Cytokinesis is the spatially and temporally regulated process by which, after chromosome segregation, eukaryotic cells divide their cytoplasm and membranes to produce two daughter cells independent of each other. In the budding yeast Saccharomyces cerevisiae cytokinesis is driven by tightly regulated pathways that coordinate cell division with nuclear division to ensure the genetic stability during cell growth. These ways promote actomyosin ring (AMR) contraction coupled to plasma membrane constriction and to centripetal deposition of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. In animal cells, the division plane is defined by the central spindle positioning and cytokinesis occurs through the contraction of the AMR, followed by the membrane furrowing. In S. cerevisiae, the first step towards cytokinesis is the assembly of a rigid septin ring, which forms at the bud neck concomitantly with bud emergence as soon as cells enter S phase and marks the position where constriction between mother and daughter cell will take place at the end of mitosis. The septin ring acts as a scaffold for the recruitment other proteins, among which Myo1, the heavy chain of the type II miosin. Myo1 forms a ring at the site of bud emergence at the onset of S phase in a septin-dependent manner. At the end of anaphase, an actin ring overlaps with that of Myo1 and the resulting contractile actomyosin ring drives primary septum deposition. The septin ring also recruits Iqg1 which is important, together with Bni1, for actin recruitment at the bud neck, Cyk3, required for proper synthesis of the septum, and Hof1, which is phosphorylated in telophase and colocalizes with the actomyosin ring during cytokinesis. The subsequent degradation of Hof1 allows efficient AMR contraction and cell separation. During mitotic exit, Chs2 localizes at the cell division site, where it drives synthesis of the primary septum, composed of chitin, simultaneously with actomyosin ring contraction. Afterwards the secondary septum, which has a similar composition to yeast cell wall, is produced on both the mother and the daughter side of the bud neck. The subsequent degradation of the primary septum from the daughter-side is ensured by the RAM pathway that is activated only in the bud. At this point mother and daughter cell separate permanently from each other leaving a chitin disk, that is the primary septum residue, called “bud scar", on the mother cell surface. In the first chapter we describe the role in cytokinesis of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2, that belong to the same ubiquitin ligase family as human Chfr and Rnf8 and Schizosaccharomyces pombe Dma1. In particular we show that both the lack of Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of Cyk3, a positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis. In the second chapter we show that the Ras-like GTPase Tem1 ubiquitylation is involved in AMR contraction regulation. Tem1 is not required for the actomyosin ring assembly but is required for its dynamics. In the first chapter we show how this protein undergoes cell cycle-regulated ubiquitylation, in particular the amount of ubiquitylated Tem1 decreases concomitantly with cells undergoing AMR contraction. Interestingly, high levels of Dma2 induce Tem1 ubiquitylation as well as inhibit AMR contraction. Analyzing the kinetics of AMR contraction in cells that express high levels of Dma2 and different Tem1 K-R variants (in which lysine residues were replaced by arginine residues, thus becoming not ubiquitylable), we show that Tem1 ubiquitylation seems to be important for Dma2’s AMR contraction inhibition. In particular lysines 112, 133 and 219 are mostly implicated in this regulation. Altogether, these findings suggest that the Dma proteins act as negative regulators of AMR contraction by indirectly influencing Tem1 ubiquitylation. In the third chapter we show that Dma1 and Dma2 are involved in the NoCut pathway, a checkpoint whose activation prevents chromosome breakage during cell division. The lack of Dma proteins affects checkpoint activation in the presence of mutations that cause chromatin persistance at the division site or spindle midzone damage. Moreover, the lack of Dma1 and Dma2 causes cell growth defect in combination with the deletion of genes involved in the NoCut pathway or in the mechanisms that permit DNA breaks repair generated when the checkpoint is not totally functional. Furthermore the lack of Dma proteins, although they do not apparently alter cell cycle progression, when combined with the lack of Boi proteins (that work as abscission inhibitors in the NoCut pathway) causes a growth defect due to an increase in chromosome missegregation. Altogether, these findings suggest that the Dma proteins act in the NoCut pathway. In the last chapther we describe the functional characterization of a new player in cell division control: Vhs2. Despite it is not essential for cells viability, we show how this protein is implicated in septins stabilizaton. The lack of Vhs2 causes cell growth defect in combination with several mutants that affect septin structure. Moreover vhs2Δ cells per se have a defect in septin stability, in fact these cells show a typical phenotype: the septins disappear from the division site before mitotic spindle disassembly, while in wild type cells septins remain until the end of cytokinesis. We also show that Vhs2 is subject to phosphorylations that decrease at the beginning of cytokinesis and that is regulated by Cdc14 phosphatase.
