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In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis., M. Ramos and J.A. Clemente-Ramos acknowledge support by fellowships from Consejo Superior de Investigaciones Científicas (CSIC, Spain) and Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain), respectively. J.C.G. Cortes was supported by a postdoctoral contract granted by the Universidad de Salamanca, Spain. This work was supported by MCIU and ERDF (European Regional Development Fund)/EU (European Union) grants BIO2015-69958-P and PGC2018-098924-B-I00, and Junta de Castilla y León, Spain, and ERDF/EU grants CSI068P17 and “Escalera de Excelencia” CLU-2017-03

Ramos et al. establish that fission yeast septation proceeds in two phases. Initially, the septum is immature and, upon F-actin depolymerization, loses the Bgs1 glucan synthase and fails to ingress. During a second phase, the mature septum can maintain Bgs1 and ingression without F-actin, and ingression becomes Cdc42 and exocyst dependent., In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division., The research reported in this publication was supported by the grants BIO2015-69958-P to JCR and PP from the Ministerio de Economía y Competitividad (http://www.mineco.gob.es/) and CSI068P17 from MINECO/FEDER and Junta de Castilla y León (https://www.jcyl.es/) to PP and JCR.

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al., Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1 (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast., MB was supported by Core funds from Temasek Life Sciences Laboratory (http://www.tll.org.sg), Wellcome Trust SIA (WT101885MA) (www.wellcome.ac.uk) and a Royal Society Wolfson Merit Award (https://royalsociety.org/). NIN was supported by Core funds from Temasek Life Sciences Laboratory (http://www.tll.org.sg). JCR was supported by Spanish Ministry of Science and Innovation (BIO2012-35372 and BIO2015-69958-P). MO was supported by Bio-imaging Center (S0991205) of Japan Women’s University established as a private university in Japan with the support of Ministry of Education, Culture, Sports Science and Technology (http://www.jiirs.org).

In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow., This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-15641 and BFU2013-39394-P) to PP. JCR was financed by the Spanish Ministry of Science and Innovation (BIO2012-35372 and BIO2015-69958-P), and the Junta de Castilla y León, Spain (CSI037U14). JCGC was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Science and Innovation.

13 páginas, 7 figuras, 5 tablas.-- El pdf del artículo es la versión post-print., Three specific β(1,3)glucan synthase (GS) inhibitor families, papulacandins, acidic terpenoids, and echinocandins, have been analyzed in Schizosaccharomyces pombe wild-type and papulacandin-resistant cells and GS activities. Papulacandin and enfumafungin produced similar in vivo effects, different from that of echinocandins. Also, papulacandin was the strongest in vitro GS inhibitor (IC50 103–104-fold lower than with enfumafungin or pneumocandin), but caspofungin was by far the most efficient antifungal because of the following. 1) It was the only drug that affected resistant cells (minimal inhibitory concentration close to that of the wild type). 2) It was a strong inhibitor of wild-type GS (IC50 close to that of papulacandin). 3) It was the best inhibitor of mutant GS. Moreover, caspofungin showed a special effect for two GS inhibition activities, of high and low affinity, separated by 2 log orders, with no increase in inhibition. pbr1-8 and pbr1-6 resistances are due to single substitutions in the essential Bgs4 GS, located close to the resistance hot spot 1 region described in Saccharomyces and Candida Fks mutants. Bgs4pbr1-8 contains the E700V change, four residues N-terminal from hot spot 1 defining a larger resistance hot spot 1-1 of 13 amino acids. Bgs4pbr1-6 contains the W760S substitution, defining a new resistance hot spot 1-2. We observed spontaneous revertants of the spherical pbr1-6 phenotype and found that an additional A914V change is involved in the recovery of the wild-type cell shape, but it maintains the resistance phenotype. A better understanding of the mechanism of action of the antifungals available should help to improve their activity and to identify new antifungal targets., This work was supported in part by Grants BIO2006-13566 from Ministerio de Educación y Ciencia, BIO2009-10597 from Ministerio de Ciencia e Innovación, and GR231 from Junta de Castilla y León, Spain. Supported by a fellowship from Fundaçâo para a Ciência e a Tecnologia, Portugal. Supported by a fellowship from Consejo Superior de Investigaciones Científicas, Spain. Supported by a fellowship from Junta de Castilla y León, Spain. Supported by a fellowship from Ministerio de Ciencia e Innovación, Spain.

24 p.-7 fig., In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast., This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-15641 and BFU2013-39394-P) to PP. JCR was financed by the Spanish Ministry of Science and Innovation (BIO2012-35372), and Junta de Castilla y León, Spain (CSI037U14).

This article is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license)., Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall ß(1,3)glucan. We show that Bgs4-derived ß(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, ß(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived ß(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular ß(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells., We thank the Ramón Areces Foundation for Instituto de Biología Funcional y Genómica financial support. J. Muñoz, M. Ramos, and J.A. Clemente-Ramos acknowledge support by fellowships from Junta de Castilla y León, Consejo Superior de Investigaciones Científicas, and Ministerio de Ciencia e Innovación (MICINN; Spain), respectively; J.C.G. Cortés by a “Juan de la Cierva” contract from MICINN; and I.M. Martins by a fellowship from Fundação para a Ciência e a Tecnologia (Portugal). This work was supported by grants BIO2009-10597, BFU2010-15641, and BIO2012-35372 (MICINN), CSI376A12-2 (Junta de Castilla y León), and OTKA 101323 (Hungarian Scientific Research Fund).

This article is distributed under the terms of a Creative Commons License Attribution–Noncommercial–Share Alike 3.0 Unported license.-- et al., Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3) glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure. © 2012 Cortés et al., We thank the Ramón Areces Foundation for IBFG financial support. J.C.G. Cortés acknowledges support from “Juan de la Cierva” program granted by Minsterio de Ciencia e Innovación (MICINN, Spain). This work was supported by grants BIO2009-10597 (MICINN) and CSI038A11-2 (Junta de Castilla y León, Spain).