Juan Carlos García Cortés

Universidad de Salamanca · Department of Microbiology and Genetics

Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthas...

In recent years, invasive fungal infections have emerged as a common source of infections in immunosuppressed patients. All fungal cells are surrounded by a cell wall that is essential for cell integrity and survival. It prevents cell death and lysis resulting from high internal turgor pressure. Since the cell wall is not present in animal cells, i...

Cytokinesis is the concluding step of the cell cycle. Coordination between multiple cellular processes is essential for the success of cytokinesis. The fission yeast, Schizosaccharomyces pombe , like other fungal cells is contained within a cell wall. During cell division, the external cell wall is extended inwards to form a special septum wall str...

Fission yeast contains three essential β(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be...

Background The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function...

Background: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viabi...

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzin...

Background: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method...

In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is abs...

The cell wall is a structure external to the plasma membrane that is essential for the survival of the fungi. This polysaccharidic structure confers resistance to the cell internal turgor pressure and protection against mechanical injury. The fungal wall is also responsible for the shape of these organisms due to different structural polysaccharide...

Background In our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA). Purpose...

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 mit...

The timing of septum deposition onset overlaps with chromosome mass separation from early anaphase B. (A) Early log-phase cells carrying GFP-Atb2 (tubulin), Hht1-RFP (histone H3) and RFP-Bgs1 were grown in YES at 28°C, stained with Calcofluor white (CW, 50 μg ml-1) and imaged by fluorescence microscopy. White arrow, first CW-stained septum synthesi...

The presence of a non-degradable version of the Cdk1-associated cyclin Cdc13 causes cell cycle arrest in anaphase B and a blockage or delay in septation onset. (A) Cells expressing an endogenous non-degradable cdc13des2 version and a 45-cdc13+ wild-type copy, were grown at 28°C in the presence of thiamine for 10–15 h to repress the expression of th...

The actomyosin ring is stably assembled in the cell middle before the recruitment of Bgs1 and septum synthesis start, and its function is required for the timely deposition of the septum in early anaphase B. (A) Timing of the major AR components and Sid2 stably localized as a ring to the division site with respect to Bgs1 ring localization and sept...

The activation of septum synthesis does not depend on the arrival of the SPB with active SIN Cdc7 to the cell tip vicinity. (A, B) The timing of septation activation depends on the SPB localization with respect to the cell middle. Cells with displaced SPBs present a delayed septation onset regardless of the location of the SPB containing the active...

Percentages of the elapsed time in each mitotic phase. (DOCX)

The timing of septation onset is not related to the asymmetry of SIN. (DOCX)

Glucan synthases Bgs1, Bgs4 and Ags1 move to the division site during both the first and second rounds of mitosis after inactivation of the SIN signaling. (A) The displacement of the glucan synthases (GSs) to the division site does not depend on the SIN pathway. SIN cdc11-119 mutant cells carrying Hht1-RFP and GFP-Bgs1 or GFP-Bgs4 were grown at 28°...

The establishment of SIN asymmetry and the timely activation of septum synthesis do not depend on each other. (A, B) Early log-phase wild-type and thermosensitive cps1-191 (Bgs1) mutant cells were grown in YES at 25°C, shifted to 28°C for 1 h (A) or 32°C for 30 min (B) to produce a gradual delay in the onset of septum synthesis of cps1-191 mutant,...

Fission yeast strains used in this study. (DOCX)

Numerical data for graphs in Fig 1, Fig 2, Fig 3, Fig 4 and Fig 6. (PDF)

Etd1 does not localize in the membrane of the incipient septum during anaphase B, instead it concentrates when the septum is advanced during telophase. Cells were grown in MM without thiamine (induced GFP-etd1+) at 32°C for 24 h and imaged as in Fig 1. Double-headed arrows, interval of septum formation without detectable Etd1 in the indicated cells...

The septum ingression onset in the absence of spindle MT coincides with the SPBs exit to the cytoplasmic face of the nuclear envelope. (A) Cells carrying the nuclear envelope nucleoporin Cut11-GFP either growing without MBC (upper panels) or with MBC as in Fig 7C (lower panels) were imaged by time-lapse fluorescence microscopy (maximum-intensity pr...

