Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42

Article Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42 Graphical abstract Highlights dOxidative stress causes Sty1-kinase-dependent inactivation of Cdc42 at cell poles dThe GAPs Rga3/6 and the GEF Gef1 are direct targets of Sty1 kinase dIncreased GAP activity at the cell poles mediates Cdc42 depolarization upon stress dGef1 delocalization from cell tips promotes stress-dependent Cdc42 inactivation Authors Cla `udia Salat-Canela, Merce `Carmona, Rebeca Martı ´n-Garcı´a, Pilar Pe ´rez, Jose ´Ayte ´, Elena Hidalgo Correspondence [email protected] (P.P.), [email protected] (J.A.), [email protected] (E.H.) In brief MAP kinases allow eukaryotic organisms to cope with environmental challenges. The fission yeast MAP kinase Sty1 couples stress signals with the Cdc42 polarity module, leading to the inhibition of polarized cell growth. Salat-Canela et al. decipher the molecular mechanism downstream of Sty1 that mediates the Cdc42 depolarization response. Salat-Canela et al., 2021, Cell Reports 37, 109951 November 2, 2021 ª2021 The Author(s). https://doi.org/10.1016/j.celrep.2021.109951 ll Article Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42 Cla `udia Salat-Canela, 1 Merce `Carmona, 1 Rebeca Martı ´n-Garcı ´a, 2 Pilar Pe ´rez, 2, *Jose ´Ayte ´, 1, *and Elena Hidalgo 1,3, * 1 Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, 08003 Barcelona, Spain 2 Instituto de Biologı ´a Funcional y Geno ´mica (IBFG), Consejo Superior de Investigaciones Cientı ´ficas, Universidad de Salamanca, 37007 Salamanca, Spain 3 Lead contact *Correspondence: [email protected] (P.P.), [email protected] (J.A.), [email protected] (E.H.) https://doi.org/10.1016/j.celrep.2021.109951 SUMMARY Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Dsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation. INTRODUCTION Ras-homologous (Rho) GTPases regulate cell polarity and growth in eukaryotes. In particular, Cdc42 has a central role in the establishment of polarized growth. In budding yeast, Cdc42 localizes exclusively at the site of bud growth during division, but this model system cannot be used to explain multiple growing areas existing in other cell types, including fission yeast. Schizosaccharomyces pombe cells display not only Cdc42 activity sites at the area of cell division but also monopolar Cdc42-dependent growth from the old end just after cell division, and they shift to a bipolar mode when reaching a certain length in a process known as NETO (new-end takeoff) (Mitchison and Nurse, 1985). Therefore, S. pombe is an excellent model to study multiple polarity sites. Furthermore, wild-type cells have a rodlike phenotype, while decreased Cdc42 levels lead to a rounded phenotype (Miller and Johnson, 1994). Cdc42 localization and activation are tightly regulated in space and in time. Cdc42 acts as a switch that can be found in a GTPor a GDP-bound state, and this modulates its ability to interact with other components. Therefore, different GEFs (guanine nucleotide exchange factors) activate Cdc42, and GAPs (GTPase-activating proteins) negatively regulate its activity. Additionally, S. pombe Cdc42 local activation promotes its accumulation at the growth regions of the plasma membrane (Bendezu ´et al., 2015;Estravı ´s et al., 2017). Two GEFs have been characterized for Cdc42: Scd1, which localizes at cell tips with Cdc42-GTP during interphase (Kelly and Nurse, 2011), and Gef1, with apparent cytoplasmic localization (Tay et al., 2018). Both GEFs are found at the cytokinetic ring during cell division (Coll et al., 2003;Hirota et al., 2003). Regarding GAPs, Rga4 was described early; it localizes to the cell sides (Das et al., 2007;Tatebe et al., 2008). Rga6 has similar lateral localization, and it has been proposed to collaborate with Rga4 to spatially restrict active Cdc42 to the cell tips (Revilla-Guarinos et al., 2016). Rga3 has been recently described as a Cdc42 GAP localized to the cell tips. Its absence does not lead to any detectable phenotype during mitotic cell growth. Instead, Rga3 does play a role during sexual reproduction by regulating Cdc42 activity at the polarity patch (Gallo Castro and Martin, 2018). How are new polarity sites established at a given position? In both Saccharomyces cerevisiae and S. pombe, the formation of a quaternary complex between Cdc42, a p21-activated kinase (PAK1/CLA4 or Pak1), a scaffolding protein (BEM1 or Scd2), and the GEF (CDC24 or Scd1) is crucial to enhance local activation of Cdc42. The recruitment of the GEF for Cdc42-GTP at the membrane favors the activation of neighboring Cdc42 molecules, creating a positive feedback loop that contributes to the creation of new polarity sites (for a review, see Chiou et al., 2017). In fission yeast, the distinct spatial distribution of GEFs and GAPs is also vital to seed sites of polarity. Therefore, the landmark established by Tea1 and Tea4, driven to the cell tips by microtubules, promotes local activation of Cdc42 at cell tips by excluding Rga4 (Kokkoris et al., 2014). Cell Reports 37, 109951, November 2, 2021 ª2021 The Author(s). 1 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ll OPEN ACCESS While positive feedback loops probably explain the concentration in space and time of the Cdc42 module at one particular position (for review, see Chiou et al., 2017;Martin, 2015), negative regulators of active Cdc42 are required to explain the oscillatory behavior of the module in post-NETO bipolar fission yeast cells (Das et al., 2012) and limit the spreading of the polarity cluster. A relevant example of the later is the inhibitory phosphorylation of S. cerevisiae CDC24, the main GEF of CDC42, by its downstream effector PAK, to downregulate the polarity sites as a negative feedback loop (Gulli et al., 2000;Kuo et al., 2014;Rapali et al., 2017;Wai et al., 2009). Stress signals mediated by the mitogen-activated protein (MAP) kinase Sty1 pathway in fission yeast cause dispersion of the Cdc42 polarity module (Mutavchiev et al., 2016). Thus, latrunculin A (LatA)-activated Sty1 promotes Cdc42-GTP dispersal from cell tips. In response to environmental stresses, Sty1 is phosphorylated and triggers a core environmental stress response, mainly based on the transcription factor Atf1. Stressdependent phosphorylation of the MAP kinase, which is kept inactive in the absence of stress by several phosphatases such as Pyp1 (Millar et al., 1995;Shiozaki and Russell, 1995b), triggers its nuclear accumulation and the phosphorylation