Littérature scientifique sur le sujet « Cytokinesis, Saccharomyces cerevisiae »

Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5ΔN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box–dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5ΔN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5ΔN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box–dependent cytokinetic pathway.

ABSTRACT The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize inmob1 mutants, suggesting that MOB1functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck requiredCDC3, MEN genes CDC5,CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent ofMYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis.

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.

A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1delta mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1delta;) morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.

ABSTRACT In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Δ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Δ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.

Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p–Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p–Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.

Abstract In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G2/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Δ defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.

Ageing in budding yeast is not determined by chronological lifespan, but by the number of times an individual cell is capable of dividing, termed its replicative capacity. As cells age they are subject to characteristic cell surface changes. Saccharomyces cerevisiae reproduces asexually by budding and as a consequence of this process both mother and daughter cell retain chitinous scar tissue at the point of cytokinesis. Daughter cells exhibit a frail structure known as the birth scar, while mother cells display a more persistent bud scar. The number of bud scars present on the cell surface is directly related to the number of times a cell has divided and thus constitutes a biomarker for replicative cell age. It has been proposed that the birth scar may be subject to stretching caused by expansion of the daughter cell; however, no previous analysis of the effect of cell age on birth or bud scar size has been reported. This paper provides evidence that scar tissue expands with the cell during growth. It is postulated that symmetrically arranged breaks in the bud scar allow these rigid chitinous structures to expand without compromising cellular integrity.

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.
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.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology." Discipline: Biology; Department/School: Biology.

Rho GTPases are highly conserved regulators of cell polarity and the actin cytoskeleton. The role of the Rho GTPase Cdc42 and its regulation during cell division is not well understood. Using biochemical and imaging approaches in budding yeast, I demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase, but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to downregulate Cdc42 during mitotic exit prevents the normal localization of key cytokinesis regulators - Iqg1 and Inn1- at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase (PAK) kinase, Ste20. Inhibition of Cdc42 and related Rho GTPases may be a general feature of cytokinesis in eukaryotes.

