Cell polarity protein Spa2 coordinates Chs2 incorporation at the division site in budding yeast

Deposition of additional plasma membrane and cargoes during cytokinesis in eukaryotic cells must be coordinated with actomyosin ring contraction, plasma membrane ingression and extracellular matrix remodelling. The process by which the secretory pathway promotes specific incorporation of key factors into the cytokinetic machinery is poorly understood. Here, we show that cell polarity protein Spa2 interacts with actomyosin ring components during cytokinesis. Spa2 directly binds to cytokinetic factors Cyk3 and Hof1. The lethal effects of deleting the SPA2 gene in the absence of either Cyk3 or Hof1 can be suppressed by expression of the hypermorphic allele of the essential chitin synthase II (Chs2), a transmembrane protein transported on secretory vesicles that makes the primary septum during cytokinesis. Spa2 also interacts directly with the chitin synthase Chs2. Interestingly, artificial incorporation of Chs2 into the cytokinetic machinery allows the localisation of Spa2 at the site of division. In addition, increased Spa2 protein levels promote Chs2 incorporation at the site of division and primary septum formation. Our data indicate that Spa2 is recruited to the cleavage site to co-operate with the secretory vesicle system and particular actomyosin ring components to promote the incorporation of Chs2 into the so-called 'ingression progression complexes' during cytokinesis in budding yeast.ASD was a recipient of a Ramon y Cajal contract (RYC-2010-06156) and received funding from the Cantabria International Campus (http://www.cantabriacampusinternacional.com/Paginas/Cantabria-Campus-de-Excelencia-Internacional.aspx) and via grants BFU2011-23193 and BFU2014-58081-P from the Spanish Ministerio de Economia y Competitividad (co-funded by the European Regional Development Fund) (http:// www.idi.mineco.gob.es/portal/site/MICINN/ menuitem.00d7c011ca2a3753222b7d1001432ea0/?vgnextoid=33881f4368aef110VgnVCM1000001034e20aRCRD) (http://ec.europa.eu/regional_policy/en/funding/erdf/). MF received a Juan de la Cierva contract from the Spanish Ministerio de Economia y Competitividad

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis

Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55 increment mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role

Studying the role of the mitotic exit network in cytokinesis

In budding yeast cells, cytokinesis is achieved by the successful division of the cytoplasm into two daughter cells, but the precise mechanisms of cell division and its regulation are still rather poorly understood. The Mitotic Exit Network (MEN) is the signaling cascade that is responsible for the release of Cdc14 phosphatase leading to the inactivation of the kinase activity associated to cyclin-dependent kinases (CDK), which drives exit from mitosis and a rapid and efficient cytokinesis. Mitotic CDK impairs the activation of MEN before anaphase, and activation of MEN in anaphase leads to the inactivation of CDK, which presents a challenge to determine the contribution that each pathway makes to the successful onset of cytokinesis. To determine CDK and MEN contribution to cytokinesis irrespectively of each other, here we present methods to induce cytokinesis after the inactivation of CDK activity in temperature sensitive mutants of the MEN pathway. An array of methods to monitor the cellular events associated with the successful cytokinesis is included.ASD is a recipient of a Ramon y Cajal contract and received funding from the Cantabria International Campus and via grant BFU2014-58081-P from the Spanish “Ministerio de Economia y Competitividad” (cofunded by the European Regional Development Fund). MF is a recipient of fellowship Juan de la Cierva (FPDI-2013-17778) from the Spanish “Ministerio de Economia y Competitividad.Peer Reviewe

Studying protein-protein interactions in budding yeast using co-immunoprecipitation

Understanding protein–protein interactions and the architecture of protein complexes in which they work is essential to identify their biological role. Protein co-immunoprecipitation (co-IP) is an invaluable technique used in biochemistry allowing the identification of protein interactors. Here, we describe in detail an immunoaffinity purification protocol as a one-step or two-step immunoprecipitation from budding yeast Saccharomyces cerevisiae cells to subsequently detect interactions between proteins involved in the same biological process.ASD is a recipient of a Ramon y Cajal contract and received funding from the Cantabria International Campus and via grant BFU2011-23193 from the Spanish “Ministerio de Economia y Competitividad” (co-funded by the European Regional Development Fund).Peer Reviewe

