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• W2896842033 abstract "•Tip growth is associated with stable polarity zones enriched in active Cdc42•Arresting growth by chemical or mechanical means destabilizes active Cdc42 domains•The influence of growth on polarity implicates actin-based transport•This process is relevant to the polar growth of many distant fungal species Polar cell growth is a conserved morphogenetic process needed for survival, mating, and infection [1Brand A. Gow N.A. Mechanisms of hypha orientation of fungi.Curr. Opin. Microbiol. 2009; 12: 350-357Crossref PubMed Scopus (101) Google Scholar, 2Merlini L. Dudin O. Martin S.G. Mate and fuse: how yeast cells do it.Open Biol. 2013; 3: 130008Crossref PubMed Scopus (145) Google Scholar]. It typically implicates the assembly and spatial stabilization of a cortical polar domain of the active form of a small GTPase of the Rho family, such as Cdc42, which promotes cytoskeleton assembly and secretion needed for local surface expansion [3Li R. Gundersen G.G. Beyond polymer polarity: how the cytoskeleton builds a polarized cell.Nat. Rev. Mol. Cell Biol. 2008; 9: 860-873Crossref PubMed Scopus (278) Google Scholar, 4Johnson J.M. Jin M. Lew D.J. Symmetry breaking and the establishment of cell polarity in budding yeast.Curr. Opin. Genet. Dev. 2011; 21: 740-746Crossref PubMed Scopus (90) Google Scholar, 5Martin S.G. Spontaneous cell polarization: feedback control of Cdc42 GTPase breaks cellular symmetry.BioEssays. 2015; 37: 1193-1201Crossref PubMed Scopus (35) Google Scholar, 6Minc N. Boudaoud A. Chang F. Mechanical forces of fission yeast growth.Curr. Biol. 2009; 19: 1096-1101Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. In multiple physiological instances, polarity domains may switch from being spatially unstable, exhibiting a wandering behavior around the cell surface, to being stable at a fixed cellular location [7Bendezú F.O. Martin S.G. Cdc42 explores the cell periphery for mate selection in fission yeast.Curr. Biol. 2013; 23: 42-47Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Bonazzi D. Haupt A. Tanimoto H. Delacour D. Salort D. Minc N. Actin-based transport adapts polarity domain size to local cellular curvature.Curr. Biol. 2015; 25: 2677-2683Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Merlini L. Khalili B. Bendezú F.O. Hurwitz D. Vincenzetti V. Vavylonis D. Martin S.G. Local pheromone release from dynamic polarity sites underlies cell-cell pairing during yeast mating.Curr. Biol. 2016; 26: 1117-1125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 11Dyer J.M. Savage N.S. Jin M. Zyla T.R. Elston T.C. Lew D.J. Tracking shallow chemical gradients by actin-driven wandering of the polarization site.Curr. Biol. 2013; 23: 32-41Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar]. Here, we show that the rate of surface growth may be a key determinant in controlling the spatial stability of active Cdc42 domains. Reducing the growth rate of single rod-shaped fission yeast cells using chemical, genetic, and mechanical means systematically causes polar domains to detach from cell tips and oscillate around the cell surface within minutes. Conversely, an abrupt increase in growth rate improves domain stabilization. A candidate screen identifies vesicular transport along actin cables as an important module mediating this process. Similar behavior observed in distant filamentous fungi suggests that this positive feedback between growth and polarity could represent a basal property of eukaryotic polarization, promoting persistent polar growth as well as growth redirection with respect to the mechanical environment of cells. Polar cell growth is a conserved morphogenetic process needed for survival, mating, and infection [1Brand A. Gow N.A. Mechanisms of hypha orientation of fungi.Curr. Opin. Microbiol. 