Mariona Ramos

Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission

The α(1-3)glucan synthase Ags1 is essential for both secondary septum formation and the primary septum structural strength needed to counter cell turgor pressure during cell separation.

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction.

β(1,3)glucan is critical for contractile ring positioning and for coupling septum synthesis to constriction of the contractile ring and plasma membrane extension during cytokinesis.

Differential activities of three families of specific β(1,3)glucan synthase inhibitors in wild-type and resistant strains of fission yeast

Three specific β(1,3)glucan synthase (GS) inhibitor families, papulacandins, acidic terpenoids, and echinocandins, have been analyzed in Schizosaccharomyces pombe wild-type and papulacandin-resistant cells and GS activities. Papulacandin and enfumafungin produced similar in vivo effects, different from that of echinocandins. Also, papulacandin was the strongest in vitro GS inhibitor (IC50 103–104-fold lower than with enfumafungin or pneumocandin), but caspofungin was by far the most efficient antifungal because of the following. 1) It was the only drug that affected resistant cells (minimal inhibitory concentration close to that of the wild type). 2) It was a strong inhibitor of wild-type GS (IC50 close to that of papulacandin). 3) It was the best inhibitor of mutant GS. Moreover, caspofungin showed a special effect for two GS inhibition activities, of high and low affinity, separated by 2 log orders, with no increase in inhibition. pbr1-8 and pbr1-6 resistances are due to single substitutions in the essential Bgs4 GS, located close to the resistance hot spot 1 region described in Saccharomyces and Candida Fks mutants. Bgs4pbr1-8 contains the E700V change, four residues N-terminal from hot spot 1 defining a larger resistance hot spot 1-1 of 13 amino acids. Bgs4pbr1-6 contains the W760S substitution, defining a new resistance hot spot 1-2. We observed spontaneous revertants of the spherical pbr1-6 phenotype and found that an additional A914V change is involved in the recovery of the wild-type cell shape, but it maintains the resistance phenotype. A better understanding of the mechanism of action of the antifungals available should help to improve their activity and to identify new antifungal targets.

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

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

The Cell Biology of Fission Yeast Septation.

SUMMARY In animal cells, cytokinesis requires the formation of a cleavage furrow that divides the cell into two daughter cells. Furrow formation is achieved by constriction of an actomyosin ring that invaginates the plasma membrane. However, fungal cells contain a rigid extracellular cell wall surrounding the plasma membrane; thus, fungal cytokinesis also requires the formation of a special septum wall structure between the dividing cells. The septum biosynthesis must be strictly coordinated with the deposition of new plasma membrane material and actomyosin ring closure and must occur in such a way that no breach in the cell wall occurs at any time. Because of the high turgor pressure in the fungal cell, even a minor local defect might lead to cell lysis and death. Here we review our knowledge of the septum structure in the fission yeast Schizosaccharomyces pombe and of the recent advances in our understanding of the relationship between septum biosynthesis and actomyosin ring constriction and how the two collaborate to build a cross-walled septum able to support the high turgor pressure of the cell. In addition, we discuss the importance of the septum biosynthesis for the steady ingression of the cleavage furrow.

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast.

Fission yeast septation

ABSTRACT In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow.

Specific detection of fission yeast primary septum reveals septum and cleavage furrow ingression during early anaphase independent of mitosis completion

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