scholarly journals The Golgi-resident protease Kex2 acts in conjunction with Prm1 to facilitate cell fusion during yeast mating

2007 ◽  
Vol 176 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Maxwell G. Heiman ◽  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Substantial Fraction ◽  
Yeast Mating ◽  
Fusion Defect ◽  
Mediate Cell ◽  
Wall Components

The molecular machines that mediate cell fusion are unknown. Previously, we identified a multispanning transmembrane protein, Prm1 (pheromone-regulated membrane protein 1), that acts during yeast mating (Heiman, M.G., and P. Walter. 2000. J. Cell Biol. 151:719–730). Without Prm1, a substantial fraction of mating pairs arrest with their plasma membranes tightly apposed yet unfused. In this study, we show that lack of the Golgi-resident protease Kex2 strongly enhances the cell fusion defect of Prm1-deficient mating pairs and causes a mild fusion defect in otherwise wild-type mating pairs. Lack of the Kex1 protease but not the Ste13 protease results in similar defects. Δkex2 and Δkex1 fusion defects were suppressed by osmotic support, a trait shared with mutants defective in cell wall remodeling. In contrast, other cell wall mutants do not enhance the Δprm1 fusion defect. Electron microscopy of Δkex2-derived mating pairs revealed novel extracellular blebs at presumptive sites of fusion. Kex2 and Kex1 may promote cell fusion by proteolytically processing substrates that act in parallel to Prm1 as an alternative fusion machine, as cell wall components, or both.

scholarly journals Prm1p, a Pheromone-Regulated Multispanning Membrane Protein, Facilitates Plasma Membrane Fusion during Yeast Mating

2000 ◽  
Vol 151 (3) ◽  
pp. 719-730 ◽  
Author(s):  
Maxwell G. Heiman ◽  
Peter Walter
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Large Fraction ◽  
Cell Contact ◽  
Yeast Mating

Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell–cell contact where fusion occurs. In matings between Δprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of ∼8 nm. Thus, the phenotype of Δprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused. This phenotype suggests a role for Prm1p in plasma membrane fusion.

scholarly journals Cell fusion during yeast mating requires high levels of a-factor mating pheromone.

1996 ◽  
Vol 135 (6) ◽  
pp. 1727-1739 ◽  
Author(s):  
V Brizzio ◽  
A E Gammie ◽  
G Nijbroek ◽  
S Michaelis ◽  
M D Rose
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Yeast Cells ◽  
Wild Type ◽  
Mating Pheromone ◽  
Gene Encoding ◽  
Fluorescence And Electron Microscopy ◽  
Identical Cell ◽  
In Trans

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.

scholarly journals The Plasma Membrane Proteins Prm1 and Fig1 Ascertain Fidelity of Membrane Fusion during Yeast Mating

2007 ◽  
Vol 18 (2) ◽  
pp. 547-556 ◽  
Author(s):  
Pablo S. Aguilar ◽  
Alex Engel ◽  
Peter Walter
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Wound Repair ◽  
Plasma Membranes ◽  
Time Lapse ◽  
Wild Type ◽  
Membrane Repair ◽  
Yeast Mating ◽  
Lysis Time

As for most cell–cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca2+ influx, yields similar cell fusion defects. Although extracellular Ca2+ is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca2+ is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca2+ binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca2+ is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca2+-dependent membrane repair.

scholarly journals The ham-2 Locus, Encoding a Putative Transmembrane Protein, Is Required for Hyphal Fusion in Neurospora crassa

Genetics ◽  
2002 ◽  
Vol 160 (1) ◽  
pp. 169-180
Author(s):  
Qijun Xiang ◽  
Carolyn Rasmussen ◽  
N Louise Glass
Keyword(s):  
Neurospora Crassa ◽  
Somatic Cell ◽  
Molecular Mechanism ◽  
Cell Fusion ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Vegetative Incompatibility ◽  
Hyphal Fusion ◽  
Aerial Hyphae

Abstract Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.

scholarly journals Inactivation of the pgmA Gene in Streptococcus mutans Significantly Decreases Biofilm-Associated Antimicrobial Tolerance

2019 ◽  
Vol 7 (9) ◽  
pp. 310 ◽  
Author(s):  
Martin Nilsson ◽  
Michael Givskov ◽  
Svante Twetman ◽  
Tim Tolker-Nielsen
Keyword(s):  
Cell Wall ◽  
Streptococcus Mutans ◽  
Congo Red ◽  
Biofilm Formation ◽  
Cell Wall Composition ◽  
Mutant Library ◽  
Wild Type ◽  
Glass Slides ◽  
Increased Sensitivity ◽  
Wall Components