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

[EN]Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central rol

TOR Complex 1: Orchestrating nutrient signaling and cell cycle progression

The highly conserved TOR signaling pathway is crucial for coordinating cellular growth with the cell cycle machinery in eukaryotes. One of the two TOR complexes in budding yeast, TORC1, integrates environmental cues and promotes cell growth. While cells grow, they need to copy their chromosomes, segregate them in mitosis, divide all their components during cytokinesis, and finally physically separate mother and daughter cells to start a new cell cycle apart from each other. To maintain cell size homeostasis and chromosome stability, it is crucial that mechanisms that control growth are connected and coordinated with the cell cycle. Successive periods of high and low TORC1 activity would participate in the adequate cell cycle progression. Here, we review the known molecular mechanisms through which TORC1 regulates the cell cycle in the budding yeast Saccharomyces cerevisiae that have been extensively used as a model organism to understand the role of its mammalian ortholog, mTORC1Funding: This research was funded by the Agencia Estatal de Investigación (AEI) of Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033), grant number PID2019-106745GB-I00. In addition, a grant from the Consejería de Universidades, Investigación, Medio Ambiente y Política Social del Gobierno de Cantabria, and another grant from Sociedad para el Desarrollo de Cantabria (SODERCAN)

Synchronization of the budding yeast Saccharomyces cerevisiae

A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.ASD is a recipient of a Ramon y Cajal contract and received funding from the Cantabria International Campus and via grant BFU2011-23193 from the Spanish “Ministerio de Economia y Competitividad” (co-funded by the European Regional Development Fund).Peer Reviewe

Cell polarity protein Spa2 coordinates chitin synthase II incorporation at the division site in budding yeast

Resumen del trabajo presentado al XL Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Barcelona del 23 al 26 de octubre de 2017.During cytokinesis cells coordinate actomyosin ring contraction, plasma membrane ingression, secretory vesicle incorporation and remodelling of the extracellular matrix (ECM). In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as important players during cytokinesis. In budding yeast the chitin synthase Chs2 makes the primary septum, a special layer of ECM that is essential for cell division. Chs2 is transported on secretory vesicles and its addition to the actomyosin ring must be coordinated with other cytokinetic steps. The process by which the secretory pathway promotes specific incorporation of key factors into the cytokinetic machinery is poorly understood. To try to understand how yeast cells coordinate late cytokinesis steps, we have purified budding yeast Chs2 and the key cytokinesis protein Inn1. We isolated the so called 'ingression progression complexes' or IPCs that contain key actomyosin rings components. We now focus on the cell polarity protein Spa2 that binds to IPCs during cytokinesis. Spa2 interacts with cytokinetic factors Cyk3 and Hof1. Interestingly, Spa2 function turns essential in cells that lack either Cyk3 or Hof1. Those lethal effects can be suppressed by expression of a hypermorphic allele of Chs2, which show the functional relationship between Spa2 and Chs2. Furthermore, Spa2 binds directly to the chitin synthase Chs2. Our findings indicate that Spa2 is recruited to the cleavage site to co-operate with the secretory pathway and actomyosin ring components to promote the incorporation of Chs2 into the IPCs during cytokinesis in budding yeast. We provide new insights into the molecular mechanism by which cells coordinate the secretory pathway, essential for cytokinesis, with the other late cytokinetic steps.Peer Reviewe

Inn1 and Cyk3 regulate chitin synthase during cytokinesis in budding yeasts

The chitin synthase that makes the primary septum during cell division in budding yeasts is an important therapeutic target with an unknown activation mechanism. We previously found that the C2-domain of the Saccharomyces cerevisiae Inn1 protein plays an essential but uncharacterised role at the cleavage site during cytokinesis. By combining a novel degron allele of INN1 with a point mutation in the C2-domain, we screened for mutations in other genes that suppress the resulting defect in cell division. In this way, we identified 22 dominant mutations of CHS2 (chitin synthase II) that map to two neighbouring sites in the catalytic domain. Chs2 in isolated cell membranes is normally nearly inactive (unless protease treatment is used to bypass inhibition); however, the dominant suppressor allele Chs2-V377I has enhanced activity in vitro. We show that Inn1 associates with Chs2 in yeast cell extracts. It also interacts in a yeast two-hybrid assay with the N-terminal 65% of Chs2, which contains the catalytic domain. In addition to compensating for mutations in the Inn1 C2-domain, the dominant CHS2 alleles suppress cytokinesis defects produced by the lack of the Cyk3 protein. Our data support a model in which the C2-domain of Inn1 acts in conjunction with Cyk3 to regulate the catalytic domain of Chs2 during cytokinesis. These findings suggest novel approaches for developing future drugs against important fungal pathogens. © 2012. Published by The Company of Biologists Ltd.This work was supported by Cancer Research UK. A.S.D. joined the University of Cantabria as a recipient of a Ramón y Cajal contract (call 2010) and receives funding from the Cantabria International Campus and from the Spanish 'Ministerio de Economía y Competitividad' (co-funded by the European Regional Development Fund) [grant number BFU2011-23193].Peer Reviewe