La citochinesi è quel processo regolato nel tempo e nello spazio tramite cui, dopo la segregazione dei cromosomi, le cellule eucariotiche dividono il loro citoplasma e le membrane per formare due cellule figlie indipendenti l’una dall’altra. Nel lievito gemmante Saccharomyces cerevisiae la citochinesi è promossa da vie finemente regolate che coordinano la divisione cellulare con la divisione nucleare al fine di garantire la stabilità genetica di cellule in crescita. Queste vie promuovono la contrazione dell’anello di actomiosina (AC) accoppiandola rispettivamente con la costrizione della membrana plasmatica e con la deposizione centripeta del setto primario (SP). Le vie che portano alla citochinesi sono parzialmente ridondanti e la loro contemporanea inattivazione causa un blocco della citochinesi e morte cellulare. Nelle cellule animali, il piano di divisione è specificato dal posizionamento del fuso mitotico e la citochinesi avviene grazie alla contrazione dell’anello di actomiosina, seguita dall’invaginazione della membrana plasmatica. In S. cerevisiae, il primo passo verso la citochinesi è l’assemblaggio di un anello rigido di septine attorno al collo della gemma contemporaneamente all’emissione della stessa, non appena le cellule entrano in fase S, definendo la posizione in cui avrà luogo la costrizione tra la cellula madre e la figlia in seguito all’uscita dalla mitosi. L’anello di septine funge da piattaforma di legame, al collo della gemma, per diverse proteine tra cui la catena pesante della miosina di tipo II, Myo1. Myo1 forma un anello al sito di emissione della gemma all’inizio della fase S in modo dipendente dalle septine. Alla fine dell’anafase un anello di actina si sovrappone con quello di Myo1 a generare il risultante anello di actomiosina la cui contrazione è strettamente accoppiata alla deposizione del setto primario. L’anello di septine permette la localizzazione anche di Iqg1 che, insieme a Bni1, è importante per il reclutamento dell’actina al collo della gemma, di Cyk3, richiesto per la corretta formazione del setto primario, e di Hof1, che colocalizza con l’anello di actomiosina durante la citochinesi. La successiva degradazione di Hof1 permette l’efficiente contrazione dell’anello di actomiosina e la separazione cellulare. Durante l’uscita dalla mitosi, la chitina sintasi Chs2 si localizza al sito di divisione e sintetizza il setto primario, composto di chitina, evento cha avviene in contemporanea con la contrazione dell’AC. Infine il setto secondario, che ha composizione simile a quella della parete di lievito, è deposto da entrambi i lati, della madre e della figlia, del setto primario. La successiva degradazione del setto primario dal solo lato della cellula figlia è garantita dal RAM pathway il quale viene attivato solo all’interno della gemma. A questo punto la cellula madre e la cellula figlia si separano definitivamente e questo processo lascia un disco di chitina (bud scar), residuo del setto primario, sulla superficie della cellula madre. Nel primo capitolo è stato descritto il ruolo delle ubiquitine ligasi Dma1 e Dma2 nella citochinesi. Queste proteine, a funzione almeno parzialmente ridondante, appartengono alla stessa famiglia FHA-RING ubiquitina ligasi di Chfr e Rnf8 umane e di Dma1 di Schizosaccharomyce pombe. In particolare abbiamo dimostrato che sia la mancanza di Dma1 e Dma2 che la moderata sovraproduzione di Dma2, nonostante non causino alterazioni nella progressione del ciclo cellulare, inficiano la contrazione dell’anello di actomiosina e la deposizione del setto primario. Inoltre, la moderata sovraproduzione di Dma2 impedisce l’interazione tra Tem1 e