A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe is a widely used model organism to study many aspects of eukaryotic cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research

Regulation of actin assembly for the formation of the fusion focus in fission yeast

The actin cytoskeleton is a complex and dynamic array of actin filament networks that provide strength to the cell for a vast range of cellular processes, such as division, migration and polarization. These actin networks emerge and self-organize from a common pool of monomeric actin to form structures of surprisingly specific architecture, allowing them to drive distinct cellular properties. How cells succeed is a very fundamental question which the actin field has been trying to tackle. Schizosaccharomyces pombe (S. pombe), a yeast distantly related to the baking yeast Saccharomyces cerevisiae, is a great model to tackle this question as its actin cytoskeleton is organized in only 4 well- segregated actin networks. These are the actin patches, which are branched, heavily cross-linked actin networks which support intake from the extracellular environment; the actin cables, long parallel bundles which serve as tracks for intracellular transport; the cytokinetic ring, an antiparallel contractile network which supports cell division; and the fusion focus, an aster which supports cell-cell fusion by allowing the concentration of hydrolase-containing vesicle delivery at the region of contact between the two cells, which leads to cell-wall digestion. The last three networks all depend on a particular class of proteins for their nucleation, the formins For3, Cdc12 and Fus1, respectively. While the first two formins are relatively well studied, Fus1 has received comparatively little attention. The work presented here sheds some light on some of the principles that govern actin network self-segregation. First, I showed that competition between proteins binding different actin networks is instrumental in maintaining the respective structural identity of each network. Capping protein is a heterodimer blocking actin filament dynamics at the barbed ends which associates with actin patches. Its deletion leads to an increase of actin incorporation within its cognate actin network, the patches, but also to weak actin cables, cytokinesis defects and fusion delay, thereby also affecting the other three actin networks. Focusing first on the organisation of S. pombe fusion focus as model, I showed that this is due to ectopic relocalization of all three formins at the now free actin filament barbed ends in actin patches. This leads to ectopic recruitment of actin binding proteins specific to linear actin structures to the branched actin patches, giving them a dual identity. Thus, insulation of actin structures from each other, for which capping protein is a key factor, is an important mechanism ensuring their unique identity. While capping protein protects branched structures from the action of formins, how do different formins assemble distinct actin networks? For Fus1, part of the answer lies in its specific actin assembly properties, such as its elongation rate or nucleation efficiency, but also in additional specific properties, which are tailored to the formin’s function in organizing its specific actin network. Indeed, I used chimeras between S. pombe’s formins to control for expression levels, expression time and regulation. However, the other formins’ specific properties couldn’t replace the cognate fusion focus formin Fus1. Building up on existing literature, I showed that this was dependent on at least three parameters: the nucleation efficiency, which has to be kept high, the elongation rate, which has to be kept low, and an additional formin property which, though I was not able to firmly identify, I was able to pinpoint to a specific amino acid within the FH2 domain of the formin. Third, I was able to show a new and exciting property of the N-terminal regulatory region of Fus1, which might be involved in its regulation. Indeed, I showed that the N-terminal regulatory region of Fus1 is forming clusters when artificially expressed in interphase. The responsible region is a low complexity region which is essential for fusion, and the phenotype resulting from its absence could be entirely rescued by replacing that region by self-assembling domains. Because the function of the fusion focus is to concentrate vesicular release at the zone of contact between the two cells, Fus1 self-aggregation could be instrumental in focalizing this release. I also showed that this self-assembling property was likely to be regulated in the native Fus1 for proper fusion. As a formin’s regulation at the proper time and place in the cell is instrumental in assembling and segregating a specific actin network, these findings are an exciting direction for future research. -- Le cytosquelette d’actine est un ensemble complexe et dynamique de réseaux de filaments d’actine qui fournissent la force nécessaire à de nombreux processus cellulaires, tels que la division, la migration ou la polarisation. Ces réseaux d’actine s’auto-organisent à partir d’une source cytosolique d’actine monomérique commune et forment des réseaux d’architecture spécifique qui leur permettent de supporter des propriétés cellulaires distinctes. Comment les cellules réussissent cette prouesse est une question fondamentale à laquelle la recherche dans le domaine de l’actine tente de répondre. Schizosaccharomyces