2009; 12: 350-357Crossref PubMed Scopus (101) Google Scholar, 2Merlini L. Dudin O. Martin S.G. Mate and fuse: how yeast cells do it.Open Biol. 2013; 3: 130008Crossref PubMed Scopus (145) Google Scholar]. It typically implicates the assembly and spatial stabilization of a cortical polar domain of the active form of a small GTPase of the Rho family, such as Cdc42, which promotes cytoskeleton assembly and secretion needed for local surface expansion [3Li R. Gundersen G.G. Beyond polymer polarity: how the cytoskeleton builds a polarized cell.Nat. Rev. Mol. Cell Biol. 2008; 9: 860-873Crossref PubMed Scopus (278) Google Scholar, 4Johnson J.M. Jin M. Lew D.J. Symmetry breaking and the establishment of cell polarity in budding yeast.Curr. Opin. Genet. Dev. 2011; 21: 740-746Crossref PubMed Scopus (90) Google Scholar, 5Martin S.G. Spontaneous cell polarization: feedback control of Cdc42 GTPase breaks cellular symmetry.BioEssays. 2015; 37: 1193-1201Crossref PubMed Scopus (35) Google Scholar, 6Minc N. Boudaoud A. Chang F. Mechanical forces of fission yeast growth.Curr. Biol. 2009; 19: 1096-1101Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. In multiple physiological instances, polarity domains may switch from being spatially unstable, exhibiting a wandering behavior around the cell surface, to being stable at a fixed cellular location [7Bendezú F.O. Martin S.G. Cdc42 explores the cell periphery for mate selection in fission yeast.Curr. Biol. 2013; 23: 42-47Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Bonazzi D. Haupt A. Tanimoto H. Delacour D. Salort D. Minc N. Actin-based transport adapts polarity domain size to local cellular curvature.Curr. Biol. 2015; 25: 2677-2683Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Merlini L. Khalili B. Bendezú F.O. Hurwitz D. Vincenzetti V. Vavylonis D. Martin S.G. Local pheromone release from dynamic polarity sites underlies cell-cell pairing during yeast mating.Curr. Biol. 2016; 26: 1117-1125Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 11Dyer J.M. Savage N.S. Jin M. Zyla T.R. Elston T.C. Lew D.J. Tracking shallow chemical gradients by actin-driven wandering of the polarization site.Curr. Biol. 2013; 23: 32-41Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar]. Here, we show that the rate of surface growth may be a key determinant in controlling the spatial stability of active Cdc42 domains. Reducing the growth rate of single rod-shaped fission yeast cells using chemical, genetic, and mechanical means systematically causes polar domains to detach from cell tips and oscillate around the cell surface within minutes. Conversely, an abrupt increase in growth rate improves domain stabilization. A candidate screen identifies vesicular transport along actin cables as an important module mediating this process. Similar behavior observed in distant filamentous fungi suggests that this positive feedback between growth and polarity could represent a basal property of eukaryotic polarization, promoting persistent polar growth as well as growth redirection with respect to the mechanical environment of cells. Polar domains of the active form of the Rho-like GTPase Cdc42 promote local growth. Studies in yeast and fungal cells have shown that polar domains can oftentimes become unstable, assembling and disassembling at successive positions around the cell surface [5Martin S.G. Spontaneous cell polarization: feedback control of Cdc42 GTPase breaks cellular symmetry.BioEssays. 2015; 37: 1193-1201Crossref PubMed Scopus (35) Google Scholar, 7Bendezú F.O. Martin S.G. Cdc42 explores the cell periphery for mate selection in fission yeast.Curr. Biol. 2013; 23: 42-47Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 11Dyer J.M. Savage N.S. Jin M. Zyla T.R. Elston T.C. Lew D.J. Tracking shallow chemical gradients by actin-driven wandering of the polarization site.Curr. Biol. 2013; 23: 32-41Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 12Howell A.S. Jin M. Wu C.F. Zyla T.R. Elston T.C. Lew D.J. Negative feedback enhances robustness in the yeast polarity establishment circuit.Cell. 2012; 149: 322-333Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Thomson D.D. Wehmeier S. Byfield F.J. Janmey P.A. Caballero-Lima D. Crossley A. Brand A.C. Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae.Cell. Microbiol. 2015; 17: 342-354Crossref PubMed Scopus (46) Google Scholar]. Motivated by the observation that faint and unstable domains are associated with slow surface growth [7Bendezú F.O. Martin S.G. Cdc42 explores the cell periphery for mate selection in fission yeast.Curr. Biol. 2013; 23: 42-47Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 12Howell A.S. Jin M. Wu C.F. Zyla T.R. Elston T.C. Lew D.J. Negative feedback enhances robustness in the yeast polarity establishment circuit.Cell. 2012; 149: 322-333Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar], we sought to dissect the causal-effect relationships between growth and polarity, and assay in a systematic manner the impact of modulating surface growth rate on polarity stability. Using the rod-shaped fission yeast Schizosaccharomyces pombe as an established model for polar growth [14Chang F. Martin S.G. Shaping fission yeast with microtubules.Cold Spring Harb. Perspect. Biol. 2009; 1: a001347Crossref PubMed Scopus (98) Google Scholar], we first manipulated growth rate by reducing turgor pressure that serves as a physical driver for growth in walled cells [15Davì V. Minc N. Mechanics and morphogenesis of fission yeast cells.Curr. Opin. Microbiol. 2015; 28: 36-45Crossref PubMed Scopus (24) Google Scholar]. We used a gpd1Δ mutant, defective in turgor adaptation, expressing the CRIB-3GFP probe to label active GTP-Cdc42, and used a defined concentration of sorbitol to reduce turgor and consequent growth rates in a reproducible manner [6Minc N. Boudaoud A. Chang F. Mechanical forces of fission yeast growth.Curr. Biol. 2009; 19: 1096-1101Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 16Ohmiya R. Yamada H. Nakashima K. Aiba H. Mizuno T. Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth.Mol. Microbiol. 1995; 18: 963-973Crossref PubMed Scopus (55) Google Scholar, 17Tatebe H. Nakano K. Maximo R. Shiozaki K. Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast.Curr. Biol. 2008; 18: 322-330Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar]. In normal medium, gpd1Δ cells exhibited the same amount of tip-associated active Cdc42 as wild-type (WT), and no gross defect in cell shape, size, or growth (Figure S1A). Using microfluidic flow chambers [18Charvin G. Cross F.R. Siggia E.D. A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells.PLoS ONE. 2008; 3: e1468Crossref PubMed Scopus (101) Google Scholar], we rapidly rinsed cells with medium containing 0.5 M sorbitol. This caused a transient cell shrinkage, followed by a stable reduction in growth rate of ∼80% within 5–10 min (Figure 1A) [6Minc N. Boudaoud A. Chang F. Mechanical forces of fission yeast growth.Curr. Biol. 2009; 19: 1096-1101Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. Strikingly, as growth rate dropped, GTP-Cdc42 domains became dimmer and more spread out, with the appearance of transient ectopic patches of activity on cell sides (Figures 1A and 1B; Video S1). Both growth and polarity changes remained stable for at least 1 hr after treatment. CRIB-3GFP polarization index (PI), computed as the ratio of membrane-associated signal at cell tips to that of the whole cell contour, decreased from a mean value of 2.02 before treatment down to 1.50 after treatment. Importantly, rinsing cells back into normal medium led to a rapid rise in growth rate and concomitant increase in CRIB PI, suggesting that this effect is reversible (Figure S1B). Similar treatment in WT cells only yielded a partial and transient reduction of both growth rate and CRIB PI (Figure S1C). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2YzUzNmFlNzlmNTUzMzk5YzJmYWI4NmIyYmM4ZjRkOSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3NzUxNjg0fQ.aRghGIRY-ownlK-ZRUqvaipvigdfM_7rkqNKft-FabMpbMcT_u6-mvfpDdIEtbgNLpe9UElobHXnfjx_AVyyJDrLZD5P0Sb-bVyGBtYx8eswYW3Eh2Ui2VuEGJPbigMPH1sipe6xfarqZFaj5GVXZbvyefxhxEyFIjiEAFs9zb9ReWO6LxG-hGwDNdCFhhzAbyA8K1H8a_EYK-iKRBZh_0KyJFEtELVay5QFf4CRP0xQxvAcXuPlmGjQC2DJ8QKkmrj4LbY59rT0NX5dKP4JEGe7utKStM9h-pLqkvNvjIZ7u6Xpb155o-Ew33fHY3BtR6BJioi6Qdy2rM3uMNcJZA Download .mp4 (0.72 MB) Help with .mp4 files Video S1. Changes in Growth Rate Act upon Polarity Domain Stability, Related to Figure 1Growth rates were dynamically changed by different means. From left to right: Addition of 0.5 M sorbitol to gpd1Δ cells, Reducing glucose content to 0.03% in wild type cells, Treatment with 50 μM Latrunculin A of wild type cells, Addition of 50 mM KCl to trk1Δtrk2Δ cells, Addition of 0.5 M sorbitol to tea1Δgpd1Δ cells. All strains express CRIB-3GFP. Time elapsed is in h:min. Scale bars are 2 μm. As another means to affect growth, we abruptly reduced the glucose content of the yeast medium to 0.03%. This caused WT cells to swell transiently and was followed by a marked growth rate decrease (Figure 1C; Video S1). CRIB PI also dropped following growth arrest, with cells featuring cap spreading and occasional domains forming on cell sides (Figure 1C). Finally, we exploited a global slow-growth phenotype of a trk1Δtrk2Δ mutant deficient in potassium import and export, which can be rescued by addition of 50 mM KCl to the medium [19Calero F. Gómez N. Ariño J. Ramos J. Trk1 and Trk2 define the major K(+) transport system in fission yeast.J. Bacteriol. 2000; 182: 394-399Crossref PubMed Scopus (39) Google Scholar]. We found that addition of KCl to these mutant cells increased their growth rate by ∼30% within a few minutes and was accompanied by a significant improvement in CRIB polarization (Figure S1D; Video S1). Together, these results suggest that growth may positively influence the stability of active Cdc42 polarity domains. We next addressed the response of other polarisome components to turgor reduction-mediated growth arrest. Landmark factors such as microtubules and tea1-3GFP did not exhibit significant changes in their spatial distribution (Figures S1E and S1F). In contrast, polarity factors downstream of Cdc42 including the actin-associated protein bud6-3GFP, the type V myosin myo52-3GFP, and exocyst components such as sec8-GFP and sec6-GFP all showed some degree of reduced polarization with occasional domain assembly on cell sides, co-localized with CRIB (Figures 1D, S1F, and S1G). F-actin visualized with a GFP-LifeAct probe [20Courtemanche N. Pollard T.D. Chen Q. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.Nat. Cell Biol. 2016; 18: 676-683Crossref PubMed Scopus (78) Google Scholar] remained mostly intact, suggesting that polarity defects were not caused by some indirect stress effects on actin polymerization. However, we noted some defects in the spatial organization of F-actin structures. At a short timescale after sorbitol addition, endocytic patches exhibited a transient increase in number (Figure S1H) [21Basu R. Munteanu E.L. Chang F. Role of turgor pressure in endocytosis in fission yeast.Mol. Biol. Cell. 2014; 25: 679-687Crossref PubMed Scopus (54) Google Scholar]. On longer timescales of more than 5–10 min, the actin network appeared disorganized as compared to controls, with the occurrence of cable elongation and endocytic patches at cell sides, consistent with the presence of GTP-Cdc42 there (Figures 1D and S1H). Finally, polarized integral membrane components transported in vesicles to cell tips, such as the cell-wall synthase GFP-bgs4 and the SNARE GFP-syb1, exhibited near-complete detachment from the membrane with consequent enrichment in internal compartments (Figures 1D, S1F, S1I, and S1J). Importantly, this complete detachment was not observed for non-polar integral membrane proteins such as GFP-psy1 [22Nakamura T. Nakamura-Kubo M. Hirata A. Shimoda C. The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein.Mol. Biol. Cell. 2001; 12: 3955-3972Crossref PubMed Scopus (122) Google Scholar], suggesting that this response was not the result of potential pleiotropic insults on membrane shape and structure (Figures S1E and S1F). These findings suggest that growth may positively influence the stability of Cdc42-based polarity as a whole. Although growth reduction did not grossly affect actin polymerization, its impact on polarity was somewhat reminiscent of cells treated with the actin-depolymerizing drug latrunculin A (LatA) [23Bendezú F.O. Martin S.G. Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast.Mol. Biol. Cell. 2011; 22: 44-53Crossref PubMed Scopus (79) Google Scholar]. Because LatA halts growth in yeast, we asked whether its effect on growth could account for some of its impact on polarity stability. Time-controlled LatA addition yielded a gradual decrease of the growth rate, eventually dropping to similar levels as with sorbitol treatment after 45 min (Figure 1E; Video S1). Interestingly, CRIB polarization dropped with similar kinetics, but LatA caused CRIB domains to completely detach from cell tips and to re-form on cell sides, as previously reported (Figure 1E) [23Bendezú F.O. Martin S.G. Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast.Mol. Biol. Cell. 2011; 22: 44-53Crossref PubMed Scopus (79) Google Scholar, 24Mutavchiev D.R. Leda M. Sawin K.E. Remodeling of the fission yeast Cdc42 cell-polarity module via the Sty1 p38 stress-activated protein kinase pathway.Curr. Biol. 2016; 26: 2921-2928Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. We reasoned that F-actin could promote some of the remnant CRIB localization at cell tips in turgor-reduced cells by mediating directly or indirectly GTP-Cdc42 domain association with tip landmarks such as tea1p. To test this, we arrested growth with sorbitol in a gpd1Δtea1Δ double mutant. This led to near-complete depolarization, which recapitulated the effect of LatA treatment on CRIB domains in turgid cells (Figure 1F; Video S1). These results suggest that landmarks retain a fraction of active Cdc42 at cell tips upon growth arrest in an actin-dependent manner. Finally, by computing the dependence of CRIB PI changes on growth rate variations using all aforementioned assays, we found a significant correlation (r = 0.7604) (Figure 1G). We conclude that growth may promote polarity stability in a dose-dependent manner. Growth in turgid cells may be influenced by external mechanical barriers such as neighboring cells, physical obstacles, or cell-encasing layers [6Minc N. Boudaoud A. Chang F. Mechanical forces of fission yeast growth.Curr. Biol. 2009; 19: 1096-1101Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13Thomson D.D. Wehmeier S. Byfield F.J. Janmey P.A. Caballero-Lima D. Crossley A. Brand A.C. Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae.Cell. Microbiol. 2015; 17: 342-354Crossref PubMed Scopus (46) Google Scholar, 25Sampathkumar A. Yan A. Krupinski P. Meyerowitz E.M. Physical forces regulate plant development and morphogenesis.Curr. Biol. 2014; 24: R475-R483Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar]. To assay this, we designed dedicated narrow channels to confine cells laterally but allow them to grow from their ends [26Goulev Y. Morlot S. Matifas A. Huang B. Molin M. Toledano M.B. Charvin G. Nonlinear feedback drives homeostatic plasticity in H2O2 stress response.eLife. 2017; 6: e23971Crossref PubMed Scopus (35) Google Scholar, 27Zegman Y. Bonazzi D. Minc N. Measurement and manipulation of cell size parameters in fission yeast.Methods Cell Biol. 2015; 125: 423-436Crossref PubMed Scopus (9) Google Scholar] (Figure 2A). WT cells growing in those channels initially proliferated with similar rates as controls outside (Figure S2A) but, as density increased, they started to hamper one another’s growth. Cells reaching high levels of confinement ceased growth or grew very slowly, and adopted small triangular or rectangular shapes (Figure 2B). Remarkably, growth-arrested cells exhibited weak CRIB domains, oftentimes dynamically exploring the cell contour away from cell tips, reminiscent of turgor reduction assays (Figure 2B; Video S2). Polar components, including bud6-3GFP, LifeAct-mCherry, sec6-GFP, sec8-GFP, and GFP-bgs4, were also spatially destabilized as in turgor reduction-mediated growth arrest and exhibited dynamic changes in localization (Figures 2C, 2D, and S2D). Importantly, dynamic assembly and disassembly of ectopic domains were still highly pronounced in mal3Δ and tea1Δ mutants in channels, ruling out a role for microtubule-based polarity reorganization following shape changes in CRIB domain instability (Figures S2B and S2C) [28Minc N. Bratman S.V. Basu R. Chang F. Establishing new sites of polarization by microtubules.Curr. Biol. 2009; 19: 83-94Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 29Terenna C.R. Makushok T. Velve-Casquillas G. Baigl D. Chen Y. Bornens M. Paoletti A. Piel M. Tran P.T. Physical mechanisms redirecting cell polarity and cell shape in fission yeast.Curr. Biol. 2008; 18: 1748-1753Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. Thus, hampering growth by external mechanical means can yield significant destabilization of polarity domains. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxOTg2NjkyMzZkZWIwNWYxNjkxYmM2MTBhNDA4YmU5MiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3NzUxNjg0fQ.I_Qp_pKEuPUz11j_f8wNXBVJ6GT3UEl2fyQcJa6gdTbGjkhRoTjkkkTC9dJfT-6TdUOOwzbB4ESlho5hFg6bzxUtS1s2UaEsnryBrYLKwvGm2Ay4Pv4JbGNqrltsPcPHZ8WsnVBpdFQDYb7nWaCN5NhpvMAmYs3t_oNStTMdvtp8Kpx3RFuqoPeFXTt1AI01ZfBxSZTn9txs1lGyFHxWPunwaEdH3hmG8GzWPwloM9Hl6aziOcw5BUC0atXksE-IGYe8amWRyoOj9S17EnqT9_iRLF2l5-z4915f03vC7JX_iRR-9wEKIv2mlvarcIjUuqq7dXlwLxdKiZ0W2IgAWw Download .mp4 (0.28 MB) Help with .mp4 files Video S2. Growth Arrest through Mechanical Confinement Triggers Polarity Domain Destabilization and Wandering, Related to Figure 2Wild-type cells expressing CRIB-3GFP were grown to confinement in microchannels. Time elapsed is in h:min. Scale bar is 2 μm. Because confinement in channels is reached as a result of multiple phases of growth and division, we devised an assay to rapidly suppress confinement and assay polarity response. We started with fully confined and depolarized cells and used UV laser ablation to kill a subset of cells in the microchannel (Figure 2E; Video S3) [9Bonazzi D. Julien J.D. Romao M. Seddiki R. Piel M. Boudaoud A. Minc N. Symmetry breaking in spore germination relies on an interplay between polar cap stability and spore wall mechanics.Dev. Cell. 2014; 28: 534-546Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. Ablated cells deflated within seconds, freeing space for adjacent cells to grow (Figure 2E). Strikingly, ∼10–20 min after ablation, neighboring cells resumed rapid growth with the concomitant formation of bright and stable CRIB-3GFP domains at growth sites. Importantly, this response was also observed in cells at a further distance from the gaps, ruling out putative effects of chemical release from ablated cells or contact-inhibition cues on polarity stability (Figure 2E). These findings demonstrate that cells can dynamically adapt their polarity behavior to different confinement states altering growth. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJmMjAzZmU3ZTZlNzI0MzRkZjk2OTJlZmUyYzNhZWViOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc3NzUxNjg0fQ.kpIYlVvJ_dvT5pOnLSOsmy_hiXD4mrMo9zAMwnOpB6IMdPDz6nU6F7kWAObpz68KBI1EADsY6w7kVo9u39qaneoX0fZbZu8BiZmXQrOU7lv-ckC0_wHOqPzerVMceiu7BRMKvAcbG8rovkTt8WVEVzpIFd0wJgkn_iP6sYPFv1zhca-Wr2DhG_57Uo12ZWJj8wGJowhs33Dh23A7_0qJg2MOv5XIIreXQjZQgpRuyrvwb9noko4PbNfSOe66-8zbdFqh-dNucfXr3AvjBFqHInABxbdCQrOtnDnxuJjoahN1Ed_xyX5iVbcuspVqA1AZM4Mn9ensGsXcXTQar1rJ-w Download .mp4 (0.66 MB) Help with .mp4 files Video S3. Confinement Lift Induces Repolarization and