anti-mouse IgG-HRP (Cell Signalling/#7076S/Lot.No. 36). Bound Gin4KA1 was calculated as (IGin4KA1–Ibackground)/(I GST– Hof1N (1-350)–Ibackground), where I is the Integrated density value of the protein bands measured using ImageJ (NIH).
Spot assay
The yeast strains were grown in liquid YPD broth overnight at 23°C. The cultures were diluted to 1.0 O.D. and was serially diluted in the order 100, 10−1, 10−2, 10−3, and 10−4. These dilutions were spotted on three YPD plates and incubated at 23°C, 30°C and 37°C. The growth was monitored, and plates were scanned after 48 hrs. of incubation.
The strains used for rescue of Δgin4Δhof1 synthetic lethality (Fig. S3J) were grown overnight in S.C. media lacking Uracil and Leucine. The culture was diluted to an O.D. of 1.0 and was serially diluted in the order 100, 10−1, 10−2, 10−3, and 10−4. These dilutions were spotted on three SC-Ura-Leu plates and were incubated at 23°C for two days. Subsequently, the colonies were patched onto the 5-FOA plates (concentration 1mg/ml) to select for Ura-cells.
Statistical analysis
The experiments were performed three independent times. The significance values for the graphs plotted were extracted using the One-way ANOVA -Kruskal-Wallis multiple comparison test (plots with residence time), and Tukey’s multiple comparison test (Bar and Stacked column plots), an inbuilt function in GraphPad Prism (v.6.04). A p-value of p<0.05 was considered statistically significant. (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: p>0.05).
Supplemental figures and tables

Dynamics of septin-associated kinases exhibiting a sequential pattern of recruitment during bud emergence and derly disassembly during the HDR transition.
(A) Representative time-lapse images and graph for kinetics of Elm1-GFP during d-emergence (t=0) (n>15 cells). (B) Representative time-lapse images and graph for kinetics of Gin4-GFP during bud-emergence 0) (n>15 cells). (C) Representative time-lapse images and graph for kinetics of Hsl1-GFP during bud-emergence (t=0) (n>15 lls). (D) Representative time-lapse images and graph for kinetics of Kcc4-GFP during bud-emergence (t=0) (n>15 cells). (E) epresentative montages of Elm1-GFP, Gin4-GFP, Hsl1-GFP and Kcc4-GFP during HDR transition (t0=septin splitting). (F) A plot normalized signal intensity at bud neck for indicated GFP-tagged proteins in (E). Cdc3-mCherry profile of Elm1 is plotted as a ference for septin splitting (n>14 cells/strain).

Septin kinases deletion differentially perturbs septin organization and alters cell morphology.
(A) Images showing the morphological defects in Δelm1, Δgin4, Δkcc4 and Δhsl1 at 23℃, 30℃ and 37℃. (B) Bar graph showing % phenotype for indicated strains of (A). (C) Growth assay for indicated strains of (A). (D) Representative images showing septin defects in kinase deletions during bud-emergence. Arrows depict mislocalised septins in Δelm1 and Δgin4 strains, scale bar-5µm. (E) The bar plot of septin mislocalisation for strains indicated in (D) (n>25 cells/strain).

Actomyosin ring dynamics is unaltered in Δhsl1 and Δkcc4 deletions, and Gin4 regulate primary septum (PS) ormation in a kinase-independent manner.
(A) Representative time-lapse images of Inn1-mNG in wildtype, Δhsl1 and Δkcc4. B) Plot of normalized signal intensity of Inn1-mNG in indicated strains of (A) (t0=spindle breakpoint). (C) Quantification for Inn1-esidence in indicated strains of (A). ns: p>0.05 (D) Representative time-lapse images of Chs2-mNG in wildtype, Δelm1 and Δgin4. E) Plot of normalized signal intensity of Chs2-mNG in indicated strains of (D); a population of cells showing asymmetric constriction n Δelm1 and Δgin4 is plotted (t0=spindle breakpoint). (F) Quantitative analysis of Chs2-residence in indicated strains of (D). *: <0.05, ns: p>0.05 (G) Representative images for the rescue of Hof1-misorganisation by Gin4FL and Gin4KD in Δgin4 cells. Scale ar-5µm. (H) Quantification for indicated strains of (G), (N=3 and n>130 cells/strain). ****: p<0.0001, ns: p>0.05 corresponds to opulation of cells with disorganized Hof1 at the bud neck. (I) Representative images of Yeast Two-Hybrid for interaction between in4-KA1 domain and different domains of Hof1. (J) Spot assay for the rescue of synthetic lethality by Gin4FL and Gin4KD in Δgin4 hof1 cells at 23℃.

