30 research outputs found
Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth
International audienceThe phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes. membrane traffic | filamentous growth | polarity | morphogenesis
On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans
International audienceCandida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells
CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae
After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, the fatty acid in sn-2 of the diacylglycerol moiety can be replaced by a C26:0 fatty acid by a deacylationâreacylation cycle catalysed by Per1p and Gup1p. Furthermore the diacylglycerol moiety of the yeast GPI anchor can also be replaced by ceramides. CWH43 of yeast is homologous to PGAP2, a gene that recently was implicated in a similar deacylation reacylation cycle of GPI proteins in mammalian cells, where PGAP2 is required for the reacylation of monoradylglycerol-type GPI anchors. Here we show that mutants lacking CWH43 are unable to synthesize ceramide-containing GPI anchors, while the replacement of C18 by C26 fatty acids on the primary diacylglycerol anchor by Per1p and Gup1p is still intact. CWH43 contains the COG3568 metal hydrolase motif, which is found in many eukaryotic and prokaryotic enzymes. The conserved His 802 residue of this motif was identified as being essential for ceramide remodelling. Ceramide remodelling is not required for the normal integration of GPI proteins into the cell wall. All remodelling reactions are dependent on prior removal of the inositol-linked fatty acid by Bst1p
Many roads to symmetry breaking: Molecular mechanisms and theoretical models of yeast cell polarity
Mathematical modeling has been instrumental in identifying common principles of cell polarity across diverse systems. These principles include positive feedback loops that are required to destabilize a spatially uniform state of the cell. The conserved small G-protein Cdc42 is a master regulator of eukaryotic cellular polarization. Here we discuss recent developments in studies of Cdc42 polarization in budding and fission yeasts and demonstrate that models describing symmetry-breaking polarization can be classified into six minimal classes based on the structure of positive feedback loops that activate and localize Cdc42. Owing to their generic system-independent nature, these model classes are also likely to be relevant for the G-proteinâbased symmetry-breaking systems of higher eukaryotes. We review experimental evidence pro et contra different theoretically plausible models and conclude that several parallel and nonâmutually exclusive mechanisms are likely involved in cellular polarization of yeasts. This potential redundancy needs to be taken into consideration when interpreting the results of recent cell-rewiring studies
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Role of Arf GTPases in fungal morphogenesis and virulence.
Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion
Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth
CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae
Arl1 regulates secretion.
<p>A) Distribution of PS is not altered in <i>arl1/arl1</i> mutant during hyphal growth. WT, <i>arl1/arl1</i> and <i>drs2/drs2</i> cells expressing the PS reporter (GFP-LactC2) were induced in the presence of FCS for 45 and 90 min, respectively and sum projections of representative images are shown. A false color look up table (LUT) is used to highlight signal intensity. Note that the <i>drs2/drs2</i> deletion strain is indicated as <i>drs2</i>. B) Distribution of PS and hyphal growth is altered in the <i>drs2/drs2</i> mutant. Time course of indicated strains expressing GFP-LactC2, incubated in the presence of FCS. Images, taken every 5 min, are sum projections (21 z-sections). C) FM4-64 endocytosis is not altered in the <i>arl1/arl1</i> mutant. WT and <i>arl1/arl1</i> cells were incubated with FM4-64 on ice for 40 min (time 0:00), and subsequently transferred to 30°C to initiate endocytosis, for the indicated times. Cells were visualized by fluorescence microscopy with the respective DIC images shown. D) Distribution of Phr2 is altered in the <i>arl1/arl1</i> mutant. WT and <i>arl1/arl1</i> cells, expressing yemCherry-Phr2 were grown in the presence of FCS for 45 and 90 min, respectively. Central Z sections of representative images are shown. E) The <i>arl1/arl1</i> mutant has increased secretion. WT and <i>arl1/arl1</i> cells, expressing HWP<sub>ss</sub>-GFP, were pelleted and BSA (100 Όg/mL) was added to the supernatant prior to TCA (10%) precipitation. Following centrifugation, precipitated proteins were washed twice with ice-cold acetone, then analyzed by SDS/PAGE and immunoblotting. The amount of secreted Hwp1<sub>ss</sub>-GFP in the WT and <i>arl1/arl1</i> mutant strains was normalized using Cdc11 and the total cellular GFP signal, quantified from the cell pellet. The equivalents (Equiv.) of cells and culture supernatants are indicated; 7 times more culture supernatant was analyzed compared to cells.</p
Arl1 is required for restricted polarized growth in hyphae.
<p>A) Actin cytoskeleton is polarized in <i>arl1/arl1</i> hyphae. WT and <i>arl1/arl1</i> cells were incubated in the presence of FCS for 90 min, prior to Alexa 568-phalloidin staining. B) The <i>arl1/arl1</i> mutant has an increased proportion of cells with 2 or more germ tubes. WT, <i>arl1/arl1</i> and <i>arf3/arf3</i> cells were incubated for 90 min in the presence of FCS and cells with 2 or more germ tubes were counted. Error bars indicate the mean +/- the SD of 4 experiments, <i>n</i> = 500 cells. C) Actin is polarized in only one of the germ tubes in <i>arl1/arl1</i> mutant. Cells were incubated for 90 min in the presence of FCS and the actin cytoskeleton was visualized using Alexa568-phalloidin. DIC and maximum projections (21 deconvolved z-sections) of representative cells are shown. D) Active Cdc42 is associated with only one of the germ tubes in the <i>arl1/arl1</i> mutant. <i>arl1/arl1</i> cells expressing a reporter for active Cdc42 or active Rho1, <i>i</i>.<i>e</i> CRIB-GFP or GFP-RID, respectively, were incubated for 45 min in the presence of FCS. DIC and sum projections (21 deconvolved z-sections) of representative cells are shown. E) Active Cdc42 is recruited sequentially to multiple sites. Time lapse of <i>arl1/arl1</i> cells expressing reporters for active Cdc42 and active Rho1, incubated in the presence of FCS. Images, taken every 5 min, are sum projections (21 deconvolved z-sections) and arrows indicate the sites of active Cdc42 or active Rho1 recruitment.</p