of Atf1 (SalatCanela et al., 2017;Wilkinson et al., 1996). Cells lacking Sty1 show reduced tolerance to many environmental stresses such as heat shock, oxidative stress, and nutritional deprivation (Toone et al., 1998;Zuin et al., 2010). Dsty1 cells display an elongated ‘‘cdc’’-like phenotype, suggesting that Sty1 may also have a role at the G2/M transition (Shiozaki and Russell, 1995a). Here, we investigated whether environmental stress regulating the Sty1 pathway could modulate the activity of Cdc42 at the growth area and whether this regulation could explain the elongated phenotype of Dsty1 cells. We show here that hydrogen peroxide (H 2 O 2 ), which transiently activates Sty1, also triggers active Cdc42 dispersal from the cell tips in a Sty1-dependent manner without promoting actin depolymerization and that stress-independent Sty1 activation is sufficient to induce these effects on polarity. We demonstrate that Sty1 activity inversely correlates with active Cdc42 at cell tips and, importantly, with cell length. Furthermore, Sty1’s inhibitory role in Cdc42 activity may be exerted through activation at cell tips of Rga3 and Rga6 GAPs and the combined Gef1 delocalization and inactivation of Scd1, with a net transient inhibition of Cdc42 and polarized growth cessation. Indeed, we demonstrate that Rga3 and Gef1 are direct targets of active Sty1. RESULTS H 2 O 2 triggers a Sty1-dependent and actin depolymerization-independent inactivation of Cdc42 at cell tips In the presence of the actin depolymerization drug LatA, the active Cdc42 polarity module is dispersed from cell tips in a Sty1-dependent manner (Mutavchiev et al., 2016). Sty1 is strongly activated by H 2 O 2 to trigger a gene expression program (Chen et al., 2003). We used GFP-tagged CRIB (Cdc42/Rac interactive binding motif) to monitor active GTP-bound Cdc42 in the presence of H 2 O 2 (Ozbudak et al., 2005;Tatebe et al., 2008). As shown in Figure 1A, treatment of wild-type cells with either LatA or H 2 O 2 causes dispersal of active Cdc42 from the cell tips to lateral patches (indicated with arrows). The loss of CRIB-GFP at cell poles follows similar kinetics upon LatA and H 2 O 2 treatments (Figure 1B). We used Lifeact-mCherry as a marker to label F-actin in living cells (Huang et al., 2012). As reported before (Mutavchiev et al., 2016), actin depolymerization upon LatA imposition is faster than CRIB-3GFP dispersal from tips (Figure 1A). H 2 O 2 treatment does not significantly perturb the actin cytoskeleton (Figure 1A) and nevertheless causes CRIB-3GFP dispersal. Both types of treatments (the irreversible inhibitor LatA and the transient natural stressor H 2 O 2 ) cause a permanent or temporal growth inhibition (respectively), as demonstrated by growth curves (Figure S1A) and measuring net cell elongation rates after stress imposition in time-lapse experiments (Figures 1C and S1B). In conclusion, oxidative stress causes inactivation of the polarity module at cell tips, which is concomitant to cell growth cessation. Stress-dependent positioning of the Cdc42 polarity module to lateral patches is dependent on the Cdc42 GEF Gef1 Stress imposition causes CRIB dispersal from the tips to lateral patches upon H 2 O 2 (see arrows in Figure 1A) or LatA treatments (Bendezu ´and Martin, 2011;Mutavchiev et al., 2016). We tested the presence of different Cdc42 module components at both cell sites before and after stress imposition. As shown in Figure 2A, the main Cdc42 GEF Scd1 and its scaffold, Scd2, are localized to cell tips prior to stress and are totally (Scd1-GFP) or partially (Scd2-GFP) dispersed from the poles upon peroxide treatment. The downstream effector kinase Pak1 is also dispersed from the poles after stress imposition, probably as a consequence of Cdc42 inactivation (Figure 2A). Upon exit from the cell tips, Scd2 and Pak1, but not Scd1, are present at the lateral patches of active Cdc42 (see white arrows in Figure2A), suggesting that a GEF other than Scd1 is responsible for the active Cdc42 present at lateral surfaces, which appear after stress imposition. In fact, the Scd1 scaffold Scd2 is not required for the formation of the active Cdc42 patches (Figure S2A). Gef1-3YFP is located at the cytosol but also decorates the tips of non-dividing cells prior to stress; the localization at tips is lost upon H 2 O 2 stress, with Gef1-3YFP clearly localizing to lateral patches 45 min after stress imposition (see white arrow in Figure 2B). Indeed, while dispersal of CRIB-3GFP from cell tips upon H 2 O 2 (Figure 2C) or LatA (Figure S2B) treatment is not blocked in strain Dgef1, the formation of the lateral patches is completely dependent on the presence of Gef1 (Figures 2Cand S2B), as shown using other stressors (Chen et al., 2019;Hercyk et al., 2019). We conclude that stress promotes redistribution of the active Cdc42 polarity module from the cell tips toward side patches, which contain and require Gef1, but not Scd1 (Figure 2D). We will use hereafter these lateral sites as a secondary hallmark of stress-dependent cell growth arrest. Active Sty1 directly promotes the inactivation of Cdc42 at cell tips To confirm that active Sty1 directly drives the redistribution of the Cdc42-dependent polarity module from the poles to the sides of the cell, we pursued three types of strategies: (1) we first tested the effect of H 2 O 2 in CRIB-3GFP dispersal in cells lacking Sty1, 2Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS (2) we analyzed whether the downstream transcription factor Atf1 is required to trigger H 2 O 2 -dependent cellular depolarization, and (3) we used genetically engineered cells expressing a stress-independent active Sty1 allele to study the effect of this Sty1 on Cdc42 activity. We expressed CRIB-tdTomato (Revilla-Guarinos et al., 2016) in wild-type and Dsty1 cells overexpressing catalase to minimize the direct toxic effect of intracellular peroxides in cells lacking the MAP kinase. Overexpression of catalase in wild-type cells does not alter the inhibitory effect of stress on active Cdc42 at cell tips (Figures 3A and S3A). Active Cdc42 is not redistributed from cell poles to cell sides upon H 2 O 2 addition in cells lacking Sty1 (Figures 3A and 3B), and polarized growth is not inhibited in these cells (Figures 3C and S3B). Second, the absence of Atf1, required to trigger the transcriptional response (Chen et al., 2003,2008), does not largely affect CRIB-3GFP dispersal from cell tips upon stress imposition (Figures S3C and S3D), which suggests that Sty1 