A GBP-based tethering screen for targeted localization of Gin4-GFP to the bud neck in Δelm1 via different bud eck proteins.
(A) Representative montages for recruitment of Gin4-GFP during bud emergence in Δelm1 background. (B) epresentative montages of the bud neck depicting the localisation of Gin4 in Δelm1. (C) Plot of raw intensity of Gin4-GFP in dicated strains of (B) (t0=spindle breakpoint). (D) Plot of normalized signal intensity of Gin4-GFP in indicated strains of (B) 0=spindle breakpoint). (E) Representative images showing artificial tethering of Gin4-GFP via Shs1-GBP, Bud4-GBP and Bni5-BP in Δelm1. Scale bar-5µm. (F) Bar graph showing % phenotype for indicated strains of (E) (N=3 and n>350/strain).

A GBP-based screen for artificially restoring Kcc4 localisation in Δelm1 cells.
(A) Representative montages for ecruitment of Hsl1-GFP during bud emergence in Δelm1 and Δgin4 background (DC* indicates Differential Contrast). (B) epresentative montages for recruitment of Kcc4-GFP during bud emergence in Δelm1 and Δgin4 backgrounds. (C) Representative ontages of the bud neck depicting the localisation of Kcc4 in Δelm1 and Δgin4 cells. (D) Plot of raw intensity of Kcc4-GFP in ndicated strains of (C) (t0=spindle breakpoint). (E) Plot of normalized signal intensity of Kcc4-GFP in indicated strains of (C) t0=spindle breakpoint). (F) Representative images showing artificial tethering of Kcc4-GFP with Shs1-GBP, Bud4-GBP, Bni5-GBP nd Gin4-GBP in Δelm1. Scale bar-5µm. (G) Bar graph showing % phenotype for indicated strains of (F) (N=3, n>350 cells/strain).

Hsl1 kinase becomes essential downstream of Elm1 to regulate cytokinesis in Δgin4.
(A) Representative montages for recruitment of Elm1-GFP during bud emergence in Δgin4 background. (B) Representative montages of the bud neck depicting the localisation of Elm1 in Δgin4. (C) Plot of raw intensity of Elm1-GFP in indicated strains of (B) (t0=spindle breakpoint). (D) Plot of normalized signal intensity of Elm1-GFP in indicated strains of (B) (t0=spindle breakpoint). (E) Representative images showing artificial tethering of Elm1-GFP with Shs1-GBP, Bud4-GBP and Hsl1-GBP in Δgin4. Scale bar-5µm. (F) Bar graph showing % phenotype for indicated strains of (E) (N=3 and n>350/strain). *: p<0.05, **: p<0.01 (G) Representative images showing tethering of Elm1-GFP with Hsl1-Δka1-GBP in Δgin4. Scale bar-5µm. (H) Bar graph showing % phenotype for indicated strains of (G) (N=3, n>300 cells/strain). ****: p<0.0001, ns: p>0.05 corresponds to population of cells with round morphology. (I) Representative images showing localisation of Myo1-3xmCherry in the Δgin4 Δhsl1 cells.




Yeast strains used in this study



Oligonucleotides used in this study


Plasmids used in this study
Acknowledgements
We thank the DST-FIST microscope facility of the Department of Biochemistry, Indian Institute of Science. We also thank the Bioimaging facility, Division of Biological Sciences. We thank Prof. Erfei Bi (University of Pennsylvania) and Prof. Gislene Periera (Centre for Organismal Studies (COS) Heidelberg) for the plasmids. We are grateful for the help of Ms. Deepthi Guturu and Ms. Spoorthi Hirematt in image acquisition at the DST-FIST microscope facility. We thank Prof. P N Rangarajan, Prof. Franz Meitinger and Prof. Ramanujam Srinivasan for their valuable suggestions on the manuscript. Schematic diagrams are created in BioRender (Palani, S. (2025) https://BioRender.com/n14d984). B.B. acknowledges the DST-INSPIRE Fellowship. A.D. and J.K. acknowledge the IISc Ph.D. fellowship from IISc. FC acknowledges the ICMR fellowship.