La cytokinèse est un processus fondamental prenant place à la fin de la mitose et permettant la séparation des deux cellules filles. Un défaut de cytokinèse peut mener à une ségrégation anormale des chromosomes et engendrer des phénomènes de cancer. Dans beaucoup d'organismes eucaryotes, la cytokinèse nécessite l'assemblage et la contraction d'un anneau d'actomyosine permettant la formation d'un sillon et la réorganisation de la membrane cellulaire au site de clivage. Dans la plupart de ces organismes, des protéines du cytosquelette appelées septines participent à la cytokinèse. Chez la levure bourgeonnante, Saccharomyces cerevisiae, cinq septines sont exprimées durant la mitose (Cdc3, Cdc10, Cdc11, Cdc12 et Shs1). Ces protéines ont la capacité de s'assembler en un anneau au niveau du site de bourgeonnement, lieu de séparation entre la cellule mère et la cellule fille. Cet anneau de septines permet la fixation et le recrutement de nombreuses protéines intervenant dans la cytokinèse. La dynamique des septines change durant le cycle cellulaire, ce qui a une importance dans la régulation de la cytokinèse. La stabilisation de cet anneau est accompagnée d'un changement du niveau de phosphorylation des septines, mais les kinases responsables de ces modifications restent inconnues. Les travaux de l'équipe de Simonetta Piatti ont mis en évidence un nouveau rôle de la GTPase Rho1 et de sa cible, la protéine kinase C (Pkc1), dans la régulation de la dynamique des septines. Le but de ce travail de thèse était de déterminer les voies moléculaires par lesquelles la protéine Pkc1 intervient dans le recrutement et la stabilisation de l'anneau de septines. Pour se faire nous avons purifié le complexe de septines chez la levure bourgeonnante en présence ou en absence de la protéine Pkc1 et nous l'avons analysé par spectrométrie de masse. Cette analyse nous a permis d'observer que le niveau de phosphorylation d'un cluster (îlot) de 5 sérines était diminué sur Shs1. L'alignement de séquence nous a permis de constater que ce domaine était conservé dans la septine Cdc11. Par ailleurs ces deux protéines sont connues pour jouer un rôle dans l'assemblage des filaments et la formation de l'anneau de septines. Il a déjà été observé qu'un mutant phosphomimétique du cluster de sérine de la septine Shs1 empêche la formation des filaments in-vitro. Nous avons voulu caractériser le rôle de ce cluster dans la protéine Cdc11 en créant un mutant non-phosphorylable (CDC11-9A) et un mutant phosphomimétique (CDC11-9D). De manière très évidente, le mutant phosphomimétique provoque des problèmes de cytokinèse dans les cellules dont le gène codant la protéine Shs1 a été supprimé. A l'inverse le mutant non-phosphorylable améliore le phénotype des cellules ne comportant pas Shs1. Ces résultats sont en parfait accord avec l'observation selon laquelle les protéines Shs1 et Cdc11 pourraient avoir des fonctions très similaires, et mettent en avant le rôle important du cluster de sérines phosphorylées de Cdc11 lors de la cytokinèse. Nous avons constaté que Pkc1 ne phosphoryle pas directement les septines, mais agit par l'intermédiaire de kinases et de phosphatases impliquées dans la régulation des septines. Nous avons pu montrer que Pkc1 régule l'interaction de Gin4 avec les septines, cette kinase étant connue pour sa capacité à phosphoryler Shs1. De plus, nous avons observé que Pkc1 impacte sur le niveau de phosphorylation des deux autres kinases de la même famille, Hsl1 et Kcc4. Par ailleurs, la délétion de PKC1 diminue drastiquement la quantité de protéines Kcc4 dans la cellule.L'absence de Pkc1 augmente également l'interaction entre les septines et Bni4, une sous-unité régulatrice de la phosphatase PP1. Nous avons également observé que Bni4-PP1 peut déphosphoryler Cdc11, expliquant la diminution de son niveau de phosphorylation en cas d'absence de la protéine Pkc1.Ces travaux mettent en évidence que Pkc1 est un nouveau régulateur majeur des septines dans la levure
Cytokinesis is the last step of mitosis and is the fundamental process leading to the physical separation of two daughter cells. Defects in cytokinesis generate polyploid cells that are prone to chromosome missegregation and cancer development. In animal cells and fungi, cytokinesis requires the formation and contraction of an actomyosin ring that drives ingression of the cleavage furrow. Additional cytoskeletal proteins called septins contribute to cytokinesis. In the budding yeast Saccharomyces cerevisiae, five different septins are expressed during the mitotic cell cycle (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1). All septins, except for Shs1, are essential for cell viability. Yeast septins form filaments that in turn organize into a ring at the bud neck, which is the constriction between the mother and the future daughter cell where cytokinesis takes place. The septin ring then expands into a rigid septin collar that acts as scaffold for cytokinesis by recruiting most cytokinetic proteins to the bud neck. Cell cycle-regulated changes in septin ring dynamics are thought to be important for its cytokinetic functions and formation of the rigid septin collar is accompanied by septin phosphorylation. However, the kinases responsible for these modifications have not been fully characterized. Unpublished data from our laboratory indicate that the Rho1 GTPase, which is essential for actomyosin ring formation and contraction, and its target protein kinase C (Pkc1) contribute to deposition and stabilization of the septin ring. Here, we have addressed how Pkc1 regulates septin ring deposition and/or stability. To this end, septin complexes were purified from yeast and analyzed by mass spectrometry, comparing wild type and pkc1Δ mutant cells. This mass spectrometry analysis clearly showed that phosphorylation of a cluster of residues in Shs1 decreased in the absence of Pkc1. Interestingly, we found that this cluster is conserved in the septin Cdc11, which together with Shs1 is known to play an important role in the assembly of high-order structures like filaments and rings. Phosphomimetic mutations of the phosphorylatable cluster in Shs1 have been previously shown to disrupt filament formation in-vitro. We therefore proceeded to mutagenise the same cluster in Cdc11, generating a phosphomimetic (CDC11-9D) and in a non-phosphorylatable mutant (CDC11-9A). Strikingly, the phosphomimetic CDC11-9D caused cytokinesis defects in cells lacking Shs1, whereas the non-phosphorylatable CDC11-9A allele partially rescued the sickness of shs1∆ mutant cells. These observations are in agreement with the notion that Cdc11 and Shs1 share overlapping functions and highlight an important role of the phosphorylatable cluster of Cdc11 for cytokinesis. We also found that Pkc1 does not phosphorylate septins directly, but rather regulates the activity of septin kinases and phosphatases. Consistently, we show that Pkc1 affects the interaction between septins and the bud neck kinase Gin4, which is known to interact with septins and to phosphorylate them. In addition, Pkc1 impacts on the phosphorylation of two additional bud neck kinases, Hsl1 and Kcc4, which are part of the same family of Nim1-related kinases as Gin4. In addition, PKC1 deletion leads to a dramatic decrease in the levels of Kcc4 , so that it is barely detected at the bud neck.Deletion of PKC1 affects also the interaction between septins and the Bni4 protein, which is a regulatory subunit for the PP1 phosphatase at the bud neck. In turn, we found that Bni4-PP1 modulates Cdc11 phosphorylation, thereby explaining how the latter is decreased in the absence of Pkc1. Altogether, our data strongly suggest that Pkc1 is a novel major regulator of septins in yeast

