Septin Filaments Research Articles

Targeted inactivation of the Septin2 and Septin9 genes in myelinating Schwann cells of mice.

The formation of axon-enwrapping myelin sheaths by oligodendrocytes in the central nervous system involves the assembly of a scaffolding septin filament comprised of the subunits SEPTIN2, SEPTIN4, SEPTIN7 and SEPTIN8. Conversely, in the peripheral nervous system (PNS), myelin is synthesized by a different cell type termed Schwann cells, and it remained unknown if septins also assemble as a multimer in PNS myelin. According to prior proteome analysis, PNS myelin comprises the subunits SEPTIN2, SEPTIN7, SEPTIN8, SEPTIN9, and SEPTIN11, which localize to the paranodal and abaxonal myelin subcompartments. Here, we use the Cre/loxP-system to delete the Septin9-gene specifically in Schwann cells, causing a markedly reduced abundance of SEPTIN9 in sciatic nerves, implying that Schwann cells are the main cell type expressing SEPTIN9 in the nerve. However, Septin9-deficiency in Schwann cells did not affect the abundance or localization of other septin subunits. In contrast, when deleting the Septin2-gene in Schwann cells the abundance of all relevant septin subunits was markedly reduced, including SEPTIN9. Notably, we did not find evidence that deleting Septin2 or Septin9 in Schwann cells impairs myelin biogenesis, nerve conduction velocity or motor/sensory capabilities, at least at the assessed timepoints. Our data thus show that SEPTIN2 but not SEPTIN9 is required for the formation or stabilization of a septin multimer in PNS myelin in vivo; however, its functional relevance remains to be established.

Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity.

Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell-cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell-cell junctions, which are composed of VE-cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell-cell contact, in curved- and scallop-shaped patterns. These membrane-associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin-rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site-specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell-cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F-actin at the cortical membrane, along with increased amounts of F-actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin-based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

Septin7 is indispensable for proper skeletal muscle architecture and function.

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.

Septin7 is indispensable for proper skeletal muscle architecture and function

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.

Preprint Highlight: Building the cytokinetic contractile ring in an early embryo: initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

Cytokinesis is driven by a contractile ring whose key components are well defined, yet how they are organized and how the contractile ring assembles and constricts remain unclear. This study combines confocal and superresolution imaging of endogenous contractile ring components in sea urchin embryos (in vivo) and isolated embryo cortices (ex vivo) to demonstrate that anillin and septin2 assemble into myosin II-positive clusters. The author surmises that these clusters are precursors to the contractile ring and observe septin filament nanostructure for the first time in an animal cell contractile ring. These insights into the organization of lesser studied yet conserved contractile ring proteins, anillin and septins, provide clues to their function and pave the way for similar studies in other animal species.

Septin filament compaction into rings requires the anillin Mid2 and contractile ring constriction.

Septin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. How septin filaments organize in such structures is still not understood. Here, we used fission yeast to explore septin filament organization during cell division and its cell cycle regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found that the anillin-like protein Mid2 is necessary to promote this compaction step, possibly acting as a bundler for septin filaments. Moreover, Mid2-driven septin compaction requires inputs from the septation initiation network as well as CR constriction and the β(1,3)-glucan synthase Bgs1. This work highlights that anillin-mediated septin ring assembly is under strict cell cycle control.

The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin.

Septins are a family of filament-forming GTP-binding proteins that regulate fundamental cellular activities, such as cytokinesis and cell polarity. In general, septin filaments function as barriers and scaffolds on the cell cortex. However, little is known about the mechanism that governs the recruitment and localization of the septin complex to the cell cortex. Here, we identified the Cdc42 GTPase-activating protein Rga6 as a key protein involved in promoting the localization of the septin complex to the cell cortex in the fission yeast Schizosaccharomyces pombe. Rga6 interacts with the septin complex and partially colocalizes with the septin complex on the cell cortex. Live-cell microscopy analysis further showed septin enrichment at the cortical regions adjacent to the growing cell tip. The septin enrichment likely plays a crucial role in confining active Cdc42 to the growing cell tip. Hence, our findings support a model whereby Rga6 regulates polarized cell growth partly through promoting targeted localization of the septin complex on the cell cortex. This article has an associated First Person interview with the first author of the paper.

Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network.

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.

An atomic model for the human septin hexamer by cryo-EM

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Protein Kinase A-Mediated Septin7 Phosphorylation Disrupts Septin Filaments and Ciliogenesis.

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

The Role of Septin7 in Skeletal Muscle Regeneration

Septins have recently been acknowledged as the fourth component of the cytoskeleton. In humans 13 isoforms have been identified and classified into 4 homology groups. While their role was extensively studied in neurons and cancer cells, their role in skeletal muscle remains unexplored. Here the Tibialis anterior muscle of mice was injected with BaCl2 to induce a mild damage and regeneration was followed for two weeks. The expression of Septin7 was upregulated on the fourth day following injection (by 60±28%) as compared to the contralateral leg; p<0.04) and its level correlated with the expression of Pax7 (r2=0.5273; p<0.01) the marker of satellite cell activation and the appearance of central nuclei of regenerating fibers. By the end of the second week the expression of both Septin7 and Pax7 decreased, in parallel, close to control levels. The migration of control and Septin7 knock-down (KD) C2C12 myoblasts was followed in a migration assay. Although the total distance covered by the cells during the migration period (3h) was identical (256±9 µm in control (n=61), while 269±7 µm in KD (n=60)), Septin7 KD cells tended not to change the direction of migration which was reflected in the larger distance between the initial and final positions (91±11, and 165±9 µm for control and KD cells, respectively; p<0.05). In parallel, KD cells displayed an altered cell shape with less protrusions. Septin filament reorganisation during migration was followed live by expressing a fluorescent Septin7 fusion protein. These findings indicate that Septin7 plays an essential role in the ability of myoblasts to migrate to the site of muscle damage and thus in the regeneration process of skeletal muscle. Supported by GINOP-2.3.2-15-2016-00044, Thematic Excellence Programme of the Ministry for Innovation and Technology in Hungary (ED_18-1-2019-0028).

The Interplay between the Self-Organization and Curvature Sensing in Septin Assemblies

The ability of cells to sense and respond to their shape is key to many cellular functions. Septins are GTP-binding nanometer-scale proteins that sense and localize to sites of positive micron-scale membrane curvature in Eukaryotic cells from yeast to humans. The underlying physical principles that control this process remain poorly understood. Septin assembly begins with the binding of short nanometer-sized rod complexes, referred as septin octamers, to the membrane. Upon binding, these octamers can diffuse and anneal (polymerize) to form larger filaments and ultimately self-organize into assemblies that can span several microns. However, it is unclear how each of these events, and hence septin self-organization as a whole, are influenced by the membrane's curvature. Here we examine the biophysical basis of curvature sensing by septins at each of these steps, using a combination of single molecule imaging, scanning electron microscopy, biophysical modeling and simulations. We find that the membrane curvature influences the kinetics of binding/unbinding of septin complexes, their diffusion on the membrane as well as their length distribution and self-organization. These data suggest that curvature sensing by septins operates at multiple length scales, rather than simply at the scale of a single protein/membrane and it provide a framework for understanding the interplay between curvature sensing and self-organization of septin filaments.

Septin Filament Compaction Into Rings Requires the Anillin Mid2 and Contractile Ring Constriction

Septin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. However, how septin filaments organize in such structures is still not well understood. In this study, we used fission yeast to explore septin filament organization during cell division and decipher key factors responsible for their regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found evidence that the anillin-like protein Mid2 is necessary to promote this novel compaction step, possibly acting as a bundler for septin filaments. We also found that Mid2-driven septin compaction requires inputs from the Septation Initiation Network (SIN) as well as CR constriction or the β-1-3 glucan synthase Bgs1. This work highlights the complex regulations that allow the coordination between septin ring assembly and cell cycle progression.

