Septins from the phytopathogenic fungus Ustilago maydis are required for proper morphogenesis but dispensable for virulence

Background

Septins are a highly conserved family of GTP-binding proteins involved in multiple cellular functions, including cell division and morphogenesis. Studies of septins in fungal cells underpin a clear correlation between septin-based structures and fungal morphology, providing clues to understand the molecular frame behind the varied morphologies found in fungal world.

Methodology/Principal Findings

Ustilago maydis genome has the ability to encode four septins. Here, using loss-of-function as well as GFP-tagged alleles of these septin genes, we investigated the roles of septins in the morphogenesis of this basidiomycete fungus. We described that septins in U. maydis could assemble into at least three different structures coexisting in the same cell: bud neck collars, band-like structures at the growing tip, and long septin fibers that run from pole to pole near the cell cortex. We also found that in the absence of septins, U. maydis cells lost their elongated shape, became wider at the central region and ended up losing their polarity, pointing to an important role of septins in the morphogenesis of this fungus. These morphological defects were alleviated in the presence of an osmotic stabilizer suggesting that absence of septins affected the proper formation of the cell wall, which was coherent with a higher sensitivity of septin defective cells to drugs that affect cell wall construction as well as exocytosis. As U. maydis is a phytopathogen, we analyzed the role of septins in virulence and found that in spite of the described morphological defects, septin mutants were virulent in corn plants.

Conclusions/Significance

Our results indicated a major role of septins in morphogenesis in U. maydis. However, in contrast to studies in other fungal pathogens, in which septins were reported to be necessary during the infection process, we found a minor role of septins during corn infection by U. maydis.     

Septin stability and recycling during dynamic structural transitions in cell division and development

Septins are conserved proteins found in hetero-oligomeric complexes that are incorporated into distinct structures during cell division and differentiation; yeast septins Cdc3, Cdc10, Cdc11, and Cdc12 form hetero-octamers and polymerize into filaments, which form a "collar" at the mother-bud neck [1]. Posttranslational modifications, nucleotide binding, and protein-protein and protein-lipid interactions influence assembly and disassembly of septin structures [2], but whether individual septins are used repeatedly to build higher-order assemblies was not known. We used fluorescence-based pulse-chase methods to visualize the fate of pre-existing (old) and newly synthesized (new) molecules of two septins, Cdc10 and Cdc12. They were recycled through multiple mitotic divisions, and old and new molecules were incorporated indistinguishably into the collar. Likewise, old and new subunits intermixed within hetero-octamers, indicating that exchange occurs at this organizational level. Remarkably, in meiosis, Cdc10 made during vegetative growth was reutilized to build sporulation-specific structures and reused again during spore germination for budding and during subsequent mitotic divisions. Although Cdc12 also persisted during sporulation, it was excluded from septin structures and replaced by another subunit, Spr3; only new Cdc12 populated the collar of germinating spores. Thus, mechanisms governing septin incorporation are specific to each subunit and to the developmental state of the cell.     

Septin localization across kingdoms: three themes with variations

Septins are GTPases that form filaments in fungi and animals. In addition to their original role in cell division, septins have been shown to have roles in coordinating nuclear division, membrane trafficking and organizing the cytoskeleton. Many recent studies have examined subcellular localization of septins in a wide range of fungi and animals. Septin localization shows three patterns, which generally correspond to function across kingdoms. Septins that localize to projections shape and compartmentalize emerging growth. Septins that localize to partitions compartmentalize pre-existing cellular material. Septins that localize to the whole cell are involved in membrane trafficking and organizing the cytoskeleton and are most often in animals. The difference in localization pattern frequency between kingdoms will probably disappear as more septins are examined in diverse organisms and tissues.     

Mutational Analysis of Essential Septins Reveals a Role for Septin-Mediated Signaling in Filamentation

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus Candida albicans has shown that septins also have vital roles in cell wall regulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.     

Actin and septin ultrastructures at the budding yeast cell cortex

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.     

Septins: the fourth component of the cytoskeleton

Septins belong to a family of proteins that is highly conserved in eukaryotes and is increasingly recognized as a novel component of the cytoskeleton. All septins are GTP-binding proteins that form hetero-oligomeric complexes and higher-order structures, including filaments and rings. Recent studies have provided structural information about the different levels of septin organization; however, the crucial structural determinants and factors responsible for septin assembly remain unclear. Investigations on the molecular functions of septins have highlighted their roles as scaffolds for protein recruitment and as diffusion barriers for subcellular compartmentalization in numerous biological processes, including cell division and host-microorganism interactions.     