Additional information
Author contributions
S.P. conceived, conceptualized and superivsed the study. S.P and B.B. designed the experiments. B.B. constructed the strains and plasmids, performed the experiments, data curation and quantitative analysis. A.D. performed image acquisition of AMR organization. B.V.T. generated the plasmids and performed yeast two hybrid screen. J.K cloned, purified and performed the in vitro binding assay. F.C. conducted spot assays for septin kinase deletion experiment and quantification. V.R. helped with image analysis, manuscript editing and formatting. S.P., A.D. and B.B. wrote and reviewed the manuscript. All the authors were involved in editing the manuscript.
Funding
This work was financially supported by a Department of Biotechnology-Wellcome Trust India Alliance intermediate fellowship (IA/I/21/1/505633), SERB SRG grant (SRG/2021/001600) and an Indian Institute of Science (IISc) start-up grant awarded to S.P.
Additional files
References
- 1.The evolutionary origins and ancestral features of septinsFront Cell Dev Biol 12:1406966https://doi.org/10.3389/fcell.2024.1406966
- 2.Evolutionary degeneration of septins into pseudoGTPases: impacts on a hetero-oligomeric assembly interfaceFront Cell Dev Biol 11:1296657https://doi.org/10.3389/fcell.2023.1296657
- 3.Membrane reshaping by micrometric curvature sensitive septin filamentsNat Commun 10:420https://doi.org/10.1038/s41467-019-08344-5
- 4.Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assemblyProc Natl Acad Sci U S A 105:8274–8279https://doi.org/10.1073/pnas.0803330105
- 5.Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiaeMol Biol Cell 23:423–432https://doi.org/10.1091/mbc.E11-10-0850
- 6.A highly ordered ring of membrane-associated filaments in budding yeastJournal of Cell Biology 69https://doi.org/10.1083/jcb.69.3.717
- 7.Architecture and dynamic remodelling of the septin cytoskeleton during the cell cycleNat Commun 5:5698https://doi.org/10.1038/ncomms6698
- 8.Here come the septins: novel polymers that coordinate intracellular functions and organizationJ Cell Sci 119:4–10https://doi.org/10.1242/jcs.02746
- 9.Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeastBiol Chem 392:813–829https://doi.org/10.1515/BC.2011.083
- 10.Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae CytokinesisJournal of Cell Biology 142https://doi.org/10.1083/jcb.142.5.1301
- 11.Spatial Coordination of Cytokinetic Events by Compartmentalization of the Cell CortexScience 305https://doi.org/10.1126/science.1099892
- 12.The septin cortex at the yeast mother–bud neckCurrent Opinion in Microbiology 4https://doi.org/10.1016/S1369-5274(01)00269-7
- 13.Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast CytokinesisJournal of Cell Biology 140https://doi.org/10.1083/jcb.140.2.355
- 14.Septin filament formation is essential in budding yeastDev Cell 20:540–549https://doi.org/10.1016/j.devcel.2011.02.004
- 15.Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: Localization of the CDC11 gene product and the timing of events at the budding siteDevelopmental Genetics 12https://doi.org/10.1002/dvg.1020120405
- 16.Immunofluorescence Localization of the Saccharomyces cerevisiae CDC12 Gene Product to the Vicinity of the 10-nm Filaments in the Mother-Bud NeckMolecular and Cellular Biology 7https://doi.org/10.1128/mcb.7.10.3678-3687.1987
- 17.Genetic control of the cell division cycle in yeast: IV. Genes controlling bud emergence and cytokinesisExperimental Cell Research 69https://doi.org/10.1016/0014-4827(71)90223-0
- 18.Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding siteJournal of Cell Biology 112https://doi.org/10.1083/jcb.112.4.535
- 19.Shs1p: A Novel Member of Septin That Interacts with Spa2p, Involved in Polarized Growth inSaccharomyces cerevisiaeBiochemical and Biophysical Research Communications 251https://doi.org/10.1006/bbrc.1998.9541
- 20.Septin assemblies form by diffusion-driven annealing on membranesProc Natl Acad Sci U S A 111:2146–2151https://doi.org/10.1073/pnas.1314138111
