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.     

Mid2p stabilizes septin rings during cytokinesis in fission yeast

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.     

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.     

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.     

The septin family of GTPases: architecture and dynamics

Septins comprise a conserved family of proteins that are found primarily in fungi and animals. These GTP-binding proteins have several roles during cell division, cytoskeletal organization and membrane-remodelling events. One factor that is crucial for their functions is the ordered assembly of individual septins into oligomeric core complexes that, in turn, form higher-order structures such as filaments, rings and gauzes. The molecular details of these interactions and the mechanism by which septin-complex assembly is regulated have remained elusive. Recently, the first detailed structural views of the septin core have emerged, and these, along with studies of septin dynamics in vivo, have provided new insight into septin-complex assembly and septin function in vivo.     

Forchlorfenuron alters mammalian septin assembly, organization, and dynamics

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.     

Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7

Mammalian septins are GTP-binding proteins the functions of which are not well understood. Knockdown of SEPT2, 6, and 7 causes stress fibers to disintegrate and cells to lose polarity. We now show that this phenotype is induced by nuclear accumulation of the adaptor protein NCK, as the effects can be reversed or induced by cytoplasmic or nuclear NCK, respectively. NCK is carried into the nucleus by SOCS7 (suppressor of cytokine signaling 7), which possesses nuclear import/export signals. SOCS7 interacts with septins and NCK through distinct domains. DNA damage induces actin and septin rearrangement and rapid nuclear accumulation of NCK and SOCS7. Moreover, NCK expression is essential for cell-cycle arrest. The septin-SOCS7-NCK axis intersects with the canonical DNA damage cascade downstream of ATM/ATR and is essential for p53 Ser15 phosphorylation. These data illuminate an unanticipated connection between septins, SOCS7, NCK signaling, and the DNA damage response.     

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae

Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.     

p62 and NDP52 proteins target intracytosolic Shigella and Listeria to different autophagy pathways

Autophagy is an important mechanism of innate immune defense. We have recently shown that autophagy components are recruited with septins, a new and increasingly characterized cytoskeleton component, to intracytosolic Shigella that have started to polymerize actin. On the other hand, intracytosolic Listeria avoids autophagy recognition by expressing ActA, a bacterial effector required for actin polymerization. Here, we exploit Shigella and Listeria as intracytosolic tools to characterize different pathways of selective autophagy. We show that the ubiquitin-binding adaptor proteins p62 and NDP52 target Shigella to an autophagy pathway dependent upon septin and actin. In contrast, p62 or NDP52 targets the Listeria ActA mutant to an autophagy pathway independent of septin or actin. TNF-α, a host cytokine produced upon bacterial infection, stimulates p62-mediated autophagic activity and restricts the survival of Shigella and the Listeria ActA mutant. These data provide a new molecular framework to understand the emerging complexity of autophagy and its ability to achieve specific clearance of intracytosolic bacteria.     

Cell cycle-regulated attachment of the ubiquitin-related protein SUMO to the yeast septins

SUMO is a ubiquitin-related protein that functions as a posttranslational modification on other proteins. SUMO conjugation is essential for viability in Saccharomyces cerevisiae and is required for entry into mitosis. We have found that SUMO is attached to the septins Cdc3, Cdc11, and Shs1/Sep7 specifically during mitosis, with conjugates appearing shortly before anaphase onset and disappearing abruptly at cytokinesis. Septins are components of a belt of 10-nm filaments encircling the yeast bud neck. Intriguingly, only septins on the mother cell side of the bud neck are sumoylated. We have identified four major SUMO attachment-site lysine residues in Cdc3, one in Cdc11, and two in Shs1, all within the consensus sequence (IVL)KX(ED). Mutating these sites eliminated the vast majority of bud neck-associated SUMO, as well as the bulk of total SUMO conjugates in G(2)/M-arrested cells, indicating that sumoylated septins are the most abundant SUMO conjugates at this point in the cell cycle. This mutant has a striking defect in disassembly of septin rings, resulting in accumulation of septin rings marking previous division sites. Thus, SUMO conjugation plays a role in regulating septin ring dynamics during the cell cycle.     