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...

Yeast strains used for yeast two hybrid analysis (PDF)

Cell wall fractionation values of the indicated strains at 24°C, after 4.5 hr (cps1-191) or 24 hr (sbg1-3) at 36°C. Numbers in parentheses indicate percentage of each component in total cell wall. (TIF)

Supplementary data. (DOCX)

Multi-copy expression of sbg1+ improves quality of septum synthesized in cps1-191 cells. (A) Quantification of the displacement of the septum from the cell middle in the indicated strains: wt+pEmpty (MBY8558) and cps1-191+pSbg1 (MBY8946) after 16 hr at 34°C. (B) Quantification of the type of septa observed with 3D analysis of confocal spinning disk...

Sbg1p is an essential integral membrane protein. (A) Cells from strain hygr-GFP-Sbg1p HA-Bgs1p (MBY9241) were lysed and proteins extracted in lysis buffer containing the compounds indicated. This was subjected to ultra-centrifugation and the supernatant (S) and pellet (P) fractions from each treatment were resolved using SDS-PAGE gels and immunoblo...

Analysis of actomyosin ring and septum synthesis machinery in sbg1-3 cells. (A) 3D projection and 3D projection tilt images obtained with Imaris for Fig 7B. Green: GFP-Bgs1p, Red: Rlc1p-tdTomato, Pcp1p-mCherry. 0min indicates time of spindle body duplication. (B) 3D projection and 3D projection tilt images obtained with Imaris for Fig 7C. Green: hy...

Genetic interaction of sbg1 with imp2 and pxl1. (A) Image of a petridish showing very slow growth of the double mutant sbg1-3 imp2Δ (MBY9400) as compared to both single mutants imp2Δ (MBY737), sbg1-3 (MBY9359) and wild type cells (MBY192) at 24°C. (B) Calcofluor white (CW) images of the medial plane of fixed cells from the indicated strains: wt (MB...

S. pombe strains used in this study. (PDF)

List of primers used in this study. (PDF)

Cell wall fractionation values of the indicated strains at 24°C and after 16 hr at 34°C. Numbers in parentheses indicate percentage of each component in total cell wall. Student’s t-test was performed for the percentage of each polysaccharide in the cell wall for the combinations indicated in the lower table. (TIF)

List of plasmids used in this study. (PDF)

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 cytokines...

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 e...

Cytokinesis is the final process of the vegetative cycle, which divides a cell into two independent daughter cells once mitosis is completed. In fungi, as in animal cells, cytokinesis requires the formation of a cleavage furrow originated by constriction of an actomyosin ring which is connected to the plasma membrane and causes its invagination. Ad...

Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosinbased ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flan...

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 stud...

Fungi have emerged worldwide as increasingly frequent causes of healthcare-associated infections. Invasive fungal infections can be life-threatening. However, the number of antifungal agents available and their use in therapy is very limited. Recently, a new family of specific fungal cell wall synthesis inhibitors has emerged as an alternative anti...

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in...

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...

Cytokinesis must be initiated only after chromosomes have been segregated in anaphase and must be terminated once cleavage is completed. We show that the fission yeast protein Etd1 plays a central role in both of these processes. Etd1 activates the guanosine triphosphatase (GTPase) Spg1 to trigger signaling through the septum initiation network (SI...

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic...

Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows r...

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28°C in minimal medium and a lethal thermosensitive phenotype at 37°C. Cell growth is completely inhibited at 28°C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the...

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and alte...

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)β-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and altered...

Sixteen convolvulaceous glycolipids selected from the tricolorin (1 - 7) and orizabin (8 - 16) series, proved to be strong in vitro inhibitors of the enzyme that catalyzes the synthesis of 1,3-beta-D-glucan, a major polymer of fungal cell-walls. Results provide an insight into function of the specific structures of these complex macrocyclic lactone...

Here we report the synthesis, in vitro antifungal evaluation and SAR study of 41 chalcones and analogues. In addition, all active structures were tested for their capacity of inhibiting Saccharomyces cerevisiae beta(1,3)-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall.

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing...

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetrahydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution as...