Abstract
Polarization is a fundamental cellular property, which is essential for the function of numerous cell types. Over the past three to four decades, research using the best-established yeast ...systems in cell biological research, Saccharomyces cerevisiae (or budding yeast) and Schizosaccharomyces pombe (or fission yeast), has brought to light fundamental principles governing the establishment and maintenance of a polarized, asymmetric state. These two organisms, though both ascomycetes, are evolutionarily very distant and exhibit distinct shapes and modes of growth. In this review, we compare and contrast the two systems. We first highlight common cell polarization pathways, detailing the contribution of Rho GTPases, the cytoskeleton, membrane trafficking, lipids, and protein scaffolds. We then contrast the major differences between the two organisms, describing their distinct strategies in growth site selection and growth zone dimensions and compartmentalization, which may be the basis for their distinct shapes.
This review compares and contrasts the mechanisms of cell polarization in budding and fission yeasts.
External signal–mediated polarized growth in fungi Bassilana, Martine; Puerner, Charles; Arkowitz, Robert A.
Current opinion in cell biology,
February 2020, 2020-Feb, 2020-02-00, 20200201, 2020-02, Letnik:
62
Journal Article
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As the majority of fungi are nonmotile, polarized growth in response to an external signal enables them to search for nutrients and mating partners, and hence is crucial for survival and ...proliferation. Although the mechanisms underlying polarization in response to external signals has commonalities with polarization during mitotic division, during budding, and fission growth, the importance of diverse feedback loops regulating external signal–mediated polarized growth is likely to be distinct and uniquely adapted to a dynamic environment. Here, we highlight recent advances in our understanding of the mechanisms that are crucial for polarity in response to external signals in fungi, with particular focus on the roles of membrane traffic, small GTPases, and lipids, as well as the interplay between cell shape and cell growth.
Flippases transport lipids across the membrane bilayer to generate and maintain asymmetry. The human fungal pathogen Candida albicans has 5 flippases, including Drs2, which is critical for ...filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to the antifungal drug fluconazole and copper ions. We show here that such a flippase mutant also has an altered distribution of phosphatidylinositol 4-phosphate PI(4)P and ergosterol. Analyses of additional lipid transporters, i.e. the flippases Dnf1-3, and all the oxysterol binding protein (Osh) family lipid transfer proteins, i.e. Osh2-4 and Osh7, indicate that they are not critical for filamentous growth. However, deletion of Osh4 alone, which exchanges PI(4)P for sterol, in a drs2 mutant can bypass the requirement for this flippase in invasive filamentous growth. In addition, deletion of the lipid phosphatase Sac1, which dephosphorylates PI(4)P, in a drs2 mutant results in a synthetic growth defect, suggesting that Drs2 and Sac1 function in parallel pathways. Together, our results indicate that a balance between the activities of two putative lipid transporters regulates invasive filamentous growth, via PI(4)P. In contrast, deletion of OSH4 in drs2 does not restore growth on fluconazole, nor on papuamide A, a toxin that binds PS in the outer leaflet of the plasma membrane, suggesting that Drs2 has additional role(s) in plasma membrane organization, independent of Osh4. As we show that C. albicans Drs2 localizes to different structures, including the Spitzenkörper, we investigated if a specific localization of Drs2 is critical for different functions, using a synthetic physical interaction approach to restrict/stabilize Drs2 at the Spitzenkörper. Our results suggest that the localization of Drs2 at the plasma membrane is critical for C. albicans growth on fluconazole and papuamide A, but not for invasive filamentous growth.
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.
In a number of elongated cells, such as fungal hyphae, a vesicle cluster is observed at the growing tip. This cluster, called a Spitzenkörper, has been suggested to act as a vesicle supply center, ...yet analysis of its function is challenging, as a majority of components identified thus far are essential for growth. Here, we probe the function of the Spitzenkörper in the human fungal pathogen Candida albicans, using genetics and synthetic physical interactions (SPI). We show that the C. albicans Spitzenkörper is comprised principally of secretory vesicles. Mutant strains lacking the Spitzenkörper component myosin light chain 1 (Mlc1) or having a SPI between Mlc1 and either another Spitzenkörper component, the Rab GTPase Sec4, or prenylated green fluorescent protein (GFP), are viable and still exhibit a Spitzenkörper during filamentous growth. Strikingly, all of these mutants formed filaments with increased diameters and extension rates, indicating that Mlc1 negatively regulates myosin V, Myo2, activity. The results of our quantitative studies reveal a strong correlation between filament diameter and extension rate, which is consistent with the vesicle supply center model for fungal tip growth. Together, our results indicate that the Spitzenkörper protein Mlc1 is important for growth robustness and reveal a critical link between filament morphology and extension rate.
Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In Candida albicans, as in many filamentous fungi, a cluster of vesicles, called a Spitzenkörper, is observed at the tip of growing hyphae that is thought to function as a vesicle supply center. A central prediction of the vesicle supply center model is that the filament diameter is proportional to the extension rate. Here, we show that mutants lacking the Spitzenkörper component myosin light chain 1 (Mlc1) or having synthetic physical interactions between Mlc1 and either another Spitzenkörper component or prenylated GFP, are defective in filamentous growth regulation, exhibiting a range of growth rates and sizes, with a strong correlation between diameter and extension rate. These results suggest that the Spitzenkörper is important for growth robustness and reveal a critical link between filament morphology and extension rate.
During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is ...established in an already asymmetric cell—a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth.
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•Photo-recruitment of active Cdc42 over the plasma membrane resets growth•Transient increase of active Cdc42 at the plasma membrane disrupts membrane traffic•Increase in plasma membrane active Cdc42 results in de novo secretory vesicle cluster•Secretory vesicle clustering can occur in the absence of directional growth
Silva et al. use light-dependent plasma membrane recruitment of active Cdc42 to reset polarity in asymmetric filamentous fungal cells. This transient increase in plasma membrane active Cdc42 disrupted membrane traffic and resulted in the formation of a striking de novo secretory vesicle cluster, in the absence of directional growth.
The initial step of a number of human or plant fungal infections requires active penetration of host tissue. For example, active penetration of intestinal epithelia by Candida albicans is critical ...for dissemination from the gut into the bloodstream. However, little is known about how this fungal pathogen copes with resistive forces upon host cell invasion.
Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate PI(4)P at the Golgi ...apparatus is required for the budding-to-filamentous-growth transition in the human-pathogenic fungus Candida albicans; however, the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e., Efr3, Ypp1, and Stt4. The amounts of plasma membrane PI(4)P in the
Δ/Δ and
Δ/Δ mutants were ∼60% and ∼40%, respectively, of that in the wild-type strain, whereas it was nearly undetectable in the
Δ/Δ mutant. All three mutants had reduced plas7ma membrane phosphatidylserine (PS). Although these mutants had normal yeast-phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity, and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal-specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the
Δ/Δ mutant had wild-type virulence, the
Δ/Δ mutant had attenuated virulence, and the
Δ/Δ mutant caused no lethality. In the mouse model of oropharyngeal candidiasis, only the
Δ/Δ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity, and virulence in C. albicans.
While the PI-4-kinases Pik1 and Stt4 both produce PI(4)P, the former generates PI(4)P at the Golgi apparatus and the latter at the plasma membrane, and these two pools are functionally distinct. To address the importance of plasma membrane PI(4)P in Candida albicans, we generated deletion mutants of the three putative plasma membrane PI-4-kinase complex components and quantified the levels of plasma membrane PI(4)P in each of these strains. Our work reveals that this phosphatidylinositol phosphate is specifically critical for the yeast-to-hyphal transition, cell wall integrity, and virulence in a mouse systemic infection model. The significance of this work is in identifying a plasma membrane phospholipid that has an infection-specific role, which is attributed to the loss of plasma membrane PI(4)P resulting in β(1,3)-glucan unmasking.
Chemical gradients of peptide mating pheromones are necessary for directional growth, which is critical for yeast mating. These gradients are generated by cell-type specific secretion or export and ...specific degradation in receiving cells. Spatial information is sensed by dedicated seven-transmembrane G-protein coupled receptors and yeast cells are able to detect extremely small differences in ligand concentration across their approximately 5-microm cell surface. Here, I will discuss our current knowledge of how cells detect and respond to such shallow chemical gradients and in particular what is known about the proteins that are involved in directional growth and the establishment of the polarity axis during yeast mating.
Ergosterol is a critical membrane lipid in fungi. In
, this essential plasma membrane amphipathic lipid is important for interactions with host cells, in particular, host immune responses. Here, we ...use a live-cell reporter for specifically visualizing ergosterol and show that apical enrichment of this sterol is not critical for budding and filamentous growth in this human fungal pathogen. Our results highlight that this live-cell reporter is likely to be a useful tool in the analyses of azole resistance and tolerance mechanisms, including alterations in drug targets and upregulation of efflux activities.
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