Asymmetric cell division is an important mechanism for the differentiation of cells during embryogenesis and cancer development. Saccharomyces cerevisiae divides asymmetrically and is therefore used as a model system for understanding the mechanisms behind asymmetric cell division. Ace2p is a transcriptional factor in yeast that localizes primarily to the daughter nucleus during cell division. The distribution of Ace2p is visualized using a fusion protein with yellow fluorescent protein (YFP) and confocal microscopy. Systems biology provides a new approach to investigating biological systems through the use of quantitative models. The localization of the transcriptional factor Ace2p in yeast during cell division has been modelled using ordinary differential equations. Herein such modelling has been evaluated. A 2-compartment model for the localization of Ace2p in yeast post-cytokinesis proposed in earlier work was found to be insufficient when new data was included in the model evaluation. Ace2p localization in the dividing yeast cell pair before cytokinesis has been investigated using a similar approach and was found to not explain the data to a significant degree. A 3-compartment model is proposed. The improvement in comparison to the 2-compartment model was statistically significant. Simulations of the 3-compartment model predicts a fast decrease in the amount of Ace2p in the cytosol close to the nucleus during the first seconds after each bleaching of the fluorescence. Experimental investigation of the cytosol close to the nucleus could test if the fast dynamics are present after each bleaching of the fluorescence. The parameters in the model have been estimated using the profile likelihood approach in combination with global optimization with simulated annealing. Confidence intervals for parameters have been found for the 3-compartment model of Ace2p localization post-cytokinesis. In conclusion, the profile likelihood approach has proven a good method of estimating parameters, and the new 3-compartment model allows for reliable parameter estimates in the post-cytokinesis situation. A new Matlab-implementation of the profile likelihood method is appended.

Cell growth and division requires delivery of new membrane and remodelling factors to the cell surface. In budding yeast, this involves actin-dependent transport of secretory vesicles to sites of growth, followed by vesicle fusion to the plasma membrane. Rho-type GTPases regulate actin polarization and vesicle fusion, but how they signal to diverse effectors controlling these separate processes is not well understood. Here, we show that the cortical proteins Boi1 and Boi2 work together with Rho GTPase signalling to regulate the exocyst, a complex that mediates the tethering of secretory vesicles to the plasma membrane. Cells lacking both Boi proteins show normal actin polarity but are defective in bud emergence, bud growth, and fusion of Bgl2-containing vesicles to the plasma membrane. A gain-of-function version of Exo70, a component of the exocyst and effector of Cdc42 and Rho3, restores growth of boi1 boi2 mutant cells. Furthermore, hyperactivation of Rho-GTPase signalling rescues boi defects, suggesting that the essential function of Boi proteins is to mediate Rho-dependent activation of the exocyst during cell growth. Finally, we find that Boi1 and Boi2-dependent inhibition of abscission in cells with chromatin bridges, via their function in the NoCut checkpoint, is bypassed by expression of gain-of-function EXO70, suggesting the NoCut pathway also involves regulation of the exocyst.
Tant el creixement com la divisió cel·lular requereix el transport de membrana i altres factors a la superfície cel·lular. En cèl·lules de S. cerevisiae, aquests processos necessiten el transport de vesícules de secreció a través dels cables d’actina fins a les zones de creixement actiu, on es fusionen. Les Rho GTPases regulen la polarització de l’actina i la fusió de vesícules, però es desconeix com les GTPases senyalitzen els diversos efectors i com regulen els dos tipus de funcions. En aquest estudi, demostrem que les proteines Boi1 i Boi2 treballen conjuntament amb la senyalització de les Rho GTPases, per tal de regular la funció del complexe “exocyst” que media els contactes inicials entre vesícules de secreció i la membrana plasmàtica. Cèl·lules sense Boi1/2 tenen el citoesquelet d’actina polaritzat, però la cèl·lula filla no pot emergir ni créixer. Un al·lel d’Exo70, una subunitat de l’”exocyst”, que és efector de les GTPases Rho3 i Cdc42, restaura el creixement de cel·lules defectuoses en la funció de Boi1/2. A més a més, l’hiperactivació de GTPases rescata defectes dels mutants de Boi1/2, suggerint que la funció essencial de Boi1 i Boi2 és promoure l’activació de l’”exocyst”, depenent de Rho, durant el creixement cel·lular. Finalment, hem demostrat que la inibició de la divisió cel·lular que controlen via NoCut els efectors Boi1/2 en cèl·lules amb defectes en la segregació de cromosomes, es rescata amb l’al·lel d’Exo70 descrit anteriorment. Aquestes observacions suggereixen que NoCut podria funcionar també a través de la regulació de l’”exocyst”.