- 21.An amphipathic helix enables septins to sense micrometer-scale membrane curvatureJ Cell Biol 218:1128–1137https://doi.org/10.1083/jcb.201807211
- 22.Subunit-dependent modulation of septin assembly: budding yeast septin Shs1 promotes ring and gauze formationJ Cell Biol 195:993–1004https://doi.org/10.1083/jcb.201107123
- 23.The step-wise pathway of septin hetero-octamer assembly in budding yeasteLife 6https://doi.org/10.7554/eLife.23689
- 24.Cell cycle control of septin ring dynamics in the budding yeastMicrobiology 147https://doi.org/10.1099/00221287-147-6-1437
- 25.The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesisJournal of Cell Science 114https://doi.org/10.1242/jcs.114.7.1379
- 26.Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constrictionNat Commun 9:4308https://doi.org/10.1038/s41467-018-06767-0
- 27.Cytokinesis in Budding Yeast: the Relationship between Actomyosin Ring Function and Septum FormationCell Structure and Function 26https://doi.org/10.1247/csf.26.529
- 28.In budding yeast, contraction of the actomyosin ring and formation of the primary septum at cytokinesis depend on each otherJournal of Cell Science 115https://doi.org/10.1242/jcs.115.2.293
- 29.Actomyosin Ring Formation and Tension Generation in Eukaryotic CytokinesisCurr Biol 26:R719–R737https://doi.org/10.1016/j.cub.2016.06.071
- 30.Central Role of the Actomyosin Ring in Coordinating Cytokinesis Steps in Budding YeastJ Fungi (Basel) 10https://doi.org/10.3390/jof10090662
- 31.Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiaeFront Cell Dev Biol 4:119https://doi.org/10.3389/fcell.2016.00119
- 32.Reciprocal regulation by Elm1 and Gin4 controls septin hourglass assembly and remodelingJ Cell Biol 223https://doi.org/10.1083/jcb.202308143
- 33.The LKB1-like Kinase Elm1 Controls Septin Hourglass Assembly and Stability by Regulating Filament PairingCurr Biol 30:2386–2394https://doi.org/10.1016/j.cub.2020.04.035
- 34.The Elm1 Kinase Functions in a Mitotic Signaling Network in Budding YeastMolecular and Cellular Biology 19https://doi.org/10.1128/MCB.19.12.7983
- 35.The Checkpoint Kinase Hsl1p Is Activated by Elm1p-dependent PhosphorylationMolecular Biology of the Cell 19https://doi.org/10.1091/mbc.e08-06-0663
- 36.Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1Mol Biol Cell 27:1764–1775https://doi.org/10.1091/mbc.E16-01-0014
- 37.Control of Mitotic Events by Nap1 and the Gin4 KinaseJournal of Cell Biology 138https://doi.org/10.1083/jcb.138.1.119
- 38.The Septins Are Required for the Mitosis-specific Activation of the Gin4 KinaseJournal of Cell Biology 143https://doi.org/10.1083/jcb.143.3.709
- 39.Phosphorylation-Dependent Regulation of Septin Dynamics during the Cell CycleDevelopmental Cell 4https://doi.org/10.1016/S1534-5807(03)00061-3
- 40.Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinaseMol Biol Cell 23:4041–4053https://doi.org/10.1091/mbc.E12-05-0395
- 41.Mechanisms of growth-dependent regulation of the Gin4 kinasebioRxiv https://doi.org/10.1101/2024.11.20.624605
- 42.Growth-Dependent Activation of Protein Kinases Suggests a Mechanism for Measuring Cell GrowthGenetics 215:729–746https://doi.org/10.1534/genetics.120.303200
- 43.Cell Cycle-dependent Assembly of a Gin4-Septin ComplexMolecular Biology of the Cell 13https://doi.org/10.1091/mbc.01-10-0500
- 44.Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiaeMol Biol Cell 27:2213–2233https://doi.org/10.1091/mbc.E16-03-0177
- 45.Roles of Hsl1p and Hsl7p in Swe1p degradation: beyond septin tetheringEukaryot Cell 11:1496–1502https://doi.org/10.1128/EC.00196-12
- 46.Coupling morphogenesis to mitotic entryProc Natl Acad Sci U S A 101:4124–4129https://doi.org/10.1073/pnas.0400641101
- 47.Cytoskeletal activation of a checkpoint kinaseMol Cell 12:663–673https://doi.org/10.1016/j.molcel.2003.08.006
- 48.A Monitor for Bud Emergence in the Yeast Morphogenesis CheckpointMolecular Biology of the Cell https://doi.org/10.1091/mbc.e03-03-0154
- 49.Molecular Dissection of the Checkpoint Kinase Hsl1pMolecular Biology of the Cell 20https://doi.org/10.1091/mbc.e08-08-0848