Iqg1, la quale è richiesta per la contrazione dell’AC, e causa la deposizione asimmetrica del setto primario nonché la delocalizzazione di Cyk3, un regolatore positivo di questo processo. Nell’insieme queste scoperte suggeriscono che le proteine Dma agiscono come dei regolatori negativi della citochinesi. Nel secondo capitolo è stato mostrato che l’ubiquitinazione della GTPasi Ras-like Tem1 è coinvolta nella regolazione della contrazione dell’anello di actomiosina. Tem1 non è richiesta per l’assemblaggio dell’AC ma per la sua dinamica. Nel primo capitolo abbiamo mostrato come questa proteina venga sottoposta ad ubiquitinazione ciclo-cellulare dipendente, in particolare la quantità di Tem1 ubiquitinata decresce quando le cellule iniziano a contrarre l’anello di actomiosina. In particolare, alti livelli di Dma2 inducono l’ubiquitinazione di Tem1, come anche l’inibizione della contrazione dell’AC. Analizzando le cinetiche di contrazione dell’AC in cellule che esprimono alti livelli di Dma2 e diverse varianti di Tem1 K-R ( in cui i residui di lisina sono stati sostituiti con arginine, rendendo così la proteina non ubiquitinabile), abbiamo dimostrato che l’ubiquitinazione di Tem1 sembra essere importante per l’inibizione Dma-dipendente della contrazione dell’anello di actomiosina. In particolare le lisine 112, 133 e 219 sembrano essere maggiormente implicate in questo processo. Nell’insieme questi dati suggeriscono che le proteine Dma agiscono come regolatori negativi della contrazione dell’AC influenzando in modo indiretto l’ubiquitinazione di Tem1. Nel terzo capitolo è stato mostrato come Dma1 e Dma2 sono coinvolte nel NoCut pathway, un checkpoint la cui attivazione previene la rottura dei cromosomi durante la divisione cellulare. La mancanza delle proteine Dma inficia l’attivazione del checkpoint in presenza di mutazioni che causano la permanenza di cromatina a livello del sito di divisione o danni alla zona centrale del fuso. Inoltre, la mancanza di Dma1 e Dma2 causa difetti di crescita se combinata con la delezione di geni coinvolti nel NoCut pathway o nei meccanismi che permettono la riparazione delle rotture del DNA che si vengono a generare quando il checkpoint non è perfettamente funzionante. Inoltre la mancanza delle proteine Dma, nonostante non sembra alterare la progressione del ciclo cellulare, quando combinata con la mancanza delle proteine Boi (le quali agiscono da inibitori dell’abscissione nel NoCut pathway) causa difetti di crescita dovuti ad un aumento della missegregazione cromosomica. Nell’insieme questi dati suggeriscono che le proteine Dma sono coinvolte nell’attivazione del NoCut pathway. Nell’ultimo capitolo è illustrata l’analisi della funzione di un nuovo fattore che regola la divisione cellulare: Vhs2. Nonostante questa proteina non sia essenziale per la vitalità cellulare, abbiamo dimostrato la sua implicazione nella stabilizzazione delle strutture generate dalle septine. La mancanza di Vhs2 causa difetti di crescita se combinata con diversi mutanti che affliggono la stabilità di queste strutture. Inoltre le cellule vhs2Δ mostrano per se un difetto nella stabilità delle septine infatti queste cellule mostrano un fenotipo tipico: le septine spariscono dal sito di divisione prima che il fuso venga disassemblato, mentre nelle cellule selvatiche le septine permangono fino alla fine della citochinesi. Abbiamo inoltre mostrato che Vhs2 è una proteina fosforilata e la sua fosforilazione decresce all’inizio della citochinesi ed è regolata dalla fosfatasi Cdc14.
(2014). Regulation of cytokinesis in saccharomyces cerevisiae. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2014).
