Fission yeast Ags1
Fission yeast cytokinesis mutant
pombe cytokinesis mutant
Fission yeast Ags1
Research
Background
The way the cells generate their polarized growth and division is of fundamental importance to understand the cell morphogenesis process. The maintenance of the cell shape requires the correct establishment of the polarized growth and division sites in the cell surface and the polarized delivery to specific sites of the cell growth components. In the case of fungi, the cells grow by addition of new material to both their plasmatic membrane and their cell wall.
The cytokinesis is the final step of the cell cycle and it is conserved in all the organisms. It is a spatially and temporally regulated process that directs the polarity components to the middle zone of the cell and establishes a physical barrier that divides the cytoplasm between the two daughter cells, which in turn will separate. Cytokinesis is a critical process for cell integrity and is very well conserved from animal to fungal cells. All require coordinated contractile actomyosin ring closure and plasma membrane extension. Our current understanding of the eukaryotic cell cycle is largely derived from studies in the two yeast species Saccharomyces cerevisiae and S. pombe.
Fission yeast S. pombe is ideally suited for the study of cytokinesis since the genes required for cytokinesis are well defined and, like animal cells, S. pombe divides by medial fission through the use of a actomyosin ring. Once the cells enter into mitosis, the cytokinesis process begins. S. pombe medial fission displays several stages: actomyosin ring positioning and assembly, recruitment of actin patches to the actomyosin ring, activation of actomyosin ring contraction and septum formation, and septum degradation and cell separation.
Fungal cytokinesis requires the additional synthesis of a special division wall termed septum, strictly coupled to actomyosin ring contraction and plasma membrane extension. The septum is a three layered structure of a middle primary septum flanked by a secondary septum on each side. The S. pombe septum grows by simultaneous synthesis of both primary septum and secondary septum. The last step of cytokinesis is cell separation by controlled cell wall and primary septum degradation. Correct septum formation and especially cell separation are critical processes for cell integrity and survival. The fission yeast cell wall contains different essential glucans. Branched β(1,6)-glucan is located in the cell wall and secondary septum; minor linear β(1,3)-glucan (L-BG) is located mainly in the primary septum and some in the cell wall; and major branched β(1,3)-glucan (B-BG) and α(1,3)-glucan are located in the cell wall and both primary septum and secondary septum. While much is established about the molecular parts of cytokinesis, it remains much less clear how these parts are coordinated to produce a functional contractile machine coupled to the septum formation.
The enzyme involved in the β(1,3)-glucan synthesis is the β(1,3)-glucan synthase (GS). This enzyme is present in all the fungi and it is composed by at least a catalytic and a regulatory subunit, the latter identified as the GTPase Rho1. The Bgs (beta(1,3)-glucan synthesis) protein family are integral plasma membrane proteins, considered putative GS catalytic subunits. S. pombe contains four Bgs subunits, which share a high identity between them, with the Fks proteins of Saccharomyces cerevisiae and rest of fungi and with the plant callose synthases CalS. The four S. pombe Bgs subunits are essential: Bgs1, Bgs3 and Bgs4 in the vegetative cycle and Bgs2 in the sexual differentiation.
β(1,3)-glucan is a major contributor to the framework of the cell wall. There are several families of antifungal drugs whose mode of action is not well known, although they clearly interfere with β(1,3)-glucan synthesis by inhibiting the GS enzyme. These inhibitors include echinocandins (lipopeptides), papulacandins (glycolipids), acidic terpenoids such as enfumafungin, aerothricins (lipopeptides), GSI578 (piperazine propanol derivative), and arborcandins (lipopeptides). To date, only the echinocandins caspofungin, micafungin and anidulafungin have been approved (in years 2002, 2005 and 2006, respectively) for treatment of invasive fungal infections . The different essential functions of Bgs proteins in cell morphogenesis make them good targets for the study of antifungal drugs that specifically inhibit β(1,3)-glucan synthesis.