Screening of a Streptococcus mutans mutant library indicated that pgmA mutants displayed a reduced biofilm-associated tolerance toward gentamicin. The biofilms formed by the S. mutans pgmA mutant also displayed decreased tolerance towards linezolid and vancomycin compared to wild-type biofilms. On the contrary, the resistance of planktonic S. mutans pgmA cells to gentamycin, linezolid, and vancomycin was more similar to wild-type levels. Investigations of biofilms grown in microtiter trays and on submerged glass slides showed that pgmA mutants formed roughly the same amount of biofilm as the wild type, indicating that the reduced antimicrobial tolerance of these mutants is not due to diminished biofilm formation. The pgmA gene product is known to be involved in the synthesis of precursors for cell wall components such as teichoic acids and membrane glycolipids. Accordingly, the S. mutans pgmA mutant showed increased sensitivity to Congo Red, indicating that it has impaired cell wall integrity. A changed cell wall composition of the S. mutans pgmA mutant may play a role in the increased sensitivity of S. mutans pgmA biofilms toward antibiotics.

scholarly journals Osmotic Balance Regulates Cell Fusion during Mating in Saccharomyces cerevisiae

1997 ◽  
Vol 138 (5) ◽  
pp. 961-974 ◽  
Author(s):  
Jennifer Philips ◽  
Ira Herskowitz
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Cell Contact ◽  
Fusion Event ◽  
Glycerol Facilitator ◽  
Fusion Defect ◽  
Osmotic Balance ◽  
Glycerol 3 Phosphate Dehydrogenase ◽  
Gpd1 Gene ◽  
Cell Cell

Successful zygote formation during yeast mating requires cell fusion of the two haploid mating partners. To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell–cell contact and only in the region of cell–cell contact. To understand how cell fusion is regulated, we identified mutants defective in cell fusion based upon their defect in mating to a fus1 fus2 strain (Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics 136:1287–1297). Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J. Albertyn, W.F. Skibbe, B.A. Prior, J. Ramos, J.M. Thevelein, and S. Hohmann. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:1360–1371). To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these mutants, we analyzed the behavior of an fps1Δ mutant with reduced intracellular glycerol levels because of a defect in the glycerol-3-phosphate dehydrogenase (GPD1) gene (Albertyn, J., S. Hohmann, J.M. Thevelein, and B.A. Prior. 1994. Mol. Cell. Biol. 14:4135– 4144): deletion of GPD1 partially suppressed the cell fusion defect of fps1 mutants. In contrast, overexpression of GPD1 exacerbated the defect. The fusion defect could also be partially suppressed by 1 M sorbitol. These observations indicate that the fusion defect of fps1 mutants results from inability to regulate osmotic balance and provide evidence that the osmotic state of the cell can regulate fusion. We have also observed that mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants. We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium. Unlike fps1 mutants, fus1 and fus2 mutants are not influenced by expression of GPD1 or by 1 M sorbitol. Their fusion defect is thus unlikely to result from altered osmotic balance.

Distribution of concanavalin a binding carbohydrates during mating in Chlamydomonas

1984 ◽  
Vol 66 (1) ◽  
pp. 223-239
Author(s):  
B.E. Millikin ◽  
R.L. Weiss
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Concanavalin A ◽  
Sodium Dodecyl ◽  
Fluorescein Isothiocyanate ◽  
Affinity Column ◽  
Wild Type ◽  
Mating Structure ◽  
Autolytic Activity ◽  
Surface Carbohydrates

Cell surface carbohydrates, detected by fluorescein isothiocyanate/concanavalin A (FITC-ConA), were identified at four locations on gametes of Chlamydomonas reinhardtii. (1) The cell wall: uniform labelling with FITC-ConA was observed; a substantial number of sites were localized in the sodium dodecyl sulphate-insoluble inner wall, which contains the flagellar collars. (2) The periplasm: a crescent-shaped area was visualized with FITC-ConA and localized by ferritin-ConA. We were able to recover autolytic activity on a ConA affinity column from the mating medium of wild-type cells after the release of these periplasmic sites. The cell-wall-less mutant CW15 displays no periplasmic sites and demonstrates a corresponding inability to release autolytic activity after mating for 60 min. A model for wall lysis is presented, which considers the involvement of these sites in the lytic process. (3) The mating structure: during mating a small fluorescent plaque-like site was observed on cells at a location corresponding to the carbohydrate-like zone of the mating type minus mating structure and may indicate the involvement of ConA binding material in gametic cell fusion. (4) Secreted products: following cell fusion zygotes begin to secrete ConA positive material at about 1 1/2 h. After 24 h a ConA positive zygote wall and pellicle appear.

