Septin organization and actomyosin ring (AMR) dynamics are defective in elm1Δ and gin4Δ cells.

(A) Domain-level architecture of Elm1, Gin4, Kcc4 and Hsl1 kinases. The KA1 domain represents the membrane-binding domain. (B) Representative time-lapse images showing Cdc3-mCherry dynamics in septin-kinase deletion strains, captured at two-minute time intervals (t0=spindle breakpoint). (C) Normalized fluorescence intensity profile showing the temporal kinetics of Cdc3-mCherry in the indicated strains shown in (B) (wild-type: n=41, elm1Δ: n=29, gin4Δ: n=33, hsl1Δ: n=34, kcc4Δ: n=36 cells). (D) Representative time-lapse images of Inn1-mNG constriction captured at two-minute time intervals in septin-kinase deletion strains (t0= appearance of Inn1-mNG at the bud neck). (E) Quantification of the residence time of Inn1-mNG during the onset of cytokinesis in the indicated strains of (D), Kruskal-Wallis nonparametric test (****: p<0.0001, ns: p>0.05). (F) Normalized fluorescence intensity graph showing the kinetics of Inn1-mNG in the indicated strains of (D). A population of elm1Δ and gin4Δ cells exhibiting asymmetric constriction is included in the plot (wild-type: n=21, elm1Δ: n=13, gin4Δ: n=23, hsl1Δ: n=21 and kcc4Δ: n=18 cells).

Gin4 regulates Hof1 organization and dynamics through interaction with the N-terminal membrane-binding F-BAR domain.

(A) Representative time-lapse images of Hof1-mNG in wild-type, elm1Δ and gin4Δ cells imaged at two-minute time intervals (t0=spindle breakpoint). (B) Quantification of the residence time of Hof1-mNG after spindle breakpoint in the indicated strains shown in (A), Kruskal-Wallis nonparametric test (**: p<0.01, ns: p>0.05). (C) Normalized fluorescence intensity profile for the temporal kinetics of Hof1-mNG during cytokinesis in the indicated strains shown in (A), (wild-type: n=33, elm1Δ: n=21, gin4Δ: n=23 cells). (D) Representative images showing the organization of Hof1-mNG at the bud neck in the indicated strains shown in (A). Scale bar-5µm. (E) Bar graph showing the percentage of cells exhibiting misorganization of Hof1 at the cell division site in the indicated strains shown in (D), one-way ANOVA with Tukey’s multiple-comparison test (****: p<0.0001, ns: p>0.05). p-values correspond to the population of cells exhibiting disorganized Hof1-mNG at the bud neck. (N=3, wild-type: n=518, elm1Δ: n=225, gin4Δ: n=384 cells). (F) Representative images from the Yeast Two-Hybrid assay depicting the interaction of full-length Gin4, Gin4-KID and Gin4-KA1 with full-length Hof1. (G) Representative Yeast Two-Hybrid images showing the interaction between Gin4-KA1 (1003-1142aa) and N-terminal F-BAR containing domain of Hof1 (1-350aa). (H) Immunoblot showing the in vitro binding assay for the interaction between 6xHis-bdSUMO-Gin4KA1 and GST-Hof1F-BAR fragments. GST-tagged Hof1 fragment is immobilized on glutathione resin, and bound 6xHis-bdSUMO-tagged Gin4 fragments were analyzed by SDS PAGE. Input (top) and pull-down fractions (middle) were probed with anti-His antibody, while GST-Hof1 fragments (bottom) were detected using anti-GST antibody, (+) present (–) absent. (I) Proposed model depicting the possible interplay between Gin4 and Hof1 at the plasma membrane interface. Created with BioRender.com.

Gin4 controls septin organization and AMR dynamics independently of its kinase activity

(A) Representative images showing septin defects or mislocalization during bud emergence in gin4Δ cells and rescue of the mislocalization by Gin4FL-GFP and Gin4KD-GFP constructs expressed under pTEF promoter. White arrows indicate mislocalized Cdc3-mcherry in the represented strains. Scale bar-5µm. (B) Quantitation of septin mislocalization and its rescue in the indicated strains shown in (A) (wild-type: n=38, gin4Δ: n=32, gin4Δ-Gin4FL-GFP: n=32, gin4Δ-Gin4KD-GFP: n=36 cells). (C) Representative time-lapse images showing Inn1-mNG dynamics acquired at two-minute time intervals in gin4Δ cells expressing Gin4FL and Gin4KD constructs cloned under endogenous promoter (t0=appearance of Inn1-mNG at the bud neck). (D) Plot of normalized fluorescence intensity of Inn1-mNG in indicated strains shown in (C). A population of gin4Δ-Empty vector cells exhibiting asymmetric constriction is plotted in the graph (t0=spindle breakpoint) (wild-type: n=38, gin4Δ-Empty vector: n=21, gin4Δ- Gin4FL: n=39, gin4Δ- Gin4KD: n=31 cells). (E) Quantification of the residence time of Inn1-mNG during cytokinesis in indicated strains of (C), Kruskal-Wallis nonparametric test (****: p<0.0001, ns: p>0.05).

Artificial tethering of Gin4-GFP to the bud neck via Hsl1-GBP restores functionality in elm1Δ cells in a Gin4 kinase-independent manner.

(A) Schematic representation of GFP-GBP artificial tethering strategy, in which the protein of interest is targeted the bud neck by C-terminal GFP tagging, while the binding partner is tagged with GBP. Created with BioRender.com. (B) Representative images showing artificial tethering of Gin4-GFP with Hsl1-GBP in elm1Δ cells. Scale bar-5µm. (C) Bar graph showing the percentage of cells exhibiting round morphology in the indicated strains shown in (B), one-way ANOVA with Tukey’s multiple-comparison test (**: p<0.01, ****: p<0.0001), (N=3, wild-type: n=407, elm1Δ: n=508, elm1Δ-Hsl1-GBP: n=400 cells). (D) Quantification of aspect ratios in the indicated strains shown in (B), Kruskal-Wallis nonparametric statistical test (****: p<0.0001), (N=3, n>150 cells/strain). (E) Graph depicting the raw fluorescence intensity of Gin4-GFP at the large bud in the indicated strains shown in (B), Kruskal-Wallis nonparametric statistical test (***: p<0.001, ****: p<0.0001) (N=3, wild-type: n=125, elm1Δ: n=125, elm1Δ-Hsl1-GBP: n=118 cells). (F) Representative images showing the localization of Gin4FL-GFP and Gin4KD-GFP constructs expressed under the pTEF promoter and their tethering to bud neck with Hsl1-GBP in elm1Δ gin4Δ double-deletion strains. Scale bar-5µm. (G) Stacked column graph showing the percentage of cells exhibiting round and elongated/clumped morphologies in the indicated strains shown in (F), one-way ANOVA with Tukey’s multiple-comparison test (****: p<0.0001, ns: p>0.05). p-values correspond to population of cells exhibiting round morphology (N=3, elm1Δ gin4Δ: n=339, elm1Δ gin4Δ-Empty vector: n=327, elm1Δ gin4Δ- Gin4FL: n=173, elm1Δ gin4Δ- Gin4KD: n=236, elm1Δ gin4Δ-Empty vector-Hsl1-GBP: n=356, elm1Δ gin4Δ-Gin4FL-Hsl1-GBP: n=365, elm1Δ gin4Δ-Gin4KD-Hsl1-GBP: n=459 cells). (H) Quantification of aspect ratios in the indicated strains shown in (F), Kruskal-Wallis nonparametric statistical test (****: p<0.0001, ns: p>0.05) (N=3, n>90 cells/strain). (I) Representative images showing artificial tethering of Gin4-GFP with Hsl1-ka1Δ-GBP in elm1Δ cells. Scale bar-5µm. (J) Stacked column graph showing the percentage of cells exhibiting round and elongated/clumped morphologies in the indicated strains shown in (I), one-way ANOVA with Tukey’s multiple comparison test (****: p<0.0001, ns: p>0.05) p-values correspond to the population of cells exhibiting round morphology (N=3, wild-type: n=674, Hsl1-ka1Δ-GBP: n=419, elm1Δ: n=572, elm1Δ Hsl1-ka1Δ-GBP: n=337 cells). (K) Quantification of aspect ratios in the indicated strains shown in (I), Kruskal-Wallis nonparametric statistical test (****: p<0.0001, ns: p>0.05), (N=3, n>150 cells/strain).