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Dynamics of septin ring and collar formation in Saccharomyces cerevisiae

Although the septin ring and collar in budding yeast were described over 20 years ago, there is still controversy regarding the organization of septin filaments within these structures and about the way in which the ring first forms and about how it converts into a collar at the mother-bud neck. Here we present quantitative analyses of the recruitment of fluorescently-tagged septins to the ring and collar through the cell cycle. Septin ring assembly began several minutes after polarity establishment and this interval was longer in daughter than in mother cells, suggesting asymmetric inheritance of septin regulators. Septins formed an initial faint and irregular ring, which became more regular as septins were recruited at a constant rate. This steady rate of septin recruitment continued for several minutes after the ring converted to a collar at bud emergence. We did not detect a stepwise change in septin fluorescence during the ring-to-collar transition. After collar formation, septins continued to accumulate at the bud neck, though at a reduced rate, until the onset of cytokinesis when the amount of neck-localized septins rapidly decreased. Implications for the mechanism of septin ring assembly are discussed.     

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.     

Assembly of mammalian septins

Septins are a conserved family of polymerizing guanine nucleotide binding proteins associated with diverse processes in dividing and non-dividing cells. In humans, 12 septin genes generate dozens of polypeptides, many of which comprise heterooligomeric complexes. Native and recombinant mammalian septin complexes are purified as approximately 8-nm-thick filaments of variable length. Ultrastructurally, a mammalian septin filament appears an irregular array of structural segments, whose polarity is obscure. The filaments have a potential to self-assemble into higher-order structures by lateral stacking and tandem annealing, eventually forming uniformly curved bundles, i.e., rings and coils. The septin filaments also undergo templated assembly along existing actin bundles containing an adapter protein, anillin. The resultant higher-order assembly of septin filaments may provide scaffolds to recruit other molecules and/or help organize the actin-based structures. The in vitro self-assembly is an irreversible process, which is not coupled with robust nucleotide exchange or hydrolysis. In contrast, septin-based structures rearrange and disassemble in cells, which might be controlled by diverse factors (e.g., the Cdc42-borg system, anillin, syntaxin, phospholipids) and covalent modifications (e.g., phosphorylation, ubiquitination, sumoylation). An immediate goal of septin biochemistry is to define the mechanisms of assembly and disassembly of this elusive cytoskeleton.     

The Anillin-Related Region of Bud4 Is the Major Functional Determinant for Bud4's Function in Septin Organization during Bud Growth and Axial Bud Site Selection in Budding Yeast

The anillin-related protein Bud4 of Saccharomyces cerevisiae is required for axial bud site selection by linking the axial landmark to the septins, which localize at the mother bud neck. Recent studies indicate that Bud4 plays a role in septin organization during cytokinesis. Here we show that Bud4 is also involved in septin organization during bud growth prior to cytokinesis, as bud4Δ shs1Δ cells displayed an elongated bud morphology and defective septin organization at 18°C. Bud4 overexpression also affected septin organization during bud growth in shs1Δ cells at 30°C. Bud4 was previously thought to associate with the septins via its central region, while the C-terminal anillin-related region was not involved in septin association. Surprisingly, we found that the central region of Bud4 alone targets to the bud neck throughout the cell cycle, unlike full-length Bud4, which localizes to the bud neck only during G₂/M phase. We identified the anillin-related region to be a second targeting domain that cooperates with the central region for proper septin association. In addition, the anillin-related region could largely mediate Bud4''s function in septin organization during bud growth and bud site selection. We show that this region interacts with the C terminus of Bud3 and the two segments depend on each other for association with the septins. Moreover, like the bud4Δ mutant, the bud3Δ mutant genetically interacts with shs1Δ and cdc12-6 mutants in septin organization, suggesting that Bud4 and Bud3 may cooperate in septin organization during bud growth. These observations provide new insights into the interaction of Bud4 with the septins and Bud3.     

The septin cortex at the yeast mother–bud neck

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.     

One ring to bind them. Septins and actin assembly

Septins are GTPases required for cytokinesis and other processes requiring spatial organization of the cell cortex, but their molecular functions in these processes are unknown. In this issue of Developmental Cell, Kinoshita et al. take an important step in elucidating the molecular functions of septins by developing an in vitro assay for septin assembly and exploring the relationship between mammalian septins and actin.     

The septin cortex at the yeast mother-bud neck.

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.