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

A novel septin-associated protein, Syp1p, is required for normal cell cycle-dependent septin cytoskeleton dynamics in yeast

Septins are a family of GTP-binding proteins whose heterooligomeric complex is the basic structural element of the septin filaments found in many eukaryotic organisms. In budding yeast, septins are mainly confined at the mother–daughter junction and are required for cell morphogenesis and division. Septins undergo assembly and disassembly in accordance with the progression of the cell cycle. In this report, we identified the yeast protein Syp1p as a new regulator of septin dynamics. Syp1p colocalizes with septins throughout most of the cell cycle. Syp1p interacts with the septin subunit Cdc10p and can be precipitated by Cdc10p and Cdc12p. In the syp1Δ mutant, both formation of a complete septin ring at the incipient bud site and disassembly of the septin ring in later stages of cell division are significantly delayed. In addition, overexpression of Syp1p causes marked acceleration of septin disassembly. The fluorescence recovery after photobleaching (FRAP) assay further showed that Syp1p promotes septin turnover in different cell cycle stages. These results suggest that Syp1p is involved in the regulation of cell cycle-dependent dynamics of the septin cytoskeleton in yeast.     

A draft of the human septin interactome

Background

Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins.

Methodology/Principal Findings

Here, we performed yeast two-hybrid screens with human septins 1–10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments.

Conclusions/Significance

If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the “group rule”, i.e. members of the same group (e.g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.     

Mammalian septins are required for phagosome formation

Septins are members of a highly conserved family of filamentous proteins that are required in many organisms for the completion of cytokinesis. In addition, septins have been implicated in a number of important cellular processes and have been suggested to have roles in regulating membrane traffic. Given the proposed role of septins in cell membrane dynamics, we investigated the function of septins during FcgammaR-mediated phagocytosis. We show that several septins are expressed in RAW264.7 and J774 mouse macrophage cell lines and that SEPT2 and SEPT11 are colocalized with submembranous actin-rich structures during the early stages of FcgammaR-mediated phagocytosis. In addition, SEPT2 accumulation is seen in primary human neutrophils and in nonprofessional phagocytes. The time course of septin accumulation mirrors actin accumulation and is inhibited by latrunculin and genistein, but not other inhibitors of phagocytosis. Inhibition of septin function by transient expression of the BD3 domain of BORG3, known to cause septin aggregation, or depletion of SEPT2 or SEPT11 by RNAi, significantly inhibited FcgammaR-mediated phagocytosis of IgG-coated latex beads. Interestingly, this occurred without affecting the accumulation of actin or the actin-associated protein coronin-1. These observations show that, although not necessary for actin recruitment, septins are required for efficient FcgammaR-mediated phagocytosis.     

The history of septin biology and bacterial infection

Investigation of cytoskeleton during bacterial infection has significantly contributed to both cell and infection biology. Bacterial pathogens Listeria monocytogenes and Shigella flexneri are widely recognised as paradigms for investigation of the cytoskeleton during bacterial entry, actin‐based motility, and cell‐autonomous immunity. At the turn of the century, septins were a poorly understood component of the cytoskeleton mostly studied in the context of yeast cell division and human cancer. In 2002, a screen performed in the laboratory of Pascale Cossart identified septin family member MSF (MLL septin‐like fusion, now called SEPT9) associated with L. monocytogenes entry into human epithelial cells. These findings inspired the investigation of septins during L. monocytogenes and S. flexneri infection at the Institut Pasteur, illuminating important roles for septins in host–microbe interactions. In this review, we revisit the history of septin biology and bacterial infection, and discuss how the comparative study of L. monocytogenes and S. flexneri has been instrumental to understand septin roles in cellular homeostasis and host defence.     

A role for septins in the interaction between the Listeria monocytogenes INVASION PROTEIN InlB and the Met receptor

Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InlB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InlB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InlB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InlB-Met interaction and the viscosity of membrane tethers at locations where the InlB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function.     

Analysis of septins across kingdoms reveals orthology and new motifs

Background  

Septins are cytoskeletal GTPase proteins first discovered in the fungus Saccharomyces cerevisiae where they organize the septum and link nuclear division with cell division. More recently septins have been found in animals where they are important in processes ranging from actin and microtubule organization to embryonic patterning and where defects in septins have been implicated in human disease. Previous studies suggested that many animal septins fell into independent evolutionary groups, confounding cross-kingdom comparison.     