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.

Animal Cell Cytokinesis: The Rho-Dependent Actomyosin-Anilloseptin Contractile Ring as a Membrane Microdomain Gathering, Compressing, and Sorting Machine.

Cytokinesis is the last step of cell division that partitions the cellular organelles and cytoplasm of one cell into two. In animal cells, cytokinesis requires Rho-GTPase-dependent assembly of F-actin and myosin II (actomyosin) to form an equatorial contractile ring (CR) that bisects the cell. Despite 50 years of research, the precise mechanisms of CR assembly, tension generation and closure remain elusive. This hypothesis article considers a holistic view of the CR that, in addition to actomyosin, includes another Rho-dependent cytoskeletal sub-network containing the scaffold protein, Anillin, and septin filaments (collectively termed anillo-septin). We synthesize evidence from our prior work in Drosophila S2 cells that actomyosin and anillo-septin form separable networks that are independently anchored to the plasma membrane. This latter realization leads to a simple conceptual model in which CR assembly and closure depend upon the micro-management of the membrane microdomains to which actomyosin and anillo-septin sub-networks are attached. During CR assembly, actomyosin contractility gathers and compresses its underlying membrane microdomain attachment sites. These microdomains resist this compression, which builds tension. During CR closure, membrane microdomains are transferred from the actomyosin sub-network to the anillo-septin sub-network, with which they flow out of the CR as it advances. This relative outflow of membrane microdomains regulates tension, reduces the circumference of the CR and promotes actomyosin disassembly all at the same time. According to this hypothesis, the metazoan CR can be viewed as a membrane microdomain gathering, compressing and sorting machine that intrinsically buffers its own tension through coordination of actomyosin contractility and anillo-septin-membrane relative outflow, all controlled by Rho. Central to this model is the abandonment of the dogmatic view that the plasma membrane is always readily deformable by the underlying cytoskeleton. Rather, the membrane resists compression to build tension. The notion that the CR might generate tension through resistance to compression of its own membrane microdomain attachment sites, can account for numerous otherwise puzzling observations and warrants further investigation using multiple systems and methods.

Molecular Recognition at Septin Interfaces: The Switches Hold the Key

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles, which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate, which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.

Septin 7 is a centrosomal protein that ensures S phase entry and microtubule nucleation by maintaining the abundance of p150glued

Septins play important roles in regulating development and differentiation. Septin 7 (SEPT7) is a crucial component in orchestrating the septin core complex into highly ordered filamentous structures. Here, we showed that genetic depletion of SEPT7 or treatment with forchlorfenuron (FCF; a compound known to affect septin filament assembly) led to reduced the S phase entry in cell models and zebrafish embryos. In addition to colocalizing with actin filaments, SEPT7 resided in the centrosome, and SEPT7 depletion led to aberrant mitotic spindle pole formation. This mitotic defect was rescued in SEPT7-deficient cells by wild-type SEPT7, suggesting that SEPT7 maintained mitotic spindle poles. In addition, we observed disorganized microtubule nucleation and reduced cell migration with SEPT7 depletion. Furthermore, SEPT7 formed a complex with and maintained the abundance of p150glued , the component of centriole subdistal appendages. Depletion of p150glued resulted in a phenotype reminiscent of SEPT7-deficient cells, and overexpression of p150glued reversed the defective phenotypes. Thus, SEPT7 is a centrosomal protein that maintains proper cell proliferation and microtubule array formation via maintaining the abundance of p150glued .

The septin cytoskeleton regulates natural killer cell lytic granule release.

Natural killer (NK) cell-mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell-mediated killing.

Cytokinesis: An Anillin-RhoGEF Module Sets the Stage for Septin Double Ring Assembly.

In many eukaryotes septin filaments form an hourglass-like structure at the division site that rearranges into a double ring at cytokinesis. A new study elucidates how an anillin-RhoGEF complex guides the assembly of the septin double ring in budding yeast.