- 50.Kinase associated-1 domains drive MARK/PAR1 kinases to membrane targets by binding acidic phospholipidsCell 143:966–977https://doi.org/10.1016/j.cell.2010.11.028
- 51.The Saccharomyces cerevisiae bud-neck proteins Kcc4 and Gin4 have distinct but partially-overlapping cellular functionsGenes & Genetic Systems 78https://doi.org/10.1266/ggs.78.113
- 52.Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeastGenes & Development 13https://doi.org/10.1101/gad.13.2.176
- 53.Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin FunctionThe Journal of Cell Biology 143https://doi.org/10.1083/jcb.143.3.719
- 54.Septin-Dependent Assembly of a Cell Cycle-Regulatory Module in Saccharomyces cerevisiaeMolecular and Cellular Biology 20https://doi.org/10.1128/MCB.20.11.4049-4061.2000
- 55.Posttranslational modifications and assembly of septin heteropolymers and higher-order structuresCurr Opin Microbiol 15:660–668https://doi.org/10.1016/j.mib.2012.09.007
- 56.Cell Cycle–Regulated Attachment of the Ubiquitin-Related Protein Sumo to the Yeast SeptinsJournal of Cell Biology 147https://doi.org/10.1083/jcb.147.5.981
- 57.Architecture, remodeling, and functions of the septin cytoskeletonCytoskeleton (Hoboken) 76:7–14https://doi.org/10.1002/cm.21475
- 58.Septins: molecular partitioning and the generation of cellular asymmetryCell Div 4https://doi.org/10.1186/1747-1028-4-18
- 59.Regulation of septin dynamics by the Saccharomyces cerevisiae lysine acetyltransferase NuA4PLoS One 6:e25336https://doi.org/10.1371/journal.pone.0025336
- 60.Smt3, a SUMO-1 Homolog, Is Conjugated to Cdc3, a Component of Septin Rings at the Mother-Bud Neck in Budding YeastBiochemical and Biophysical Research Communications 259https://doi.org/10.1006/bbrc.1999.0821
- 61.Septins, under Cla4p regulation, and the chitin ring are required for neck integrity in budding yeastMol Biol Cell 14:2128–2141https://doi.org/10.1091/mbc.e02-08-0547
- 62.Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence MicroscopyFront Cell Dev Biol 5:42https://doi.org/10.3389/fcell.2017.00042
- 63.Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiaeJ Cell Biol 185:995–1012https://doi.org/10.1083/jcb.200903125
- 64.Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeastNat Cell Biol 10:395–406https://doi.org/10.1038/ncb1701
- 65.Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and abscission during yeast cytokinesisJ Cell Biol 202:311–329https://doi.org/10.1083/jcb.201302001
- 66.Cyk3 acts in actomyosin ring independent cytokinesis by recruiting Inn1 to the yeast bud neckMol Genet Genomics 282:437–451https://doi.org/10.1007/s00438-009-0476-0
- 67.Inn1 and Cyk3 regulate chitin synthase during cytokinesis in budding yeastsJ Cell Sci 125:5453–5466https://doi.org/10.1242/jcs.109157
- 68.Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeastNat Commun 15:3383https://doi.org/10.1038/s41467-024-47709-3
- 69.Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding YeastPLoS Genet 12:e1005864https://doi.org/10.1371/journal.pgen.1005864
- 70.A septin-Hof1 scaffold at the yeast bud neck binds and organizes actin cablesMol Biol Cell 31:1988–2001https://doi.org/10.1091/mbc.E19-12-0693
- 71.Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesisMol Biol Cell 24:1290–1304https://doi.org/10.1091/mbc.E12-08-0608
- 72.Dual Function of Cyk2, a cdc15/PSTPIP Family Protein, in Regulating Actomyosin Ring Dynamics and Septin DistributionJournal of Cell Biology 143https://doi.org/10.1083/jcb.143.7.1947
- 73.Roles of Hof1p, Bni1p, Bnr1p, and Myo1p in Cytokinesis inSaccharomyces cerevisiaeMolecular Biology of the Cell 11https://doi.org/10.1091/mbc.11.2.593
- 74.Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatusCytoskeleton (Hoboken) 67:13–22https://doi.org/10.1002/cm.20412
- 75.Effects of Bni5 Binding on Septin Filament OrganizationJ Mol Biol 428:4962–4980https://doi.org/10.1016/j.jmb.2016.10.024
- 76.Bni5 tethers myosin-II to septins to enhance retrograde actin flow and the robustness of cytokinesisbioRxiv https://doi.org/10.1101/2023.11.07.566094