Regulation of cytokinesis in saccharomyces cerevisiae
CASSANI, CORINNE
2014
Abstract
Cytokinesis is the spatially and temporally regulated process by which, after chromosome segregation, eukaryotic cells divide their cytoplasm and membranes to produce two daughter cells independent of each other. In the budding yeast Saccharomyces cerevisiae cytokinesis is driven by tightly regulated pathways that coordinate cell division with nuclear division to ensure the genetic stability during cell growth. These ways promote actomyosin ring (AMR) contraction coupled to plasma membrane constriction and to centripetal deposition of the primary septum, respectively. These pathways can partially substitute for each other, but their concomitant inactivation leads to cytokinesis block and cell death. In animal cells, the division plane is defined by the central spindle positioning and cytokinesis occurs through the contraction of the AMR, followed by the membrane furrowing. In S. cerevisiae, the first step towards cytokinesis is the assembly of a rigid septin ring, which forms at the bud neck concomitantly with bud emergence as soon as cells enter S phase and marks the position where constriction between mother and daughter cell will take place at the end of mitosis. The septin ring acts as a scaffold for the recruitment other proteins, among which Myo1, the heavy chain of the type II miosin. Myo1 forms a ring at the site of bud emergence at the onset of S phase in a septin-dependent manner. At the end of anaphase, an actin ring overlaps with that of Myo1 and the resulting contractile actomyosin ring drives primary septum deposition. The septin ring also recruits Iqg1 which is important, together with Bni1, for actin recruitment at the bud neck, Cyk3, required for proper synthesis of the septum, and Hof1, which is phosphorylated in telophase and colocalizes with the actomyosin ring during cytokinesis. The subsequent degradation of Hof1 allows efficient AMR contraction and cell separation. During mitotic exit, Chs2 localizes at the cell division site, where it drives synthesis of the primary septum, composed of chitin, simultaneously with actomyosin ring contraction. Afterwards the secondary septum, which has a similar composition to yeast cell wall, is produced on both the mother and the daughter side of the bud neck. The subsequent degradation of the primary septum from the daughter-side is ensured by the RAM pathway that is activated only in the bud. At this point mother and daughter cell separate permanently from each other leaving a chitin disk, that is the primary septum residue, called “bud scar", on the mother cell surface. In the first chapter we describe the role in cytokinesis of the functionally redundant FHA-RING ubiquitin ligases Dma1 and Dma2, that belong to the same ubiquitin ligase family as human Chfr and Rnf8 and Schizosaccharomyces pombe Dma1. In particular we show that both the lack of Dma1 and Dma2 and moderate Dma2 overproduction affect actomyosin ring contraction as well as primary septum deposition, although they do not apparently alter cell cycle progression of otherwise wild type cells. In addition, overproduction of Dma2 impairs the interaction between Tem1 and Iqg1, which is thought to be required for AMR contraction, and causes asymmetric primary septum deposition as well as mislocalization of Cyk3, a positive regulator of this process. In agreement with these multiple inhibitory effects, a Dma2 excess that does not cause any apparent defect in wild-type cells leads to lethal cytokinesis block in cells lacking the Hof1 protein, which is essential for primary septum formation in the absence of Cyk3. Altogether, these findings suggest that the Dma proteins act as negative regulators of cytokinesis. In the second chapter we show that the Ras-like GTPase Tem1 ubiquitylation is involved in AMR contraction regulation. Tem1 is not required for the actomyosin ring assembly but is required for its dynamics. In the first chapter we show how this protein undergoes cell cycle-regulated ubiquitylation, in particular the amount of ubiquitylated Tem1 decreases concomitantly with cells undergoing AMR contraction. Interestingly, high levels of Dma2 induce Tem1 ubiquitylation as well as inhibit AMR contraction. Analyzing the kinetics of AMR contraction in cells that express high levels of Dma2 and different Tem1 K-R variants (in which lysine residues were replaced by arginine residues, thus becoming not ubiquitylable), we show that Tem1 ubiquitylation seems to be important for Dma2’s AMR contraction inhibition. In particular lysines 112, 133 and 219 are mostly implicated in this regulation. Altogether, these findings suggest that the Dma proteins act as negative regulators of AMR contraction by indirectly influencing Tem1 ubiquitylation. In the third chapter we show that Dma1 and Dma2 are involved in the NoCut pathway, a checkpoint whose activation prevents chromosome breakage during cell division. The lack of Dma proteins affects checkpoint activation in the presence of mutations that cause chromatin persistance at the division site or spindle midzone damage. Moreover, the lack of Dma1 and Dma2 causes cell growth defect in combination with the deletion of genes involved in the NoCut pathway or in the mechanisms that permit DNA breaks repair generated when the checkpoint is not totally functional. Furthermore the lack of Dma proteins, although they do not apparently alter cell cycle progression, when combined with the lack of Boi proteins (that work as abscission inhibitors in the NoCut pathway) causes a growth defect due to an increase in chromosome missegregation. Altogether, these findings suggest that the Dma proteins act in the NoCut pathway. In the last chapther we describe the functional characterization of a new player in cell division control: Vhs2. Despite it is not essential for cells viability, we show how this protein is implicated in septins stabilizaton. The lack of Vhs2 causes cell growth defect in combination with several mutants that affect septin structure. Moreover vhs2Δ cells per se have a defect in septin stability, in fact these cells show a typical phenotype: the septins disappear from the division site before mitotic spindle disassembly, while in wild type cells septins remain until the end of cytokinesis. We also show that Vhs2 is subject to phosphorylations that decrease at the beginning of cytokinesis and that is regulated by Cdc14 phosphatase.File | Dimensione | Formato | |
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