Growth, Related to Figure 3Wild type cells expressing CRIB-3GFP were grown in microchannels to full confinement and two cells were subsequently killed using UV-laser ablation to generate space for neighbors to grow. Arrowheads label reforming stable CRIB domains. Images are maximum intensity projections. Time elapsed is in h:min. Scale bars are 2 μm. To identify potential factors mediating these effects, we then designed a candidate screen using the gpd1Δ-sorbitol growth arrest assay (Figure 3A). We selected candidates from different classes of sensing and regulating systems, including components of the cell-wall integrity pathway, factors feeding into polarity, and a set of globally acting protein kinases and modulators that have been implicated in growth or polarity before (Figures 3B and S3A–S3D). We crossed candidate mutants into a gpd1Δ background expressing CRIB-3GFP or CRIB-3Citrine [24Mutavchiev D.R. Leda M. Sawin K.E. Remodeling of the fission yeast Cdc42 cell-polarity module via the Sty1 p38 stress-activated protein kinase pathway.Curr. Biol. 2016; 26: 2921-2928Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar], and computed the ratio of CRIB PI after and before sorbitol addition (Figures S3A–S3D). This screen identified suppressors, in which CRIB PI after growth reduction was maintained at a higher level than gpd1Δ controls. Mutants in actin cable-dependent transport, such as a mutant in the formin for3 and in the myosin type V myo52, had the most pronounced suppressing effect [14Chang F. Martin S.G. Shaping fission yeast with microtubules.Cold Spring Harb. Perspect. Biol. 2009; 1: a001347Crossref PubMed Scopus (98) Google Scholar]. Mutants in signaling components including the stress-related MAP kinase sty1 and in the TOR-activating Rab family GTPase ryh1 exhibited a lower, yet significant, suppression (Figures 3B–3D and S3D) [30Millar J.B. Buck V. Wilkinson M.G. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.Genes Dev. 1995; 9: 2117-2130Crossref PubMed Scopus (310) Google Scholar, 31Tatebe H. Morigasaki S. Murayama S. Zeng C.T. Shiozaki K. Rab-family GTPase regulates TOR complex 2 signaling in fission yeast.Curr. Biol. 2010; 20: 1975-1982Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar]. This screen also identified enhancers including mutants for the Cdc42 GAP rga4 and in the landmark tea1, as discussed above (Figures 1F and 3B). Because both factors have established roles in confining GTP-Cdc42 to cell tips, this result could reflect that spatial landmarks outcompete the basal destabilizing impact of growth arrest on polarity. Finally, a mutant in the endocytosis-promoting factor end4 and in the Cdc42 GEF gef1 also exhibited milder enhancing effects (Figures 3B–3D). To test the epistatic relationships between suppressing pathways, we compared the responses of gpd1Δfor3Δ, gpd1Δsty1Δ, and for3Δsty1Δ with gpd1Δ alone (Figures 3E, 3F, and S3E). We resorted to using a for3Δsty1Δ mutant, because we were unable to obtain a viable gpd1Δfor3Δsty1Δ triple mutant, but noted that this double mutant, as in gpd1Δ background strains, did not adapt growth upon sorbitol treatment, due to the absence of sty1. Interestingly, in for3Δsty1Δ cells, we found that polarization upon growth arrest remained nearly indistinguishable from the pre-treatment state (Figures 3E and 3F). Although this analysis cannot fully rule out unknown direct effects of" @default.
• W2896842033 created "2018-10-26" @default.
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• W2896842033 date "2018-10-01" @default.
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• W2896842033 title "A Positive Feedback between Growth and Polarity Provides Directional Persistency and Flexibility to the Process of Tip Growth" @default.
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• W2896842033 doi "https://doi.org/10.1016/j.cub.2018.09.022" @default.
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