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae

1983 ◽  
Vol 3 (5) ◽  
pp. 922-930
Author(s):  
R L Roberts ◽  
B Bowers ◽  
M L Slater ◽  
E Cabib
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Permissive Temperature ◽  
Wild Type ◽  
Chitin Synthesis ◽  
Specific Staining ◽  
Septal Region ◽  
Wall Components ◽  
Generalized Distribution

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

scholarly journals Maintenance of Mating Cell Integrity Requires the Adhesin Fig2p

2002 ◽  
Vol 1 (5) ◽  
pp. 811-822 ◽  
Author(s):  
Mingliang Zhang ◽  
Daniel Bennett ◽  
Scott E. Erdman
Keyword(s):  
Cell Wall ◽  
Cell Fusion ◽  
Large Family ◽  
Cell Integrity ◽  
Wild Type ◽  
Cell Agglutination ◽  
Spindle Positioning ◽  
Fungal Adhesins ◽  
Cell Cell

ABSTRACT Fungal adhesins represent a large family of serine/threonine-rich secreted glycoproteins. Adhesins have been shown to play roles in heterotypic and homotypic cell-cell adhesion processes, morphogenetic pathways and invasive/pseudohyphal growth, frequently in response to differentiation cues. Here we address the role of the Saccharomyces cerevisiae mating-specific adhesin Fig2p. Cells lacking FIG2 possess a variety of mating defects that relate to processes involving the cell wall, including morphogenetic defects, cell fusion defects, and alterations in agglutination activities. We found that mating-specific morphogenetic defects caused by the absence of FIG2 are suppressible by increased external osmolarity and that, during mating, fig2Δ cells display reduced viability relative to wild-type cells. These defects result from alterations in signaling activated by the mating and cell integrity pathways. Finally, we show that fig2Δ zygotes also have defects in zygotic spindle positioning that are osmoremedial, whereas the requirements for FIG2 in normal cell-cell agglutination and cell fusion during mating are insensitive to changes in the extracellular osmotic environment. We conclude that FIG2 performs distinct functions in the mating cell wall that are separable with respect to their ability to be suppressed by changes in external osmolarity and that a fundamental role of FIG2 in mating cells is the maintenance of cell integrity.

scholarly journals 1727. Candida albicans Phosphatidylinositol-(4,5)-Bisphosphate (PIP2) Directs Aberrant Cytokinesis and Septation in Response to Echinocandins, Which Correlates with Fungicidal Activity and Attenuated Virulence

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S633-S633
Author(s):  
Hassan Badrane ◽  
Minh-Hong Nguyen ◽  
Cornelius J Clancy
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Live Cell Imaging ◽  
Plasma Membranes ◽  
Cell Imaging ◽  
Fungicidal Activity ◽  
Wild Type ◽  
Cell Wall Integrity Pathway ◽  
Dividing Cells ◽  
In Cells

Abstract Background We previously showed that highly dynamic PIP2, septin, and PKC-Mkc1 cell wall integrity pathway responses correlate with echinocandin activity against C. albicans and attenuated virulence during invasive candidiasis. Our objectives were to determine whether PIP2 dysregulation in response to an echinocandin results in aberrant localization of the septation and cytokinesis apparatus, and to quantitate aberrant localization. Methods Live cell imaging (LCI) was performed for 3 hours (Nikon A1 confocal microscope, NIS Elements software; Tokyo) on C. albicans irs4 mutant and wild-type SC5314 expressing fluorescently labeled PIP2 and Cdc10 (septin), Act1 (actin), or Myo1 (myosin). Results C. albicans irs4, in which PIP2 5’-phosphatase is disrupted, mislocalizes PIP2 and septins, and over-activates the PKC-Mkc1 pathway in a manner similar to echinocandin-exposed C. albicans SC5314. LCI revealed that PIP2 co-localized with Act1 and Myo1 at aberrant sites in C. albicans irs4, similar to PIP2-Cdc10 co-localization. 83% of co-localizing patches were in cells undergoing active cytokinesis. 78% of patches were at sites of cytokinesis, which reflected both normal budding and abnormal, wide-necked budding; 5% of patches localized to aberrant plasma membrane sites during cytokinesis. 17% of co-localizing patches were in cells that were not undergoing active cytokinesis. 6% of patches were at old cytokinesis sites; 11% of patches were at aberrant plasma membrane sites. Similar PIP2-septin-actin-myosin dysregulation was observed in C. albicans SC5314 immediately upon 4x MIC caspofungin exposure (Figure; videos). Conclusion Dysregulated C. albicans PIP2 recruits the septation and cytokinesis apparatus, including septins, actin, and myosin, to sites of incomplete cytokinesis at bud necks and to sites of aberrant, ectopic septation in plasma membranes of both dividing and non-dividing cells. Our data support a model in which a dysregulated PIP2 response is triggered immediately upon echinocandin exposure, over-activates the PKC-Mkc-1 pathway, and correlates with the extent of fungicidal activity and attenuated virulence. PIP2-septation-cytokinesis dysregulation is likely to lead to C. albicans death by promoting cell lysis, or selecting cells to undergo apoptosis. Disclosures All authors: No reported disclosures.

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