kinase activity is itself required at cell tips. In a third strategy, we monitored CRIB-3GFP in cells expressing the analog-sensitive mutant Sty1.AS from the endogenous sty1 locus (Gregan et al., 2007;Zuin et al., 2010) and lacking Pyp1, the main Sty1 phosphatase (Millar et al., 1995;Shiozaki and Russell, 1995a)(Figure 3D). In the Sty1.AS protein, the T97A mutation Figure 1. Activation of the Sty1 MAP kinase by oxidative stress leads to Cdc42 depolarization from cell tips (A) CRIB dispersal from cell tips after oxidative stress imposition follows similar kinetics than in LatA treatment. Images depicting GTP-bound Cdc42 and F-actin at several points from timelapse experiments in YE cultures of CS208 (expressing CRIB-3GFP and Lifeact-mCherry) during DMSO, 1 mM H 2 O 2 ,or50mM LatA treatments. Arrows point to the formation of Cdc42-GTP lateral patches. (B) Quantification of CRIB-3GFP intensity at cell tips in time-lapse experiments as described in (A). Data are represented as a percentage with respect to time zero. The shaded area represents SD. At least nine cells were analyzed for each condition from three independent biological replicates. (C) Cell growth ceases after treatment with LatA or H 2 O 2 . Kymographs showing CRIB-3GFP cells from time-lapse experiments as shown in (A). See also Figure S1. in the kinase ATP pocket facilitates the entry of the bulky inhibitory ATP analog 3MBPP1, which fully blocks H 2 O 2 -dependent Sty1.AS activity without affecting wildtype Sty1 (Figures 3E and S3E). Cells lacking Pyp1 display basal activation of Sty1 under unstressed conditions (Shiozaki and Russell, 1995a), but addition of 3MBPP1 keeps Sty1.AS inactive and downstream Atf1 dephosphorylated, until the analog is withdrawn (Figure 3E). In the presence of the kinase inhibitor, CRIB3GFP expressed in Dpyp1 sty1.AS cells localizes to cell tips, but removal of the analog quickly triggers CRIB dispersal from the poles (Figure 3F), with kinetics similar to the effect of H 2 O 2 addition to CRIB-3GFP-expressing wild-type cells (compare Figure S3F and Figure 1B). We conclude that direct activation of the Sty1 kinase, either genetically or upon stress imposition, is sufficient to redistribute the Cdc42 polarity module away from the cell tips. Cell length and activity of Cdc42 at the tips inversely correlate to Sty1 activity The rod-shaped morphology and tip elongation growth of fission yeast has enabled the isolation of mutations advancing or delaying cell-cycle progression based on cell length at division. In fact, a genetic interaction was described more than two decades ago between Sty1 and the cell-cycle machinery (Shiozaki and Russell, 1995a). We have measured cell length and CRIB intensity (as a measure of active Cdc42 levels) at cell poles of different MAP kinase pathway mutants before cell division (Figures 4A– 4C). While Dsty1 cells are longer than wild-type cells, Dpyp1 cells (in which the Sty1 pathway is constitutively activated) are shorter (Figures 4A and 4B). Importantly, CRIB-3GFP expression does not affect cell size in the different backgrounds (compare Figures 4A and 4B with Figure S4A). It is worth noting that Datf1 cells are Cell Reports 37, 109951, November 2, 2021 3 Article ll OPEN ACCESS similar in size to wild type, confirming our previous observation that the effect of Sty1 in Cdc42 regulation is independent of transcription (Figure 4B). Regarding the levels of active Cdc42 (CRIB-3GFP) at cell tips (Figure 4C), we determined a 2-fold increase in CRIB-3GFP fluorescence intensity in Dsty1 cells; in contrast, we detected a significant decrease of active Cdc42 at the tips of Dpyp1 cells, enforcing the idea that Sty1 activity is required to control Cdc42 at cell tips also under unstressed conditions. We did not observe enhanced CRIB levels in cell-cycle mutants with elongated phenotypes, such as cdc25-22 (data not shown), suggesting that cell size per se does not control active Cdc42 at cell tips. A consequence of active Cdc42 deposition at cell tips may be an increase in the growth rate. We measured cellular growth rates of wild-type, Dsty1,Datf1, and Dpyp1 strains by incubating cultures with fluorescein isothiocyanate (FITC)-lectin, which stains cell surface carbohydrates in green; the dye was washed out, and growth proceeded for 90 min to expose unlabeled growth areas, which were counterstained with calcofluor (visualized in red) to highlight new growth surfaces (see STAR Methods for details) (Figure S4B). In order to compare strains pairwise, we labeled wild-type cells with a nuclear fluorescent reporter, Hta1mRFP. This tag did not affect the growth rate of wild-type cells (Figure 4D, left panel). While wild-type cells (labeled with Hta1mRFP) elongated at a rate of 3mm/h, Dsty1 cells did it much faster (R4mm/h), and Dpyp1 cells elongated slower (%2mm/h) (Figure 4D). As expected, Datf1 cells grew at the same rate as wild-type cells. To completely separate cytosolic from nuclear Sty1 activity, we generated a Sty1 chimera fused to the Tea1 polarity marker, which localizes to cell tips (Mata and Nurse, 1997). This Tea1Sty1-GFP chimera localized at cell tips in a wild-type or Dsty1 background (Figure 4E). We also confirmed that this chimera is unable to activate the transcriptional cascade downstream of Sty1 using two different approaches: (1) in the absence of endogenous Sty1, Atf1 was not phosphorylated upon H 2 O 2 treatment, indicating that the chimera lacks Sty1 nuclear functions (Figure S4C); (2) Dsty1 cells expressing Tea1-Sty1-GFP are as sensitive to H 2 O 2 in liquid growth as cells lacking Sty1 (Figure S4D). Altogether, these results demonstrate than Tea1-Sty1GFP lacks nuclear functions. We then tested whether the chimera, which is constitutively trapped at cell tips, contributed to regulate cell length at septation. As can be observed in Figure 4F, expressing Tea1-Sty1-GFP slightly, but significantly, Figure 2. Formation of active Cdc42 lateral patches is dependent on Gef1 (A) Scd2 and Pak1 proteins are localized at Cdc42-GTP lateral patches after stress imposition. Images from logarithmic YE cultures of PPG56.66 (Scd1-GFP), PPG142.42 (Scd2-GFP), and PPG69.03 (Pak1-GFP), treated or not with 1 mM H 2 O 2 . White arrows point to lateral patches. (B) Gef1 is the unique GEF detected at lateral patches. Images from logarithmic YE cultures of FV1218 (Gef1-3YFP), treated or not with 1 mM H 2 O 2 . White arrows point to lateral patches. (C) Activation of Cdc42 at cortical regions exclusively depends on Gef1. Images of GTP-bound Cdc42 from logarithmic YE cultures of PPG65.60 (wild-type strain expressing CRIB-3GFP) and PPG70.07 (Dgef1 strain expressing CRIB-3GFP), treated or not with 1 mM H 2 O 2 . Arrowheads point to the presence of Cdc42-GTP at lateral patches. (D) Scheme depicting the localization of several Cdc42 polarity module components before and after stress imposition. Scale bar, 5 mm. See also Figure S2. 4Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS reduced cell length at division in wild-type cells. More noticeably was the effect in a Dsty1 background, where expressing Tea1Sty1-GFP reduced the length at septation from R20 mmto 15 mm(Figure 4F); importantly, CRIB levels in Dsty1 cells were also reduced by expression of Tea1-Sty1 (Figure S4E). We propose that Sty1 activity inversely correlates with Cdc42 activity at cell tips (Figure 4G). Cells devoid of the Cdc42 GAPs Rga3 and Rga6 are insensitive to stress-dependent cell polarity inhibition In the search for downstream effectors of Sty1 in the polarity module, we first interrogated the putative GAPs of Cdc42. Rga4 and Rga6 localize to cell sides and contribute to the spatial restriction of active Cdc42 at cell poles (Revilla-Guarinos et al., 2016;Tatebe et al., 2008), while Rga3 co-exists with Cdc42-GTP at growth sites Figure 3. Cdc42 depolarization from cell tips after oxidative stress depends on Sty1 activity (A) CRIB signal is not dispersed form cell tips upon H 2 O 2 treatment in Dsty1 cells. Images of GTPbound Cdc42 from time-lapse experiments of YE cultures of CS245 (wild-type cells expressing CRIB-tdTomato and Ctt1) and CS238 (Dsty1 cells expressing CRIB-tdTomato and Ctt1) untreated (unt) or treated with 1 mM H 2 O 2 . (B) Quantification of CRIB-tdTomato signal at cell tips in time-lapse experiments as described in (A). Data representation was done as in Figure 1B. (C) Cell growth is not halted in Dsty1 cells after stress imposition. Kymographs showing CRIBtdTomato cells from time-lapse experiments as described in (A). (D and E) Schematic representation of the sty1.AS Dpyp1 strain. In wild-type cells, Sty1 activation is counteracted by the phosphatase Pyp1, eventually leading to the shutoff of the cascade. In the sty1.AS Dpyp1 mutant, Sty1 can be artificially activated by removing the ATP analog. (F) Stress-independent activation of Sty1 leads to Cdc42-GTP dispersal from cell tips. Images of GTP-bound Cdc42 staining from YE cultures of CS147 (sty1.AS Dpyp1 expressing CRIB-3GFP) in the presence of or after the removal of 10 mM of the inhibitor 3MB-PP1. See also Figure S3. (Gallo Castro and Martin, 2018). We first monitored the localization of Rga3, Rga4, and Rga6 after environmental stress. We co-expressed CRIB-3mCherry and GFPtagged versions of each GAP. While a weak signal of Rga3-GFP can be detected at cell tips during unstressed conditions, the green fluorescence of the GFP protein is significantly increased after treatment with H 2 O 2 , inversely correlating with the CRIB-3mCherry fluorescence (Figures 5A and 5B). Rga4-GFP remains in lateral patches before and after stress imposition (Figure 5C), and Drga4 cells are sensitive to CRIB-GFP tip depolarization after H 2 O 2 stress (Figure S5A), discarding any role of this GAP in Sty1-dependent Cdc42 inhibition. After H 2 O 2 imposition, Rga6-GFP still localizes at the lateral membranes but it is also present at the cell tips (Figure 5C), as reported before upon LatA treatment (Figure S5B) (Revilla-Guarinos et al., 2016). Moreover, in Dsty1 cells, Rga6-GFP is excluded from cell tips even after LatA treatment (Figure S5B), indicating that Rga6 localization may be somehow regulated by Sty1. Therefore, two GAPs are present at cell tips after stress (Rga3 and Rga6) and are candidates to trigger Cdc42 inactivation (Figure 5D). We then tested the effect of single and double deletions of GAP-coding genes on stress-dependent inactivation of cell polarity. Cells lacking Rga3 retain active Cdc42-GTP at cell tips 60 min after H 2 O 2 imposition (Figures 5E and 5F), while Drga6 cells display H 2 O 2 inactivation kinetics similar to wild-type cells Cell Reports 37, 109951, November 2, 2021 5 Article ll OPEN ACCESS (Figures S5C and S5D). The growth poles of double Drga3 Drga6 mutant cells retain higher Cdc42 activity than single Drga3 cells and did not show lateral patches after H 2 O 2 imposition (Figures 5E and 5F). We conclude that cells lacking the Cdc42 GAPs Rga3 and Rga6 do not display two important hallmarks linked to stress-dependent cell polarity inhibition: dispersal of CRIB-3GFP from poles and appearance of Cdc42-GTP lateral patches. Unlike cells lacking Sty1, cell growth is still inhibited by stress in Drga3 Drga6 cells (Figure 5G), and the main GEF Scd1 is still dispersed from the tips upon stress in cells lacking Rga3 (Figure S5E); all of this suggests that other components of the polarity module may be also targets of the active kinase. Figure 4. Cell length and Cdc42 activation at the poles are inversely correlated with Sty1 activity (A) CRIB levels at cell tips are inversely correlated with Sty1 activation. Images of from logarithmic YE cultures of PPG65.60 (WT), CS137 (Dsty1), CS133 (Datf1), and CS207 (Dpyp1) expressing CRIB3GFP. (B) Cell length at septation of logarithmic YE cultures of strains as in (A). Mean, SD, and number of cells analyzed (n) are indicated. Statistical significance was assessed by ANOVA test followed by Bonferroni post hoc analysis (*p < 0.05; ***p < 0.001). (C) Quantification of CRIB intensity at cell tips of the strains shown in (A). Whiskers indicating minimum to maximum are shown, horizontal line represents mean, and n indicates the number of cells analyzed. Statistical significance was calculated as in (B) (ns, not significant). (D) Cdc42 activity is directly related to the growth rate. Cell growth rates of MM cultures of wild-type (WT), JA1329 (wild-type cells expressing Hta1mRFP), AV18 (Dsty1), MS98 (Datf1), and EP48 (Dpyp1) were quantitated. Graphs show values distribution as in (B); statistical significance was determined using unpaired t test (***p < 0.001; n.s. not significant). (E) Tea1-Sty1-GFP delivers Sty1 to cell tips. Images from MM cultures of CS309 (wild-type cells expressing the Tea1-Sty1-GFP under the nmt81 integrative promoter) and CS310 (Dsty1 cells expressing the same construct) are shown. (F) Trapping Sty1 to cell tips is sufficient to restore wild-type cell size. Cell length distribution at septation of strains as in (E) was measured and is represented as in (B). Statistical significance to wild-type strain was assessed as in (B) (***p < 0.001). (G) Schematic representation indicating the inverse proportionality between Sty1 activity and cell length and Cdc42-GTP activation at cell poles. Scale bar, 5 mm. See also Figure S4. Inhibition of GEF activity mediates the stress-dependent effect on growth polarity We next tested whether inactivation or delocalization of the two S. pombe GEFs, Gef1 or Scd1, could mediate the Sty1-dependent inhibition of Cdc42-GTP. Cells lacking Scd1 are rounded due to its essential role in establishing sites of polarity and display weak patches of active Cdc42 (Figure S6A). In an attempt to recover cell polarity of Dscd1 cells through the artificial tethering of either Gef1 or Scd1 to cell tips, we expressed in this background chimeras of Gef1 or Scd1 fused to Tea1. As shown in Figure 6A, both GEFbased fusion proteins, Tea1-Scd1-GFP and Tea-Gef1-GFP, when expressed in cells lacking Scd1, are localized to cell tips and able to promote the accumulation of CRIB-mCherry at the growing poles. They partially suppress the rounded phenotype ofstrainDscd1 both inlength(Figure6B)and in width (FigureS6B). With this constitutively tethered GEF-based chimeras in cells 6Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS lacking Scd1, we tested the effect of stress on polarized growth. Probably due to the different conformation of the large chimeras, the fluorescence levels of Tea1-Scd1-GFP and Tea1-Gef1-GFP are different, but both proteins are retained at cell poles after stress imposition, as expected (Figures 6C and 6D), and the same occurs with Tea1-GFP (Figures S7A and S7B). H 2 O 2 causes dispersal of active Cdc42 from the cell tips to lateral patches in Dscd1 cells expressing Tea1-Scd1-GFP (Figures 6C and 6E), indicating that tethering Scd1 at cell poles does not prevent the inhibition of Cdc42 activity upon stress (see Figure 2A). In contrast, Dscd1 cells expressing Tea1-Gef1-GFP are blind to stress imposition; CRIB-mCherry remains at cell tips, and lateral patches are not formed (Figures 6C and 6E). Importantly, inverFigure 5. Cells lacking the Cdc42 GAPs Rga3 and Rga6 are insensitive to stressdependent cell polarity inhibition (A) Rga3 is enriched at cell poles after stress imposition. Images from logarithmic MM cultures of CS391 (expressing CRIB-3mCherry and a GFPtagged version of Rga3 from its own locus), treated or not with 1 mM H 2 O 2 . (B) Quantification of Rga3-GFP intensity along the cell tips of cells as in (A). Shaded areas represent SD, and n indicates the number of tips analyzed. (C) Rga6 spreads to cell tips after stress imposition. Images with merged staining of GTP-bound Cdc42 (in magenta) and GFP-tagged versions of Rga4 or Rga6 (in green). YE cultures of CS361 (expressing CRIB-3mCherry and Rga4-GFP) and CS362 (expressing CRIB-3mCherry and Rga6GFP) were treated or not with 1 mM H 2 O 2 . (D) Schematic representation of GAPs cellular distribution before and after H 2 O 2 imposition. See text for details. (E) CRIB dispersal after stress imposition is blocked in cells lacking Rga3 and Rga6. Images of GTP-bound Cdc42 from YE cultures of PPG65.60, CS244, and CS315 (expressing CRIB-3GFP in wild-type, Drga3, and Drga3 Drga6 backgrounds, respectively) treated or not with 1 mM H 2 O 2 . White arrows point to lateral patches. (F) Quantification of CRIB-3GFP intensity at cell tips of strains as in (E). Whiskers represent minimum to maximum values, horizontal lines indicate the mean, and n indicates the number of tips analyzed for each condition. Statistical analysis was performed as in Figure 4B (ns, not significant; **p < 0.01; ***p < 0.001). (G) Cells lacking Rga3 and Rga6 do not elongate after stress imposition. Kymographs showing CRIB-3GFP cells from time-lapse experiments as in (E). Scale bar, 5 mm. See also Figure S5. sion of the fluorescent tags (Tea1-Gef1mCherry and CRIB-3GFP) in Dscd1 cells (Figures S6C and S7C) or in a Dgef1 background (Figure S6D) also yields stressinsensitive strains. This experiment suggests that removal of Gef1 from cell tips could be one of the molecular events leading to the stress-dependent Cdc42 inactivation in wild-type cells. In concordance, cells lacking Gef1 suffer faster stress-dependent inhibition of active Cdc42 at the poles than wild-type cells (compare 15 min after H 2 O 2 imposition in both strain backgrounds in Figure 2C), and exit of Gef1-3YPF from cells tips upon stress depends on Sty1 (Figure S7D), suggesting a possible participation of this GEF in the inactivation of Cdc42 at cell tips. Rga3, Rga6, and Gef1 are direct targets of the MAP kinase Sty1 To identify direct molecular targets of Sty1 in the polarity module, we developed an in vitro phosphorylation assay with Cell Reports 37, 109951, November 2, 2021 7 Article ll OPEN ACCESS recombinant proteins. We purified several recombinant chimeras fused to glutathione S-transferase (GST) in Escherichia coli, including the MAP kinase Sty1, a kinase-dead version Sty1.K49R (carrying a mutation in the ATP binding site), and the upstream constitutively activated version of the MAP kinase Wis1.DD (Shiozaki et al., 1998). We used as a control for the kinase reaction a bona fide substrate of Sty1, the transcription factor Atf1 (Wilkinson et al., 1996), that presents 11 MAP kinase sites (Lawrence et al., 2007). Since the GST-fused full-length Atf1 is degraded during protein purification (Figure S8A), we tested in parallel a truncated version carrying eight of the MAP kinase sites (Atf1 71–291 in Figure S8A). We also tested as substrates full-length Atf1 carrying mutations at the MAP kinase Figure 6. Trapping Gef1 to cell tips creates stress-blinded poles (A) Trapping GEFs to cell tips restores cell morphology and GTP-Cdc42 levels in Dscd1 cells. Images of CRIB and GFP from MM cultures of CS357, CS359 (wild-type and Dscd1 strains, respectively, expressing CRIB-3mCherry), CS365, and CS360 (Dscd1 strains expressing CRIB3mCherry with Tea1-Scd1-GFP or Tea1-Gef1GFP fusion proteins, respectively). (B) Cell-length distribution at septation of strains as in (A). Mean, SD, and number of cells analyzed (n) are indicated. Statistical differences were calculated as in Figure 4B (*p < 0.05; ***p < 0.001). (C) Trapping Gef1 to cell tips, but not Scd1, retains GTP-Cdc42 at tips after stress imposition. Images of CRIB and GFP from MM cultures of CS365 and CS360 treated with 1 mM H 2 O 2 . (D) The Tea1-GEF-GFP fusion protein is anchored at cell tips after stress imposition. Quantification