Structure and composition of fungal cell wall. The upper panel shows an scheme of the organization and composition of the main fungal cell wall layers with the cell wall synthases embedded in the plasma membrane. Transmission electron micrographs (TEM) of a fission yeast cell and a detail of the cell wall are presented in the lower panel
Models of the septation process and alternative septations of fission yeast. WT septation (top). Simultaneous coordinated synthesis (arrow) of primary septum (PS) (perpendicular to cell wall) and secondary septum (SS) (parallel to PS) form a three-layered septum. Septum maturation proceeds by PS anchorage into the cell wall (yellow arrow) and a second round of SS synthesis. Septation in Bgs1 absence (middle). The SS is synthesized parallel to the cell wall. The septum grows by successive parallel SS depositions (orange arrow). The matériel triangulaire dense (MTD) changes to a septum medial position, forming a dotted line of matériel dense (MD) in the SS layers. Septation in Bgs4 absence (bottom). CAR and septum are oblique positioned in the cell middle. The septum grows as a weak twisted and misdirected PS (wavy arrow) that is delayed and uncoupled from contractile actomyosin ring (CAR) and plasma membrane (PM) ingression (red arrow). After septum completion, the defective middle region is repaired with new PS (orange arrow) and the PS is retracted from the cell wall
Role of cell wall glucan in the control of actomyosin ring contraction and morphogenesis.
During cell division the plasma membrane ingresses centripetally from the cell surface at the medial division site, with the extracellular cell wall just outside the plasma membrane, and the actomyosin ring attached to the membrane on the cytoplasmic side. The S. pombe cell wall contains an essential α(1,3)-glucan and three different essential β-glucans: a B-BG, which is the major contributor to the cell wall and septum structure; a minor L-BG, concentrated in the primary septum, with minor amounts in the cell wall, and a minor branched β(1,6)-glucan in cell wall and secondary septum. α(1,3) and B-BG are present in cell wall, primary septum and secondary septum and both are essential for cell shape maintenance. Before our studies, little was established about the importance of the different glucans for cell wall and septum structure and function; and about the biosynthetic enzymes responsible for the septum and cell wall synthesis.
Bgs1 is responsible for the synthesis of L-BG and the structure of primary septum that it forms.
The gene bgs1 was the first identified coding for a putative GS catalytic subunit. It was cloned by complementation of the cps1-12 mutant, hypersensitive to a mitotic spindle poison. Bgs1 localizes first to the growing poles and moves to the actomyosin ring at the start of septation, remaining in the septum and reappearing later on at the poles, before the two daughter cells separate. This Bgs1 localization at the medial zone depend on the ring formation and SIN pathway. All these data suggested that Bgs1 is present and could play a role in every process of the S. pombe life cycle in which synthesis of b-glucan takes place. next I focused in the study of the function of Bgs1 during cytokinesis. We demonstrated that Bgs1 is the GS subunit responsible for the L-BG synthesis and the primary septum that this glucan originates. The bgs1 repression generates branching and multiseptated cells, denoting the essential Bgs1 role in septation and polarity control. In addition, transmission electron (TEM) and immunoelectron (IEM) microscopy studies of bgs1 repressed cells showed a considerable difference between primary septa absence (50%) and L-BG absence (15%), indicating that the L-BG is necessary but not sufficient for primary septum formation. bgs1Δ deletion is lethal, but the bgs1Δ spores are able to originate large multiseptated and branching cells, similar to those observed during bgs1 repression. However, only the bgs1Δ cells permitted to observe that the Bgs1 absence produces the complete absence of L-BG, primary septum and Calcofluor staining, a fluorochrome that binds with high affinity to the septa of wild type cells. All these data demonstrated that 1) the primary septum is not necessary for septum synthesis. In the absence of primary septum, the cell is able to form remedial septa and complete cytokinesis but not cell abscission. 2) Bgs1 is responsible for the L-BG synthesis, which in turn is responsible for the primary septum structure. 3) The L-BG is the cell wall polysaccharide that specifically and with high affinity interacts with the fluorochrome Calcofluor. Remarkably, these results show that S. pombe presents similarities with plant cytokinesis. Plant septum synthesis is different from that of fungal and animal cells because of the septum is not made centripetally but by synthesis of a medial cell plate that will constitute the primary septum. However as in S. pombe, the plant cell plate is made of callose (L-BG) and the protein involved is the Bgs homologue CalS1. In this sense, fission yeast presents a mechanism of septum formation intermediate between chitin-containing yeasts and plants, similar to that of budding yeast in the strategy that it has adopted and similar to that of plant cells in the synthase involved, in the polysaccharide formed and in the primary septum structure.