Table summarizing the rescue of elongated or clumped morphology upon redirecting the Nim1-related kinases to the bud neck in elm1Δ cells and Elm1-GFP in gin4Δ cells using GFP-GBP artificial tethering strategy.

Redirecting Gin4 to the bud neck via Hsl1 in elm1Δ cells rescues septin organization and AMR dynamics.

(A) Representative time-lapse montages showing the rescue of mislocalization of both Gin4-GFP and Cdc3-mCherry upon artificial tethering with Hsl1-GBP in elm1Δ cells. Scale bar-5µm. (B) Stacked column graph showing the percentage of cells exhibiting normal septin localization or septin mislocalization to bud cortex during the bud emergence in the indicated strains shown in (A) (wild-type: n=39, Hsl1-GBP: n=39, elm1Δ: n=39, elm1Δ-Hsl1-GBP: n=42 cells). (C) Quantification of Gin4-GFP localization rescue in the indicated strains shown in (A) (wild-type: n=39, Hsl1-GBP: n=39, elm1Δ: n=39, elm1Δ-Hsl1-GBP: n=42 cells). (D) Representative montages of the bud neck showing the constriction kinetics of Myo1-ymScarletI in elm1Δ strains in which Gin4-GFP is artificially tethered to bud neck via Hsl1-GBP. (E) Quantitative analysis for normal localization and mislocalization of Myo1-ymScarletI in the indicated strains shown in (D) (wild-type: n=41, Hsl1-GBP: n=37, elm1Δ: n=39, elm1Δ-Hsl1-GBP: n=44 cells). (F) Graph showing the constriction dynamics of Myo1-ymScarletI in the indicated strains shown in (D). Disappearance of Gin4 from the bud neck is used to normalize the initial time point for Myo1 constriction. Kruskal-Wallis nonparametric statistical test (****: p<0.0001, ns: p>0.05) (wild-type: n=39, Hsl1-GBP: n=30, elm1Δ: n=32, elm1Δ-Hsl1-GBP: n=39 cells).

Gin4 tethering to the bud neck via Hsl1 bypasses the requirement for Elm1 independently of the morphogenetic checkpoint kinase Swe1.

(A) Time-lapse montages representing the dynamics of Cdc3-mCherry in both elm1Δ and elm1Δ swe1Δ strains upon artificial tethering of Gin4-GFP to bud neck with Hsl1-GBP (t0 = bud emergence). Scale bar-5µm. (B) Stacked column graph showing the percentage of cells exhibiting normal localization and mislocalization of Cdc3-mCherry in the indicated strains shown in (A) (wild-type: n=55, swe1Δ: n=49, elm1Δ: n=58, elm1Δ swe1Δ: n=51, elm1Δ-Gin4-GFP: n=55, elm1Δ swe1Δ-Gin4-GFP: n=55, elm1Δ-Gin4-GFP-Hsl1-GBP: n=58, elm1Δ swe1Δ-Gin4-GFP-Hsl1-GBP: n=52 cells). (C) Representative time-series montages showing the localization of Gin4-GFP in both elm1Δ and elm1Δ swe1Δ strains upon artificial tethering of Gin4-GFP to the bud neck via Hsl1-GBP (t0 = bud emergence). Scale bar-5µm. (D) Quantification graphs depicting the normal localization and mislocalization of Gin4-GFP at the bud cortex during bud emergence in the indicated strains shown in (C) (wild-type: n=57, swe1Δ: n=52, Hsl1-GBP: n=54, Hsl1-GBP swe1Δ: n=51, elm1Δ-Gin4-GFP: n=55, elm1Δ swe1Δ-Gin4-GFP: n=55, elm1Δ-Gin4-GFP-Hsl1-GBP: n=58, elm1Δ swe1Δ-Gin4-GFP-Hsl1-GBP: n=52 cells).