of GFP intensity at cell tips of strains as in (C), represented as in Figure 5F. Statistical analysis was performed as in Figure 4B (ns, not significant; ***p < 0.001). (E) Quantification of CRIB-3mCherry intensity at cell tips of strains as in (C), represented as in (D). Scale bar, 5 mm. See also Figures S6 and S7. sites (10M and 10D in Figure S8A). In the kinase reaction, we first activated Sty1 with Wis1.DD in the presence of cold ATP to then add the purified substrates, Atf1 and derivatives, and Ɣ32 P-ATP. As shown in Figures 7A and S8A, when Atf1 was incubated with pre-activated Sty1, but not with the kinase-dead Sty1.K49R, the transcription factor was unambiguously phosphorylated by Sty1. Full-length or truncated Atf1 are both phosphorylated to similar extents, while the 10M and 10D mutants are barely phosphorylated in our assay at nonspecific sites, as previously reported in vivo (Salat-Canela et al., 2017;Sa ´nchez-Mir et al., 2020). Recombinant GST-GFP was used as a negative substrate control (Figure S8A). We then purified several recombinant proteins of the polarity module, including the GAPs and GEFs of Cdc42 fused to GST. The schemes of the proteins tested are shown in Figures S8B and S9C. Purified GST-tagged Rga3 1–447 ,containing17outof 20 putative MAP kinase sites (Figure S8B), and full-length Rga6 were first tested for their phosphorylation by wild-type GST-Sty1 or its catalytically dead version GST-Sty1.K49R. As shown in Figure 7B, GST-tagged Rga3 1–447 , and, to a lesser extent, Rga6, are phosphorylated by wild-type GST-Sty1, but not by GSTSty1.K49R. Based onthe genetic evidences and the in vitro results shown above, we propose that Rga3 is a direct target of Sty1 and that its phosphorylation promotes its activation to initiate Cdc42 dispersion from the cell tips upon stress imposition. 8Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS RESOURCE AVAILABILITY Lead contact Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Elena Hidalgo (elena. [email protected]). Materials availability All unique reagents generated in this study are available from the Lead Contact without restriction. Data and code availability dAll images included in the main and supplemental figures have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table. dThis paper does not report original code. dAny additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Continued REAGENT or RESOURCE SOURCE IDENTIFIER p701.81’ pnmt81’:Tea1-(linker)-GFP This study p712.81’ pnmt81’:Tea1-Sty1-(linker)-GFP This study p717.81’ pnmt81’:Tea1-Gef1-(linker)-GFP This study p734.81’ pnmt81’:Tea1 This study p747.81’ pnmt81’:Tea1-Scd1-(linker)-GFP This study p748.81’ pnmt81’:Tea1-mCherry This study p755 pGEX-4T-1-Rga3 1-447 This study p758 pGEX-4T-1-Scd1 10-872 This study p761 pGEX-4T-1-Gef1 1-304 This study p759 pGEX-4T-1-Rga6 This study p771 pGEX-4T-1-Atf1 This study p771.10D pGEX-4T-1-Atf1.10D This study p771.10M pGEX-4T-1-Atf1.10M This study p772 pGEX-4T-1-GFP This study p773 pGEX-4T-2-Wis1.DD This study p775.8D’ pgef1’:gef1.8D-HA This study p775.8M’ pgef1’:gef1.8M-HA This study p777 pGEX-4T-1-Ras1 3-217 This study p780 pGEX-4T-1-Scd2 4-534 This study Software and algorithms Adobe Illustrator 2021 Adobe N/A FiJi-ImageJ NIH https://imagej.net/software/fiji/ Gen5 software Biotek N/A GraphPad Prism (6.0c) GraphPad Software https://www.graphpad.com/ Metamorph 7.8.13 Gataca Systems N/A R studio N/A https://www.rstudio.com SMART EMBL Heidelberg http://SMART.embl-heidelberg.de SoftWoRx 5.5.0 Applied Precision N/A Other 8-chamber coverglass slides Thermo Scientific 155411 35 mm Dish coverslip MatTek P35G-1.5-14-C 1 ml transfer pipette FisherBrand 13469118 Cell Reports 37, 109951, November 2, 2021 e2 Article ll OPEN ACCESS EXPERIMENTAL MODEL AND SUBJECT DETAILS Fission yeast strains were grown in rich medium (yeast extract, YE) or minimal medium (MM) at 30C as described previously (Alfa et al., 1993). The genotypes of strains used in this study are shown in Table S1. METHOD DETAILS Yeast strains, plasmids and molecular biology Yeast strains were constructed by standard genetic methods. Origins and genotypes of strains used in this study are described in Table S1. Strains under the control of thiamine-repressible nmt81 promoter were grown under the presence of 0.02 mg/ml thiamine until saturation. In order to induce gene expression, a small volume of this culture was diluted in MM media and cells were let to grow for 16 hours. E. coli DH5awas used as a host for propagation and construction of the plasmids used in this study. Bacteria were grown in LB medium supplemented with 0.1 mg/ml ampicillin. Plasmid p4220(Paulo et al., 2014), containing the ctt1 gene under the control of the constitutive sty1 promoter, was linearized and integrated by homologous recombination at the leu1-32 locus of different strain backgrounds. To create Tea1 protein fusions, we PCR-amplified genomic tea1 and cloned it into an episomal nmt81-driven background, yielding p696.81. Next, a PCR product containing a linker region followed by a GFP molecule was amplified from pARC2080, which was kindly provided by Paul Nurse (Kelly and Nurse, 2011) and cloned into the previous plasmid generating the episomal plasmid p697.81. The resulting nmt81-tea1-(linker)-GFP was cloned into an integrative background yielding plasmid p701.81’. A BamHI restriction site located in frame between tea1 and the (linker)-GFP coding sequence (CDS) was used to clone PCR-amplified sty1 CDS, yielding p712.81’, and gef1 CDS, yielding p717.81’. Since scd1 CDS contains an internal BamHI site, compatible ends were generated by BglII digestion and cloned into BamHI -linearized p701.81, yielding the plasmid p747.81’. All these constructions were linearized and integrated by homologous recombination at the leu1-32 locus of different strain backgrounds. Constructions were checked by sequencing. tea1 CDS contains codon changes causing I359V and T983A mutations that were generated during PCR amplification. Tea1-mCherry constructions were created using a similar approach. tea1 CDS was isolated from p696.81 and cloned into an integrative vector, yielding p734.81’. mCherry CDS was amplified by PCR and cloned into the previous plasmid using BamHI/ SmaI enzymes. The resulting plasmid codes for integrative nmt81-driven Tea1-mCherry. As previously described, a BamHI restriction site located in frame between tea1 and mCherry was used to clone gef1 CDS, yielding p748.81’, which was linearized and inserted by homologous recombination