Bgs1p localizes to the sites of active growth: one or both cell ends during polar growth, and in the actomyosin ring and septum during cytokinesis. (A) Calcofluor staining and localization of Bgs1 during the cell cycle. (B) Magnification of the Calcofluor staining and localization of Bgs1 in the CAR and along the plasma membrane during septum formation and cell separation.
Morphology and septum structure of WT and cells from germinated bgs1Δ spores. Differently from the WT septum, the bgs1Δ septa do not contain a primary septum and its L-BG and are not stained with Calcofluor.
Bgs4 synthesizes the main cell wall β(1,3)-glucan which is responsible for the secondary septum structure and cell integrity, and is required to couple septation to actomyosin ring contraction.
We have characterized Bgs4 as the first Bgs subunit found to be part of the GS catalytic subunit. We discovered that the mutants cwg1-1, which displays reduced cell wall β(1,3)-glucan and GS activity , and orb11-59, which was described as rounded and showing a complete loss of polarity, were allelic to bgs4. Bgs4 is crucial for maintaining the cell integrity, as its absence promotes cell lysis at the poles and septum. Bgs4 localizes to the growing poles, actomyosin ring and septum, and to each site of cell wall synthesis during the sexual differentiation. Bgs4 showed for the first time in a GS protein that it depends on the F-actin for its delocalization from and relocalization to, but not for its stable maintenance at the growing sites, poles and septum. All the above data suggested that Bgs4 played a role in each cell wall synthesis process of the S. pombe life cycle by synthesizing a β(1,3)-glucan that is essential for preserving the cell integrity and reinforce its importance as an essential target for the design of specific antifungal drugs.
Despite our knowledge of cytokinesis, little is known about how extracellular signals communicate with intracellular events. The extracellular cell wall must be connected to the plasma membrane and actomyosin ring. In fact, proteins and polysaccharides of the animal extracellular matrix (functional equivalent of fungal cell wall) have been shown to be critical for cytokinesis. However, how the extracellular matrix is coupled to the actomyosin ring and how it affects the actomyosin ring function remain largely unknown. Our data showed for the first time that a extracellular polysaccharide has a role in the actomyosin ring function. In the absence of B-BG the actomyosin ring is often oblique assembled, sliding along the cell middle until septum synthesis initiation fixes the actomyosin ring and septum position to the cell wall. With these results we showed a surprising separation between a slower primary septum synthesis and a faster actomyosin ring and plasma membrane ingression, indicating that the pushing force of primary septum synthesis is not necessary for actomyosin ring and plasma membrane progression.
More surprisingly, we found that the ingression rate of the uncoupled actomyosin ring and plasma membrane is faster than normal, suggesting that in a normal coupled cytokinesis the synthesis rate of the attached primary septum restricts the actomyosin ring and plasma membrane ingression rates. Moreover, these data showed that septation can progress with a defective actomyosin ring pulling force. In the absence of B-BG, the initial septa are twisted and bent and larger septa appear misdirected, indicating a relaxed actomyosin ring without the tensile pulling force necessary to maintain straight the growing septum. These results suggest that cytokinesis can progress and be completed without or with a defective pushing force of septum synthesis and/or pulling force of actomyosin ring contraction, just with the help of plasma membrane extension by the addition of membrane vesicles. In fact, it has been described that cytokinesis can be completed with defective actomyosin ring, but to date this has not been reported with delayed primary septum deposition. These observations reveal important convergent similarities between fungal and animal cytokinesis, with a fungal cell wall B-BG required for connecting cell wall and actomyosin ring during cytokinesis and extracellular matrix polysaccharides required for animal cytokinesis, suggesting that the extracellular cell wall is an evolutionarily highly conserved component of eukaryotic cytokinesis.
(A,B) Lysis of Bgs4-depleted cells at poles and septum region at the start of cell separation, and Bgs4 localization during cell growth and magnification of GFP-Bgs4 localization during septum synthesis. (C) Bgs4 and its β(1,3)-glucan are required for correct and stable actomyosin ring (CAR) positioning in the cell middle. (D,E) Bgs4 and its β(1,3)-glucan are required for correct straight CAR constriction, and for coupling septum synthesis to CAR contraction and plasma membrane extension. (F) Bgs4 is responsible for the secondary septum (SS) formation and necessary for the last steps of the primary septum (PS) synthesis.
Bgs4 is required for primary septum completion but not for general septum closure. The defect in open primary septum does not correspond to a general defect in septum synthesis or CAR contraction. Progression (bracket) of WT PS coincides with that of septum membrane (Bgs1, Bgs3, Psy1) and CAR (Rlc1) proteins but not in the absence of Bgs4, in which the CAR stays attached to the PM, whereas the PS formation is uncoupled and delayed.
The α(1,3)-glucan synthase Ags1 confers the essential septum strength needed for safe gradual cell abscission.
Once septation is accomplished, subsequent cell separation requires the selective degradation of the primary septum. Because of the high turgor pressure, even a minor rupture of the cell wall structure might lead to cell lysis and cell death. Therefore, the correct assembly and structural integrity of the cell wall and the specialized septum structure are vital for cell survival. S. pombe is an attractive model for morphogenesis whose growth pattern likely diverged from filamentous fungi in response to the loss of hyphal growth. Like fission yeast, the cell wall of filamentous fungi also contains β- and α-glucans.
To date, the detailed study of Ags1 localization has been impossible because of the unsuccessful attempts for the GFP tagging of a functional Ags1. Similarly, the study of α(1,3)-glucan localization in the cell wall and septum by IEM has been unsuccessful. Ags1 was described as a putative α(1,3)-glucan synthase essential for cell integrity, but a specific role for Ags1 in the cell wall and septum architecture has never been described. We succeeded in generating a fully functional Ags1-GFP permitting us to find Ags1 localized, as Bgs proteins, in all sites of cell wall synthesis. Interestingly, we found a tight co-localization and physical interaction with Bgs1 at the septum, both cooperating in the essential early steps of cell wall and septum construction. In addition, we studied the Ags1 function by constructing ags1 repression strains and studying the lethal effect induced by the absence of Ags1. We showed that α(1,3)-glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during separation. As result, absence of Ags1 and therefore α(1,3)-glucan, generate a special and unique side-explosive separation caused by an abrupt tear of a weak primary septum that cannot support the physical forces generated by the internal pressure of the cells during abscission.
Together these findings reveal a new function of the primary septum keeping attached the secondary septum for a steady and safe cell separation, allowing its progressive and slow curvature only in the site where the primary septum has gradually been degraded. As we advance in our knowledge of fission yeast septation, I find new and surprising evolutionary convergent structural and functional similarities between fission yeast and plant septa: (i) as in S. pombe, the plant cell plate is made of callose (L-BG) and the protein involved is the Bgs homologue CalS1; (ii) S. pombe α-glucan and plant pectin (an α-linked polysaccharide) assume the same function in a similar structure. Both are essential for wall adhesion and safe cell separation, maintaining the linkages between polysaccharides in equivalent structures, the fission yeast primary septum and the plant mature cell plate (middle lamella).
(A, B) Ags1 and Bgs1 appear simultaneously and interact at the division site before septum synthesis. (C,D) Ags1 is responsible for the a(1,3)glucan synthesis and the septum adhesion strength required for safe cell separation. (E) Model of the septation apparatus. Top: Septation and cell separation in WT cells. A balance between the osmotic pressure that curves the secondary septum to the stable spherical conformation and the degradation of the primary septum ensures a symmetrical and steady separation. Bottom: Alternative septation and side-explosive cell separation in the absence of Ags1 and the corresponding α-glucan. Asymmetrical septum-edging degradation and mechanical tear of a weak primary septum that cannot hold the turgor pressure, leading to an instantaneous side-explosive separation to adopt the stable spherical conformation in both new ends. The cells stay attached by the septum-edging for the next cell cycle (botton). CW: cell wall; Fs: fission scar; ICW: remedial internal cell wall layer; Mtd: materiel triangulaire dense; Ne: new end; Pr: turgor pressure; Pm: plasma membrane; Ps: primary septum; RSs: remedial secondary septum; Se: septum-edging; Ss: secondary septum.