- 77.Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin IIJ Cell Biol 191:1333–1350https://doi.org/10.1083/jcb.201005134
- 78.The role of Bni5 in the regulation of septin higher-order structure formationBiological Chemistry 396https://doi.org/10.1515/hsz-2015-0165
- 79.The septin-associated kinase Gin4 recruits Gps1 to the site of cell divisionMol Biol Cell 28:883–889https://doi.org/10.1091/mbc.E16-09-0687
- 80.Direct phosphorylation and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeastJ Biol Chem 281:27090–27098https://doi.org/10.1074/jbc.M601483200
- 81.The septins function in G1 pathways that influence the pattern of cell growth in budding yeastPLoS One 3:e2022https://doi.org/10.1371/journal.pone.0002022
- 82.Critical Roles of a RhoGEF-Anillin Module in Septin Architectural Remodeling during CytokinesisCurr Biol 30:1477–1490https://doi.org/10.1016/j.cub.2020.02.023
- 83.Septins: the fourth component of the cytoskeletonNat Rev Mol Cell Biol 13:183–194https://doi.org/10.1038/nrm3284
- 84.Mammalian Septins Are Required for Phagosome FormationMolecular Biology of the Cell 19https://doi.org/10.1091/mbc.E07-07-0641
- 85.Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoaDev Cell 8:343–352https://doi.org/10.1016/j.devcel.2004.12.005
- 86.Balancing the length of the distal tip by septins is key for stability and signalling function of primary ciliaThe EMBO Journal 41https://doi.org/10.15252/embj.2021108843
- 87.Regulation of cytokinesis by the Elm1 protein kinase in Saccharomyces cerevisiaeJournal of Cell Science 113https://doi.org/10.1242/jcs.113.8.1435
- 88.Control of Saccharomyces cerevisiae filamentous growth by cyclin-dependent kinase Cdc28Mol Cell Biol 19:1369–1380https://doi.org/10.1128/MCB.19.2.1369
- 89.A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettesYeast 21:947–962https://doi.org/10.1002/yea.1142
- 90.Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routinesYeast 15:963–972https://doi.org/10.1002/(sici)1097-0061(199907)15:10b<963::Aid-yea399>3.0.Co;2-w
- 91.Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeastJ Cell Biol 200:271–286https://doi.org/10.1083/jcb.201208030
- 92.Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiaeTraffic 16:773–786https://doi.org/10.1111/tra.12276
- 93.Mitosis in Living Budding Yeast: Anaphase A But No Metaphase PlateScience 277https://doi.org/10.1126/science.277.5325.574
- 94.A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiaeGenetics 122https://doi.org/10.1093/genetics/122.1.19
- 95.Nat Methodsan open-source platform for biological-image analysis: Fiji pp. 676–682https://doi.org/10.1038/nmeth.2019
- 96.Analysis of local protein accumulation kinetics by live-cell imaging in yeast systemsSTAR Protoc 2:100733https://doi.org/10.1016/j.xpro.2021.100733
- 97.The spindle pole body component Spc98p interacts with the gamma-tubulin-like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachmentThe EMBO Journal 15https://doi.org/10.1002/j.1460-2075.1996.tb00764.x
- 98.Cdc14-dependent dephosphorylation of Inn1 contributes to Inn1-Cyk3 complex formationJ Cell Sci 125:3091–3096https://doi.org/10.1242/jcs.106021
- 99.Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2Cell 75https://doi.org/10.1016/0092-8674(93)90498-F
- 100.Modes of spindle pole body inheritance and segregation of the Bfa1p–Bub2p checkpoint protein complexThe EMBO Journal 20https://doi.org/10.1093/emboj/20.22.6359
- 101.The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4J Cell Biol 193:1033–1048https://doi.org/10.1083/jcb.201101056
- 102.mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cellsJ Cell Sci 135https://doi.org/10.1242/jcs.260288
- 103.Asgard archaea shed light on the evolutionary origins of the eukaryotic ubiquitin-ESCRT machineryNat Commun 13:3398https://doi.org/10.1038/s41467-022-30656-2
- 104.Lre1 directly inhibits the NDR/Lats kinase Cbk1 at the cell division site in a phosphorylation-dependent mannerCurr Biol 23:1736–1745https://doi.org/10.1016/j.cub.2013.07.032
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