at the leu1-32 locus. The plasmid pJK148-Gef1-HA (Coll et al., 2003) was linearized with XbaI to be inserted in the gef1 locus. To generate the phosphomutant versions Gef1.8D (containing the substitutions of S14D, T97E, T100E, T140E, S167D, S246D, T284E and T480E) and Gef1.8M (S14A, T97I, T100I, T140I, S167A, S246A, T284I and T480I), the whole mutated ORF sequences were obtained from Integrated DNA Technologies (IDT) and cloned into pJK148-Gef1HA using BglII and NotI, yielding p775’.8D and p775’.8M. In order to express GST-tagged proteins, full-length wild-type version of Atf1, the phosphomutants Atf1.10D and Atf1.10M or a truncated version (from amino acid 71 to 291), the CDSs were amplified by PCR and cloned into pGEX-4T-1 using BglII/SmaI, yielding p771, p771.10D, p771.10M and GST-Atf171-291. Similarly, the wild-type version of sty1 gene and the kinetic mutant version sty1.K49R were PCR-amplified and cloned into pGEX-4T-1 using BamHI/EcoRI, yielding p109 and p109.K49R. The CDS coding for the constitutive active version of Wis1, Wis1.DD, was amplified by PCR and cloned into pGEX-4T-2 using XhoI, yielding p773. In order to be used as a negative control, the GFP coding sequence preceded by a linker region was amplified from pARC2080 and cloned into pGEX-4T-1 using BamHI/SmaI, yielding p772. The fulllength ORF of rga6 was PCR-amplified and cloned into pGEX-4T-1 using EcoRI/SmaI, yielding p759. The CDSs coding for fragments of Rga3 1-447 , Scd1 10-872 , Gef1 1-304 and Gef1.7M 1-304 were amplified by PCR and cloned into pGEX-4T-1 using BamHI/SmaI, yielding p755, p758 and p761, respectively. The CDSs coding for fragments Ras1 3-219 and Scd2 4-536 were amplified by PCR and cloned into pGEX-4T-1 using BamHI/SmaI, yielding p777 and p780, respectively. The plasmid coding for GST-Pak1.K415R was kindly provided by James Moseley (Magliozzi et al., 2020). pAV0756 coding for stable-integrative version of CRIB-3mCherry was obtained from the Japanese National BioResource Project (NBTP; https://yeast.nig.ac.jp/yeast ) and cloned at the wild-type his5 locus as described elsewhere (Vje stica et al., 2020). Drug treatments Cells were incubated with H 2 O 2 and LatA purchased from Sigma (H1009 and L5163, respectively) at the indicated concentrations. ATP-analogs 1-NM-PP1 and 3-MB-PP1 were obtained from Toronto Research Chemicals (A603003; A602960). Microscopy techniques and image processing Cells were grown to logarithmic phase in either YE or MM, as indicated in each figure legend. For time-lapse experiments, 8-chamber coverglass slides (155411, Thermo Scientific) or Petri dishes with glass bottom coverslip (MatTek, P35G-1.5-14-C) were coated with 1 mg/ml of lectin from glycine max soybean (L1395, Sigma) and left for 30 min. Excess of lectin was washed with appropriate medium and 200 ml of cell culture was added to each chamber and left to settle for 2 min. Next, the cells were washed once with appropriate media and the same volume of pre-warmed fresh media was added. The preparations were placed at the microscope chamber at e3 Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS 30C, except for experiments involving pretreatment with 3MB-PP1. In that case, media containing the drug was added and the chamber was left for 60 min in a 30C incubator. We realized that the use of other ATP analogs such as the 1NM-PP1 or 3Br-PP1 had an inhibitory effect on the wild-type version of the Sty1 kinase, and probably other kinases (data not shown), as described elsewhere (Bohnert et al., 2020). For that reason, we strongly recommend the use of 3MB-PP1 with the analog sensitive version of Sty1 kinase, Sty1.T97A. For time-lapse experiments (as in Figures 1A, 1C, 2A, 2C, 5G, and S3C) we used a spinning disk confocal microscope (Revolution XD; Andor Technology) with a Plan Apochromat 100 3, 1.45 NA objective equipped with a dual-mode electron-modifying charge-coupled device camera (iXon 897 E; Andor Technology). Seventeen z stacks along 4 mm were acquired every 3 min for CRIB-GFP cells and every 6 min for CRIB-dTomato cells. iQ Live Cell Imaging software (Andor Technology) was used for image acquisition. For the addition of either LatA or H 2 O 2 during imaging, media was removed carefully using a 1 ml transfer pipette (13469118, FisherBrand) and the same amount of media containing the appropriate compound was added. This process was completed within a 3 min time interval so the acquisition timing was not disrupted. For time-lapse experiments (as shown in Figure 3F), Petri dishes were kept at 30C using an Okolab stage top chamber on a Nikon Eclipse Ti2 microscope fused to an Andor Dragonfly spinning disk unit equipped with a 488nm laser, using a U Plan Apo 60x 1.4 oil objective and an iXON-EMCCD Du-897 camera. A 2x camera zoom was used to reach Nyquist sampling. In this case, three z stacks along 2 mm were acquired every 6 min. Fiji (ImageJ, National Institutes of Health) (Schindelin et al., 2012) was used for image processing. Still images of time-lapse experiments are presented as z stacks at maximum intensity. Kymographs were generated using Fiji imaging software. Briefly, the stack of interest was aligned using StackReg plugin. Then a montage consisting of the different time-points was done from a horizontal rectangle of 5 mm located along the cell cortex. For DIC and conventional fluorescence microscopy images (as shown in Figures 2A, 2B, 4A, 4E, 5A, 5C, 5E, 6A, 6C, S2,S4B, S5, and S6) we used a Nikon Eclipse 90i microscope equipped with differential interference contrast optics, a PLAN APO VC 100x 1.4 oil immersion objective, an ORCA-II-ERG camera (Hamamatsu) image acquisition software Metamorph 7.8.13 (Gataca Systems) and a LED illumination Cool LED pE-300lite. Fluorescence single plane images shown in Figure 2B were acquired on an inverted microscope (model IX71; Olympus) equipped with a PlanApo 100x/1.40 IX70 objective, a Personal DeltaVision system, a Solid State Illumination System InsightSSI TM Spectra7 (Applied Precision) a CoolSnap HQ2 monochrome camera (Photometrics) and softWoRx 5.5.0 imaging software (Applied Precision). Images were then corrected by 3-D deconvolution (conservative ratio, 10 iterations and medium noise filtering) using the softWoRx imaging software. For cell length and cell width measurements, cells were stained with calcofluor (Sigma, F3543) and examined under the microscope. Elongation rates assay using FITC Novel growth zones were measured as described previously (Revilla-Guarinos et al., 2016). Briefly, yeast cells were grown to an OD 600 of 0.3. To minimize the effects of the media and other technical conditions, we co-cultured each mutant strain (lacking fluorescent markers) with the wild-type strain (expressing Hta1-mRFP) in MM containing 5 mg/ml of FITC-lectin (L9381, Sigma-Aldrich). After 15 min of incubation, cells were pelleted by mild centrifugation and washed twice with MM. Then cells were further incubated with pre-warmed stain-free media for 90 min. Finally, cells were counterstained with calcofluor white and observed under the microscope. H 2 O 2 sensitivity assay To monitor cell growth upon stress imposition, growth curves were performed as described elsewhere (Calvo et al., 2009). Briefly, MM cultures at an initial OD 600 of 0.1 were treated or not with the drugs and inoculated by duplicate in 96-well non-coated polystyrene microplates with an adhesive plate seal. Plates were incubated in a Power Wave microplate scanning spectrophotometer (Bio-Tek) at 30C with continuous shaking. The OD 600 was automatically recorded every 10 min for the indicated hours using Gen5 software. TCA extracts and immuno blot analysis Modified trichloroacetic acid (TCA) protocol was used for protein extraction as previously described (Jara et al., 2007). Briefly, TCA to a final concentration of 10% was added to S. pombe cultures at an OD 600 of 0.5. Cells were pelleted and washed in 20% TCA. The pellets were resuspended in 100 ml of 12.5% TCA and lysed by vortexing in presence of glass beads. Cell lysates were pelleted, washed in acetone and dried. Pellets were resuspended in a Tris buffer (0.1 M Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS), loading buffer was added and samples were boiled for 5 min at 100C. Samples were separated by SDS-PAGE and detected by immunoblotting with polyclonal anti-Atf1 (Sanso ´et al., 2008), house-made monoclonal anti-HA antiserum (12CA5), polyclonal anti-Sty1 (Jara et al., 2007), monoclonal anti-GFP (Takara) and monoclonal anti-tubulin (T5168, Sigma-Aldrich) antibodies. Protein production in E. coli To produce and purify GST-tagged proteins, the protease deficient E. coli strain FB810 was transformed with the corresponding plasmids. A liquid culture of 200 ml was grown up to OD 595 of 0.6, then 100 mM of IPTG was added to the culture and further incubated during 20 h at 18C. Cells were collected and resuspended in STET buffer (50 mM Tris HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 5% Triton X-100, 1 mM DTT). Cell lysis was obtained by 5 pulses of sonication of 15 s at 40% amplitude. Soluble fraction was prepared by centrifugation at 10,000 rpm for 10 min at 4C. A volume of 2.5 ml of soluble fraction was incubated with 100 ml of Glutathione Cell Reports 37, 109951, November 2, 2021 e4 Article ll OPEN ACCESS Sepharose beads (GE Healthcare, 17-0756-01) for 1 h at 4C. Beads were washed three times with NET-N buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40). A final washing in Elution Buffer (100 mM Tris-HCl pH 8.0, 120 mM NaCl) was performed. Finally, the desired protein was eluted form the beads by incubation with 100 ml of Elution Buffer containing 6 mg/ml of GSH (Sigma, 4251) during 30 min at 4C. Recombinant proteins were checked and quantified in a polyacrylamide gel and stained with Coomassie Brilliant Blue (Amresco, 0472). Purified proteins were stored at 80C. In vitro kinase assay To perform kinase assays, two sequential enzymatic reactions were prepared. First of all, recombinant GST-Sty1 kinase should be activated by pre-incubation with its MAP kinase kinase, GST-Wis1.DD. To prepare reaction A, 0.2 mg of either GST-Sty1 or GSTSty1.K49R was mixed with 0.02 mg of GST-Wis1.DD in 1x Kinase buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM DTT) containing 0.5 mM of freshly added ATP (Promega, E6011) in 10 ml reactions. Reaction A was incubated at 30C for 5 min. Then it was mixed with an equal volume of reaction B containing 2 mg of the GST-substrate in 1x Kinase buffer and 2 mCi g32 P-ATP (PerkinElmer, BLU002Z250UC). The mix was further incubated at 30C for 20 min. The enzymatic reaction was stopped by adding SDS-PAGE sample buffer. Proteins were boiled and separated into a polyacrylamide gel. Gels were dried overnight and the signal of g32 P-ATP was detected on an autoradiogram. Protein sequence analysis Protein domains as in Figure S8B and S9C were obtained using a simple modular architecture research tool (SMART) (http://SMART. embl-heidelberg.de). Canonical S/T-P phosphorylation sites were mapped manually. QUANTIFICATION AND STATISTICAL ANALYSIS Unless otherwise stated, all experiments were performed at least three times and representative experiments were shown. Several approaches were used for quantification of microscopy images. To quantify Cdc42 depolarization from time-lapse experiments (as in Figure 1B, 3B, S3A, S3D, and S3F), the mean value of each cell cytoplasm was subtracted from the image in order to obtain pure CRIB signal at tips. Tip signal was narrowly selected using the square tool of Fiji software and integrated density of each tip was measured along the different time points. To obtain CRIB signal per cell, the values of both tips were added and the percentage of CRIB intensity was calculated by referencing all the values to the time zero. For quantification of CRIB intensity at cell tips (as shown in Figure 4C, 5F, 6D, 6E, and S5D) we obtained the intensity profile from a 5-pixel wide line along the x axis of the cell. The two peak values, corresponding to the maximum pixel value of each tip, were added obtaining CRIB intensity per cell. For line-scans quantification (as shown in Figure 5B) a 5-pixel line was drawn along the cell tip and profiles for the GFP and mCherry channels were obtained. For the untreated cells Rga3-GFP profiles were centered to the maximum value of CRIB-3mCherry. In the case of peroxide treated cells, profiles were centered on the maximum value of the GFP channel. For cell length and cell width measurements (as shown in Figure 4B, 4F, 6B, S4A, and S6B) only septated cells were analyzed. All cell measurements were performed manually using the line tool from Fiji software. Elongation rates (as in Figure 4D) were calculated by dividing the novel length by the incubation time. Graphs and statistical analysis were performed with either Prism (GraphPad Software) or the R package Ggplot2. Details of the statistical test used in each case can be found in the figure legend. e5 Cell Reports 37, 109951, November 2, 2021 Article ll OPEN ACCESS