Membrane binding controls ordered self-assembly of animal septins.

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

The hierarchical assembly of septins revealed by high-speed AFM.

Septins are GTP-binding proteins involved in diverse cellular processes including division and membrane remodeling. Septins form linear, palindromic heteromeric complexes that can assemble in filaments and higher-order structures. Structural studies revealed various septin architectures, but questions concerning assembly-dynamics and -pathways persist. Here we used high-speed atomic force microscopy (HS-AFM) and kinetic modeling which allowed us to determine that septin filament assembly was a diffusion-driven process, while formation of higher-order structures was complex and involved self-templating. Slightly acidic pH and increased monovalent ion concentrations favor filament-assembly, -alignment and -pairing. Filament-alignment and -pairing further favored diffusion-driven assembly. Pairing is mediated by the septin N-termini face, and may occur symmetrically or staggered, likely important for the formation of higher-order structures of different shapes. Multilayered structures are templated by the morphology of the underlying layers. The septin C-termini face, namely the C-terminal extension of Cdc12, may be involved in membrane binding.

Dynamic assembly of a higher-order septin structure during appressorium morphogenesis by the rice blast fungus.

The rice blast fungus Magnaporthe oryzae differentiates a specialized infection structure called an appressorium, which is used to break into plant cells by directed application of enormous turgor force. Appressorium-mediated plant infection requires timely assembly of a higher-order septin ring structure at the base of the appressorium, which is needed to spatially orchestrate appressorium repolarization. Here we use quantitative 4D widefield fluorescence imaging to gain new insight into the spatiotemporal dynamics of septin ring formation, and septin-mediated actin re-organization, during appressorium morphogenesis by M. oryzae. We anticipate that the new knowledge will provide a quantitative framework for dissecting the molecular mechanisms of higher-order septin ring assembly in this devastating plant pathogenic fungus.

A revised order of subunits in mammalian septin complexes.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP-SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2-6-7 is therefore established to be SEPT2-SEPT6-SEPT7-SEPT7-SEPT6-SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Membrane reshaping by micrometric curvature sensitive septin filaments.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.

An amphipathic helix enables septins to sense micrometer-scale membrane curvature.

Cell shape is well described by membrane curvature. Septins are filament-forming, GTP-binding proteins that assemble on positive, micrometer-scale curvatures. Here, we examine the molecular basis of curvature sensing by septins. We show that differences in affinity and the number of binding sites drive curvature-specific adsorption of septins. Moreover, we find septin assembly onto curved membranes is cooperative and show that geometry influences higher-order arrangement of septin filaments. Although septins must form polymers to stay associated with membranes, septin filaments do not have to span micrometers in length to sense curvature, as we find that single-septin complexes have curvature-dependent association rates. We trace this ability to an amphipathic helix (AH) located on the C-terminus of Cdc12. The AH domain is necessary and sufficient for curvature sensing both in vitro and in vivo. These data show that curvature sensing by septins operates at much smaller length scales than the micrometer curvatures being detected.

In vitro reconstitution of septin assemblies on supported lipid bilayers.

Septins are polymerizing eukaryotic proteins that play conserved roles in cell cortex organization and are essential in many cell types. How septin dynamics and protein-protein interactions determine their function at the plasma membrane remains a mystery. Here, we present a method for recapitulating septin polymerization and lipid interaction utilizing supported lipid bilayers to mimic the eukaryotic plasma membrane. Septins on supported lipid bilayers can be visualized with single-molecule sensitivity using total internal reflective fluorescence microscopy. Microscopy-based in vitro assays have revolutionized our understanding of actin, microtubules, and bacterial cytoskeletal systems, and will likely immediately advance our understanding of the septin proteins. As such, we hope that this technique will be adopted and widely utilized by those interested in uncovering septin properties and functions of septin interacting proteins.

Visualization of in vivo septin ultrastructures by platinum replica electron microscopy.

Septins are cytoskeletal proteins involved in diverse biological processes including cytokinesis, cell morphogenesis, motility, and ciliogenesis. Septins form various filamentous structures in vitro and in vivo, but the higher-order architecture of septin structures in vivo remains poorly defined. The best understood system in this respect is the budding yeast Saccharomyces cerevisiae, where septins form a ring structure that undergoes multiple stages of remodeling during the cell cycle. In this chapter, we describe a method for visualizing supramolecular septin structures in yeast at high spatial resolution using platinum replica electron microscopy. This approach can be applied to further understand the regulation of assembly and remodeling of septin higher-order structures, as well as the relationship between septin architecture and function.

Live cell imaging of septin dynamics in Ustilago maydis.