Restoring Hsl1-GFP localization to the bud neck via septins or its related kinases can bypass the requirement of Elm1.

(A) Representative images showing artificial tethering of Hsl1-GFP to the bud neck via Shs1-GBP, Bud4-GBP, Kcc4-GBP, Bni5-GBP and Gin4-GBP in elm1Δ cells. Scale bar-5µm. (B) Bar graph showing the percentage of cells exhibiting round morphology in the indicated strains shown in (A), one-way ANOVA with Tukey’s multiple comparison test (****: p<0.0001, ns: p>0.05) (N=3, wild-type: n=724, elm1Δ: n=458, elm1Δ-Shs1-GBP: n=726, elm1Δ-Bud4-GBP: n=717, elm1Δ-Kcc4-GBP: n=862, elm1Δ-Bni5-GBP: n=358 and elm1Δ-Gin4-GBP: n=754 cells). (C) Quantification of aspect ratios in the indicated strains shown in (A), Kruskal-Wallis nonparametric statistical test (***: p<0.001, ****: p<0.0001) (N=3, n>135 cells/strain). (D) Representative montages of the bud neck depicting the localization of Hsl1-GFP in elm1Δ and gin4Δ cells during cytokinesis. DC*=Differential contrast. (E) Plot of raw fluorescence intensity of Hsl1-GFP in the indicated strains shown in (D) (t0=spindle breakpoint) (wild-type: n=29, elm1Δ: n=24, gin4Δ: n=38 cells). (F) Plot of normalized fluorescence intensity of Hsl1-GFP in the indicated strains shown in (D) (t0=spindle breakpoint).

A non-canonical role for Hsl1-Kinase in regulating septin organization and AMR dynamics.

(A) Representative images showing Cdc3-mCherry mislocalization during bud emergence in gin4Δ and gin4Δ hsl1Δ cells in which Elm1-GFP is artificially tethered to bud neck via Shs1-GBP. White arrows indicate mislocalized Cdc3-mCherry in the represented strains. Scale bar-5µm. (B) Representative time-lapse montages of the bud neck showing the constriction profile of Inn1-3xmCherry in gin4Δ and gin4Δ hsl1Δ cells in which Elm1-GFP is artificially tethered to bud neck via Shs1-GBP. (C) Bar graph showing the percentage of cells exhibiting round and elongated morphologies in the indicated strains shown in (A), one-way ANOVA with Tukey’s multiple-comparison test (**: p<0.01, ***: p<0.001, ns: p>0.05). p-values correspond to population of cells exhibiting round morphology, (N=3, gin4Δ: n=689, gin4Δ-Shs1-GBP: n=639, gin4Δ hsl1Δ: n=492, gin4Δ hsl1Δ-Shs1-GBP: n=590 cells). (D) Quantification of aspect ratios in the indicated strains shown in (A), Kruskal-Wallis nonparametric statistical test (****: p<0.0001, ns: p>0.05), (N=3, n>165 cells/strain). (E) Stacked column graph representing the percentage of cells exhibiting normal septin localization and septin mislocalization in the indicated strains of (A) (gin4Δ: n=44, gin4Δ-Shs1-GBP: n=41, gin4Δ hsl1Δ: n=38, gin4Δ hsl1Δ-Shs1-GBP: n=44 cells). (F) Quantification of normal localization and mislocalization of Elm1-GFP in the indicated strains shown in (A) (gin4Δ-Shs1-GBP: n=37, gin4Δ hsl1Δ-Shs1-GBP: n=44 cells) (G) Quantification of the residence time of Inn1-3xmcherry during cytokinesis in the indicated strains shown in (B), Kruskal-Wallis nonparametric statistical test (*: p<0.05, **: p<0.01, ****: p<0.0001, ns: p>0.05) (N=2, n>60 cells/strain).

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

Created with BioRender.com.

Yeast strains used in this study

Oligonucleotides used in this study

Plasmids used in this study