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).

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.

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.

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).

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

Representative model for the role of Hsl1 kinase in septin organization and actomyosin ring constriction downstream of Gin4 and Elm1.

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