Septins are highly conserved cytoskeletal proteins involved in a variety of biological processes such as cell polarization and cytokinesis. In humans, functional defects in these proteins have been linked to cancer and neuronal diseases. In recent years, substantial progress has been made in studying the structure of septin subunits and the formation of defined heteromeric building blocks. These are assembled into higher-order structures at distinct subcellular sites. An important microscopic approach in studying septin assembly and dynamics is the use of septins tagged with fluorescent proteins. This revealed, eg, that septins form rings during cytokinesis and that septins build extended filaments partially colocalizing with actin cables and microtubules. Here, we describe extensive live cell imaging of septins in the model microorganism Ustilago maydis. We present techniques to study dynamic localization of protein and septin mRNA on shuttling endosomes as well as colocalization of proteins at these highly motile units. Moreover, FLIM-FRET experiments for analyzing local protein interactions are presented. Importantly, these imaging approaches transfer well to other fungal and animal model systems for in vivo analysis of septin dynamics.

Ashbya gossypii as a model system to study septin organization by single-molecule localization microscopy.

Heteromeric complexes of GTP-binding proteins from the septin family assemble into higher order structures that are essential for cell division in many organisms. The correct organization of the subunits into filaments, gauzes, and rings is the basis of septin function in this process. Electron microscopy and polarization fluorescence microscopy contributed greatly to the understanding of the dynamics and organization of such structures. However, both methods show technical limitations in resolution and specificity that do not allow the identification of individual septin complexes in assemblies in intact cells. Single-molecule localization-based fluorescence superresolution microscopy methods combine the resolution of cellular structures at the nanometer level with highest molecular specificity and excellent contrast. Here, we provide a protocol that enables the investigation of the organization of septin complexes in higher order structures in cells by combining advantageous features of the model organism Ashbya gossypii with single-molecule localization microscopy. Our assay is designed to investigate the general assembly mechanism of septin complexes in cells and is applicable to many cell types.

Visualizing septins in early Drosophila embryos.

Functional studies in Drosophila have been key for establishing a role for the septin family of proteins in animal cell division and thus extending for the first time observations from the budding yeast to animal cells. Visualizing the distribution of specific septins in different Drosophila tissues and, in particular, in the Drosophila embryo, together with biochemical and mutant phenotype data, has contributed important advances to our understanding of animal septin biology, suggesting roles in processes other than in cytokinesis. Septin localization using immunofluorescence assays has been possible due to the generation of antibodies against different Drosophila septins. The recent availability of lines expressing fluorescent protein fusions of specific septins further promises to facilitate studies on septin dynamics. Here, we provide protocols for preparing early Drosophila embryos to visualize septins using immunofluorescence assays and live fluorescence microscopy. The genetic tractability of the Drosophila embryo together with its amenability to high-resolution fluorescence microscopy promises to provide novel insights into animal septin structure and function.

Fluorescence microscopy of actin- and microtubule-associated septins in mammalian cells.

Septins are a major component of the mammalian cytoskeleton. Septins associate with filamentous actin (F-actin) and microtubules, but the nature and significance of these interactions are not well understood. Fluorescence microscopy of F-actin- and microtubule-associated septins in fixed and living cells has been instrumental in uncovering septin functions in cellular morphogenesis and cytoskeleton-dependent processes (eg, cell division, cell migration). Here, we provide a detailed methodology for the visualization of endogenous septins by immunofluorescence microscopy, discussing sample preparation and reagents that are critical for optimal staining. In addition, we review approaches for the construction and expression of fluorescent septins and their time-lapse imaging with F-actin and microtubules. The recommended methodology is adaptable for high- and superresolution imaging of mammalian cells with various instrumentation, including wide-field and confocal microscopy as well as total internal reflection fluorescence and structured illumination microscopy.

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.

Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae.

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

Self assembly of human septin 2 into amyloid filaments.

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in ß-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.

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.

Septin structure and function in yeast and beyond.

Septins are conserved GTP-binding proteins that assemble into hetero-oligomeric complexes and higher-order structures such as filaments, rings, hourglasses or gauzes. Septins are usually associated with a discrete region of the plasma membrane and function as a cell scaffold or diffusion barrier to effect cytokinesis, cell polarity, and many other functions. Recent structural studies of septin complexes have provided mechanistic insights into septin filament assembly, but key questions concerning the assembly, dynamics, and function of different septin structures remain to be answered.

Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization.

Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order structures. In budding yeast (Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin-membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation in vitro via atypical Cdc12-Cdc12 and Cdc3-Cdc3 interactions, respectively.