Les septines forment une famille de GTPases conservée chez les champignons et dans les cellules animales [Kinoshita et al., 2003]. Pendant la division cellulaire, elles se localisent aux sites de cytocinèse et sont essentielles pour ce processus dans la levure bourgeonnante, les embryons de drosophile et les cellules de mammifère en culture [Faty et al., 2002]. Dans les levures bourgeonnantes, les septines, composées de réseaux parallèles de 1laments [Byers et al., 1976], forment un anneau au cou mère-fille. Cet anneau est étroitement associé à la membrane plasmique et constitue un échafaudage pour le recrutement de la myosine II et d'autres facteurs cytocinétiques au futur site de clivage [Longtine et al., 2003]. De plus, l'anneau de septines contribue à la formation d'une barrière de diffusion latérale sur la membrane plasmique, qui aide à maintenir les facteurs de la polarité cellulaire dans le bourgeon [Barral et al., 2000; Takizawa et al., 2000]. Chez les métazoaires, les septines sont aussi requises pour la compartimentation du cortex cellulaire [Schmidt et al., 2004; Joo et al., 2005] et sont impliquées dans une myriade de processus cellulaires, y compris l'assemblage et l'orientation du corps polaire du fuseau [Kusch et al., 2002; Spiliotis et al., 2005], l'exocytose et le transport vésiculaire [Hsu et al., 1998;Beites et al., 1999], la migration cellulaire [Finger et al., 2003], et l'apoptose Larisch et al., 2000; Gottfried et al., 2004].[...]Dans ce mémoire, après avoir passé en revue les fonctions essentielles des septines chez la levure bourgeonnante Saccharomyces cerevisiae, je présente une étude dans laquelle j’ai démontré que UNC-59 et UNC-61 forment un complexe hétérotétramérique capable de s’associer en structures d’ordre supérieur et en filaments dans les cellules et que le complexe hétérotétramérique de septines est assemblé à partir d’hétérodimères UNC-59/UNC-61. De plus, au cours d’un projet visant à développer un outil moléculaire pour l’étude des fonctions des septines in vivo dans des cellules sauvages de Saccharomyces, j’ai mis au point une méthode d’isolement d’intrabodies à partir de la banque d’anticorps recombinants humains en phage display ETH-2 et démontré leur activité d’inhibition de la formation de l’anneau de septines au col du bourgeon dans les cellules de Saccharomyces cerevisiae. L’ensemble des résultats présentés met en lumière l'arrangement moléculaire des monomères dans le complexe de septines et suggère que les complexes de septines s’assemblent dans les filaments et les structures d'ordre supérieur au col du bourgeon chez Saccharomyces cerevisiae. Ainsi, nous proposons que les septines forment la quatrième composante du cytosquelette
Septins form a family of GTPases conserved in fungi and animal cells [Kinoshita etal., 2003]. During cell division, they localize at cytokinesis sites and are essential for this process in budding yeast, Drosophila embryos and cultured mammalian cells [Fatyet al., 2002].In budding yeasts, septins, composed of parallel networks of filaments [Byers et al.,1976], form a mother-daughter neck ring. This ring is closely associated with the plasma membrane and constitutes a scaFold for the recruitment of myosin II and other cytokinetic factors at the future cleavage site [Longtine et al., 2003]. In addition, the septin ring contributes to the formation of a lateral diffusion barrier on the plasma membrane, which helps maintain the factors of cell polarity in the bud [Barral et al.,2000; Takizawa et al., 2000].In metazoans, septins are also required for compartmentalization of the cellular cortex [Schmidt et al., 2004; Joo et al., 2005] and are involved in a myriad of cellular processes, including assembly and orientation of the polar body of the spindle [Kusch etal., 2002; Spiliotis et al., 2005], exocytosis and vesicular transport [Hsu et al., 1998;Beites et al., 1999], cell migration [Finger et al., 2003], and apoptosis [Larisch et al.,2000; Gottfried et al., 2004].[...]The set of results presented highlights the molecular arrangement of the monomersin the septin complex and suggests that the septin complexes assemble in filaments and higher order structures at the bud neck in Saccharomyces cerevisiae. Thus, wepropose that septins form the fourth component of the cytoskeleton