Septin Form and Function at the Cell Cortex

Septins are GTP-binding proteins that form filaments and higher-order structures on the cell cortex of eukaryotic cells and associate with actin and microtubule cytoskeletal networks. When assembled, septins coordinate cell division and contribute to cell polarity maintenance and membrane remodeling. These functions manifest themselves via scaffolding of cytosolic proteins and cytoskeletal networks to specific locations on membranes and by forming diffusional barriers that restrict lateral diffusion of proteins embedded in membranes. Notably, many neurodegenerative diseases and cancers have been characterized as having misregulated septins, suggesting that their functions are relevant to diverse diseases. Despite the importance of septins, little is known about what features of the plasma membrane influence septin recruitment and alternatively, how septins influence plasma membrane properties. Septins have been localized to the cell cortex at the base of cilia, the mother-bud neck of yeast, and branch points of filamentous fungi and dendritic spines, in cleavage furrows, and in retracting membrane protrusions in mammalian cells. These sites all possess some degree of curvature and are likely composed of distinct lipid pools. Depending on the context, septins may act alone or in concert with other cytoskeletal elements to influence and sense membrane properties. The degree to which septins react to and/or induce changes in shape and lipid composition are discussed here. As septins are an essential player in basic biology and disease, understanding the interplay between septins and the plasma membrane is critical and may yield new and unexpected functions.     

Differential localization patterns of septins during growth of the human fungal pathogen Aspergillus fumigatus reveal novel functions

Septins, a conserved family of GTPases, are heteropolymeric filament-forming proteins that associate with the cell membrane and cytoskeleton and serve essential functions in cell division and morphogenesis. Their roles in fungal cell wall chitin deposition, septation, cytokinesis, and sporulation have been well established and they have recently been implicated in tissue invasion and virulence in Candida albicans. Septins have never been investigated in the human pathogenic fungus, Aspergillus fumigatus, which is a leading cause of death in immunocompromised patients. Here we localize all the five septins (AspA–E) from A. fumigatus for the first time, and show that each of the five septins exhibit varied patterns of distribution. Interestingly AspE, which is unique to filamentous fungi, and AspD, belonging to the CDC10 class of septins, localized prominently to tubular structures which were dependent on actin and microtubule networks. Localization of AspD and AspE has never been reported in filamentous fungi. Taken together these results suggest that septins in A. fumigatus might have unique functions in morphogenesis and pathogenicity.     

The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules

Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.     

Septins: traffic control at the cytokinesis intersection

The physical division of one cell into two requires the highly orchestrated separation of genetic and cytoplasmic contents during M phase of the cell cycle. Mitosis, the physical segregation of the genetic material of a cell into two daughter cells, has traditionally received more attention than cytokinesis, the partitioning of the cytoplasmic contents, yet clearly the two processes must be intimately co-ordinated and tightly regulated. While plant cells divide by the formation of a membranous cell barrier called the phragmoplast, animal cell division is largely driven by contraction of an actomyosin ring. However, recent evidence has suggested that membranes derived from one or more intracellular compartments are also required to break the cytoplasmic bridge connecting two dividing cells during late telophase. In this review, we focus on studies of animal cell cytokinesis that support a requirement for specific endomembrane fusion during fission, define molecular components of the membrane fusion apparatus that may be involved and point to possible roles for an emerging family of cytoskeletal proteins, the septins, in this process.     

Septins and a formin have distinct functions in anaphase chiral cortical rotation in the Caenorhabditis elegans zygote

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the Caenorhabditis elegans zygote, actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and cross-link the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior–posterior axis and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.     

Vhs2 is a novel regulator of septin dynamics in budding yeast

In budding yeast, septins are assembled into structures that undergo dramatic changes during the cell cycle. The molecular mechanisms that drive these remodelings are not fully uncovered. In this study, we describe a characterization of Vhs2, a nonessential protein that revealed to be a new player in septin dynamics. In particular, we report that Vhs2 is important to maintain the stability of the double septin ring structure until telophase. In addition, we show that Vhs2 undergoes multiple phosphorylations during the cell cycle, being phosphorylated during S phase until nuclear division and dephosphorylated just before cell division. Importantly we report that cyclin-dependent protein kinase Cdk1 and protein phosphatase Cdc14 control these Vhs2 post-translational modifications. These results reveal that Vhs2 is a novel Cdc14 substrate that is involved in the control of septin organization.