pombe (S. pombe), une levure lointainement apparentée à la levure de boulanger Saccharomyces cerevisiae, est un très bon modèle pour adresser cette question puisque son cytosquelette d’actine n’est constitué que de 4 réseaux, qui sont bien ségrégés dans la cellule. Ce sont les patches d’actine, qui sont des réseaux d’actine dont les filaments sont ramifiés et hautement interconnectés, et qui contrôlent l’internalisation de nutriments à partir du milieu extracellulaire ; les câbles d’actine, longs faisceau de filaments parallèles qui servent de rails pour le transport intracellulaire ; l’anneau cytokinetique, un réseau contractile de filaments antiparallèles qui sous-tend la division cellulaire ; et le focus de fusion, un aster de filaments d’actine qui contrôle la fusion cellulaire en permettant la concentration à la région de contact entre les deux cellules de vésicules contenant des hydrolases, ce qui mène à la digestion du mur cellulaire. Les trois derniers réseaux d’actine dépendent tous d’une classe spécifique de protéines pour leur nucléation, les formines For3, Cdc12 et Fus1, respectivement. Alors que ces deux premières formines sont relativement bien étudiées, Fus1 a reçu comparativement peu d’attention. Le travail présenté dans ce manuscrit permet d’apporter quelques éléments de réponse sur les principes qui gouvernent l’auto-organisation des réseaux d’actines. En premier lieu, j’ai pu montrer que la compétition entre des protéines se liant à différents réseaux d’actine était déterminante pour maintenir l’identité propre de chaque réseau. La protéine de coiffe est un hétéro-dimère qui bloque la polymérisation et la dépolymérisation de l’extrémité barbelée des filaments d’actine dans les patches d’actine. Sa suppression conduit à une augmentation de la quantité d’actine incorporée dans son propre réseau, les patches, mais également à une déstabilisation des cables d’actine, à des défauts de cytokinèse et à un délai de fusion. Ainsi, sa suppression affecte tous les réseaux d’actine, pas seulement celui dans lequel la protéine de coiffe est impliquée. En prenant d’abord l’organisation du focus de fusion chez S. pombe comme modèle d’étude, j’ai montré que cela était dû à une relocalisation ectopique des trois formines de S. pombe aux extrémités barbelées des patches d’actine, libérées par l’absence des protéines de coiffe. En conséquence, des protéines accessoires spécifiques des réseaux d’actine linéaires sont relocalisées dans le réseau dendritique des patches d’actine, ce qui leur confère une identité double. Ainsi, l’isolation entre les différents réseaux d’actine, pour laquelle la protéine de coiffe est clé, est un important mécanisme qui assure que chaque réseau conserve son identité propre. Tandis que les protéines de coiffe protègent les réseaux dendritiques de l’action des formines, comment différentes formines assemblent-elles des réseaux d’identité distincte ? Pour Fus1, une partie de la réponse repose sur ses propriétés spécifiques d’assemblage de filaments d’actine, telles que sa vitesse d’élongation ou son efficacité de nucléation, mais aussi sur une propriété spécifique à Fus1, l’ensemble étant adapté à construire le réseau d’actine spécifique qui sous-tend sa fonction. En effet, j’ai utilisé des chimères entre les différentes formines de S. pombe pour controler le niveau d’expression de la chimère impliquée dans la formation du focus de fusion, le moment auquel elle était exprimée ainsi que sa régulation, variant seulement les autres propriétés de ces chimères. Cependant, aucune des autres formines n’était capable de remplacer Fus1 dans son rôle d’organisatrice du focus de fusion. En me basant sur la littérature existante, j’ai montré que cela dépendait d’au moins 3 paramètres : l’efficacité de nucléation, qui doit être maintenue haute, la vitesse d’élongation, qui doit rester basse, et une propriété additionnelle qui, bien qu’elle n’ait pas pu être formellement identifiée, a pu être restreinte à un acide aminé spécifique dans le domaine FH2 de la formine Fus1. Troisièmement, j’ai découvert une nouvelle et passionnante propriété de la région N-terminale de Fus1, qui pourrait être impliquée dans sa régulation. En effet, j’ai montré que la région N-terminale de Fus1, lorsqu’exprimée artificiellement en interphase, dictait la formation de clusters. La région responsable est une région de faible complexité qui est essentielle pour la fusion cellulaire. Les effets de son absence peuvent être complètement annihilés par le remplacement de cette région par des séquences dictant des propriétés d’auto-association. Puisque la fonction du focus de fusion est de concentrer le transport vésiculaire à la région de contact entre les deux cellules, les propriétés d’auto-association de Fus1 pourraient être cruciales pour focaliser ce transport. J’ai également montré que cette propriété d’auto-assemblage était probablement régulée dans la protéine native. Puisque la régulation d’une formine au bon endroit et au bon moment dans la cellule est déterminante pour ségréger dans le temps et l’espace une structure spécifique d’actine, ces résultats fournissent une direction fascinante pour la recherche future

Genomic contacts reveal the control of sister chromosome decatenation in E. coli

Abstract In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links. We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome

International audienceAbstract In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo