During cytokinesis, budding yeast assemble a double septin ring composed of circumferential filaments that compartmentalize the membrane at the bud neck. This structure’s integrity depends on two proteins, Bud3 and Bud4. Using in vitro reconstitution assays, we demonstrate that Bud3 and Bud4 organize septin filaments in distinct ways and cooperate to assemble complex, higher order networks. They bind to different septins and require septins to associate with each other, suggesting that they modulate septin architecture independently but synergistically. Furthermore, both proteins possess lipid-binding domains that interact directly with membranes and are involved in their recruitment to the bud neck and in septin organization. In vivo experiments using bud3 bud4 double mutants reveal that the double ring is essential for concentrating at the division site proteins that mark cytokinetic remnants, while other proteins landing at the bud neck at cytokinesis are unaffected. We propose that the septin double ring serves as a selective spatial memory, preserving a molecular track of cytokinesis across subsequent cell divisions.

Abstract Budding yeast septins assemble into filamentous networks bound to the inner plasma membrane. In situ or in vitro , septins are implicated in membrane deformations. We therefore suspected that septins might alter membrane mechanical properties both directly or indirectly. To decipher whether septins directly tune the rigidity of membranes, we used a cell free in vitro approach. To this end, using AFM, we measured the mechanical response of reconstituted GUVs pre-incubated with septins. Unexpectedly, we find that large GUVs (typically tens of µm diameter size) are more deformable in the presence of septins. Theoretical modeling suggests that this peculiar behavior is likely due to initial micrometer membrane “wrinkled” deformations imposed by septins. Conversely, small GUVs (1 to 2 microns in diameter) cannot undergo any micrometric deformations and are thereby less deformable with septin filaments bound. Our findings suggest that, in specific cellular context, septins could provide a membrane reservoir and eventually facilitate membrane deformations. Significance statement Filamentous cytoskeletal septins, interacting with membranes would be expected to enhance membrane rigidity. Upon mechanical stress, GUVs larger than tens of microns appear, more deformable in the presence of septins. Septins’ initial membrane reshaping is responsible for this unexpected behavior, as shown by theoretical modeling. However smaller non deformable vesicles are more rigid, with septins bound.

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. Significance:We provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

Septins are cytoskeletal filaments bound to the inner leaflet of the plasma membrane, essential for cell division. They are involved in the formation of diffusion barriers for membrane-bound components. Whether septins directly function as barriers or as part of a broader regulatory cascade of events remains unclear. We address this using in vitro reconstituted assay of biomimetic synthetic membranes. We probe whether the diffusion of biomimetic fluorescent objects with tunable steric hindrance – mimicking membrane bound proteins – is constrained by septins. Our results indicate that (i) lipids and transmembrane proteins lacking cytosolic domains diffuse freely in the presence of septins (ii) membrane-bound objects with large cytosolic domains experience size-dependent diffusion constraints; and (iii) the ability of septins to act as diffusion barriers is finely tuned by their intrinsic filament organization. These findings demonstrate that septins can directly impose size-selective diffusion barriers and that their filament organization critically tunes this function. In vitro reconstitution assays show that cytoskeletal septins act as diffusion barriers, at the membrane. Septins restrict the diffusion of large membrane bound items and their efficiency depends on their filamentous ultrastructural organization

At the time of cytokinesis, a double septin ring is assembled at the division site in many eukaryotic cells. In budding yeast the double ring is made by two arrays of circumferential septin filaments at the two sides of the bud neck, which are thought to compartmentalize the membrane around the cleavage site. Integrity of the double septin ring requires the anillin Bud4 and its presumed partner Bud3, which associate with septins in mitosis and have separate, yet unknown, roles in stabilizing septin circumferential filaments. Through in vitro reconstitution assays using purified proteins, we show that Bud3 and Bud4 organise septin filaments in distinct ways, while together they cooperate to assemble higher-order septin networks. In agreement with their separate roles in septin organization, Bud3 and Bud4 bind to different septins and require septins to associate with each other, indicating that they modulate septin architecture independently but synergistically. We also provide evidence that Bud3 and Bud4 bind membranes in vivo and in vitro , consistent with the presence of lipid-binding domains in their primary sequence. Using bud3 bud4 double mutants that lack the septin double ring, we show that in vivo proteins marking cytokinetic remnants require the double ring to efficiently concentrate at the division site, while other proteins landing at the bud neck at cytokinesis are unaffected. Thus, we propose that a septin double ring may imprint a selective spatial memory for cytokinesis that is transmitted throughout subsequent cell divisions. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, SEPTORG ANR-18-CE13-0015-01, CONSTRICT ANR-24-CE13-2422 Labex EpiGenMed

The phosphoinositidyl lipid (PIP) family is key to spatial regulation of cellular processes, and their appearance, disappearance, and interconversion provide key signals for membrane remodeling at the plasma membrane and beyond. These signals are connected to recruitment of various peripheral membrane proteins, including sorting nexin 9 (SNX9), an important regulator of late stage endocytic and macropinocytic remodeling. Using a combination of live cell imaging, in vitro reconstitution, and atomistic molecular dynamics simulation, we probe the question of whether SNX9 is selective for PI(3,4)P2 over PI(4,5)P2, and what the physiological implications of this selectivity may be. We find that SNX9 recruitment to the plasma membrane is coincident with PI(3,4)P2 but not PI(4,5)P2 during macropinocytic ruffle maturation, and that SNX9 differentially remodels GUVs containing PI(3,4)P2 over PI(4,5)P2. Atomistic simulations further corroborate these findings and suggest that the primary mediator of this difference is a non-canonical binding interface in the fourth helix of the PX domain, which is further probed with mutational assays. We therefore hypothesize that SNX9 binding to PI(3,4)P2 over PI(4,5)P2 not only helps with modulating the spatiotemporal recruitment of SNX9 during plasma membrane remodeling processes, but also acts as a check to prevent the premature conversion of PI(3,4)P2 into PI(3)P, thus acting as a regulator of these processes. ### Competing Interest Statement The authors have declared no competing interest.

Septins are essential cytoskeletal proteins involved in key cellular processes and have also been implicated in diseases from cancers to neurodegenerative pathologies. However, they have not been as thoroughly studied as other cytoskeletal proteins. In vivo, septins interact with other cytoskeletal proteins and with the inner plasma membrane. Hence, bottom-up in vitro cell-free assays are well suited to dissect the roles and behavior of septins in a controlled environment. Specifically, in vitro studies have been invaluable in describing the self-assembly of septins into a large diversity of ultrastructures. Given that septins interact specifically with membrane, the details of these septin-membrane interactions have been analyzed using reconstituted lipid systems. In particular, at a membrane, septins are often localized at curvatures of micrometer scale. In that context, in vitro assays have been performed with substrates of varying curvatures (spheres, cylinders or undulated substrates) to probe the sensitivity of septins to membrane curvature. This Review will first present the structural properties of septins in solution and describe the interplay of septins with cytoskeletal partners. We will then discuss how septins interact with biomimetic membranes and induce their reshaping. Finally, we will highlight the curvature sensitivity of septins and how they alter the mechanical properties of membranes.

In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.

The fine regulation of actin polymerization is essential to control cell motility and architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Different models have been extrapolated to describe the mole-cular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using negative stain electron microscopy, we directly identified for the first time two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the speculated open and closed conformations of the "stair-stepping" model. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end while they interact with three actin subunits in the closed conformation. In addi-tion, we identified and characterized the structure of single FH2 dimers encircling the core of actin filaments, and reveal their ability to spontaneously depart from barbed ends.

ABSTRACT Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.

This Special Issue of Cytoskeleton was inspired by the 2021 EMBO Workshop entitled ‘Molecular and Cell Biology of Septins’ held in Berlin, Germany (https://meetings.embo.org/event/20-septins). As described in a meeting report (Baillet et al., 2021), the 2021 EMBO Workshop was transformative, enabling septin enthusiasts from around the globe to exchange ideas during a highly isolating period because of the COVID-19 pandemic. Septins, an evolutionarily conserved protein family essential for cell division, have been implicated in numerous cellular processes including cellular organisation, intracellular transport and morphogenesis. Although septins are a relatively poorly understood components of the cytoskeleton (as compared with actin or microtubules), the field of septin biology is blooming as much as ever. As our knowledge of septin structure and assembly improves, and more innovative technologies become available, new domains of study promise to illuminate septin roles in fundamental cellular processes and pathogenesis. This Special Issue of Cytoskeleton is a collection of 12 primary research articles and reviews serving to highlight the latest insights on the molecular and cell biology of septins. Septins were discovered by Leland Hartwell screening for cell division cycle (cdc) mutants in the budding yeast Saccharomyces cerevisiae. Septin genes have since been found in fungi and higher eukaryotes, yet their number ranges from 2 in Caenorhabditis elegans to 5 in Drosophila melanogaster to 13 in humans. Septins form heterooligomeric complexes, which assemble to form non-polar filaments and higher order structures including rings, collars and gauzes. In this Special Issue, Grupp et al. (2023) describe an in silico approach to determine septin subunit affinities based on their conserved guanine nucleotide-binding (G) domain, proposing a novel mechanism that helps to stabilise hetero-oligomeric complexes and filaments. de Freitas Fernandes et al. (2023) report the crystal structure of the heterodimer formed between G-domains of D. melanogaster septins Sep1 and Sep2. In sum, these two studies significantly enhance our understanding of molecular interactions involved in septin assembly and disassembly. The role of post-translational modifications (including ubiquitylation, SUMOylation, phosphorylation and acetylation) in septin biology has been of great interest for many years. In a review, Sharma and Menon (2023) discuss post-translational modifications of human septins and how this may impact their function. The BORG/Cdc42EP family, composed of five Rho GTPase binding proteins, is of historical interest to the field because of their fundamental interplay with the septin cytoskeleton. Here, Tomasso and Padrick (2023) review the role of BORG family proteins in cellular and disease physiology. Septins are widely recognised as components of the membrane cortex. Despite recent insights, the mechanisms underlying septin interactions with membranes are not fully understood. Benson and McMurray (2023) work with S. cerevisiae septins Cdc3 and Cdc10 to suggest what may restrict septin localisation to specific membranes. Perry et al. (2023) study C. elegans septins UNC-59 and UNC-61, implicate septins in the architecture of the germline syncytium (where many germs cells share a common cytoplasm), and propose different roles for the two different septins. Kim et al. (2023) discuss the role of septins and actin at endothelial cell–cell junctions and how they impact monolayer integrity. Taken together, we hope these three articles will inspire future studies concerning septin–membrane interactions in a wide variety of model systems. In the case of rice blast fungus Magnaporthae orzyae, septins are crucial for appressorium morphogenesis and plant invasion. Eisermann et al. (2023) review the role of septins in fungal pathogenesis and discuss septins as important targets for anti-fungal plant protection strategies. Septin–bacteria interactions have been the subject of intense investigation for more than 15 years. Using Shigella flexneri infection to stimulate septin-mediated immunity in human epithelial cells, Van Ngo et al. (2023) discover a new role for septins in caspase activity and apoptotic cell death. Next, Torraca et al. (2023) generate zebrafish (Danio rerio) null mutants of Sept6 and Sept15, showing that they are developmentally viable but more susceptible to S. flexneri infection than wild-type zebrafish. In the future, it will be important to study S. flexneri infection in vivo using these zebrafish models, and to discover new roles for septins in host defence from the single cell to whole animal level. Septins are enriched in the mammalian nervous system where they play key roles in neuronal development and function. Werner and Yadav (2023) provide an overview of kinases that interact with and phosphorylate mammalian septins, and how phosphoregulation may impact septin function in neuronal development and disease. In the peripheral nervous system, myelin is synthesised by Schwann cells. Using state-of-the-art mouse models, Martens et al. (2023) perform targeted inactivation of Sept2 and Sept9 in myelinating Schwann cells, discovering that SEPT2 (but not SEPT9) controls septin hetero-oligomeric complex formation and localisation in peripheral nervous system myelin in vivo. In conclusion, we thank all of our colleagues for their innovative and exciting contributions to this Special Issue of Cytoskeleton. We next look forward to the next gathering of our dynamic field. Received: 9 May 2023 Accepted: 10 May 2023

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane, we have used a combination of methods to assay their ultrastructural organization, their curvature sensitivity as well as their role in membrane reshaping. In contrast to budding yeast septins, on membranes, human septins systematically organize into a two-layered mesh of orthogonal filaments instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is curvature sensitive and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarsegrained computed simulation to understand their mechanisms. Our results highlight the specificity of animal septins as opposed to fungal proteins.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.

Septins are ubiquitous cytoskeletal filaments that interact with the inner plasma membrane and are essential for cell division in eukaryotes. In cellular contexts, septins are often localized at micrometric Gaussian curvatures, where they assemble onto ring-like structures. The behavior of budding yeast septins depends on their specific interaction with inositol phospholipids, enriched at the inner leaflet of the plasma membrane. Septin filaments are built from the non-polar self-assembly of short rods into filaments. However, the molecular mechanisms regulating the interplay with the inner plasma membrane and the resulting interaction with specific curvatures are not fully understood. In this report, we have imaged dynamical molecular assemblies of budding yeast septins on PIP2-containing supported lipid bilayers using a combination of high-speed AFM and correlative AFM-fluorescence microscopy. Our results clearly demonstrate that septins are able to bind to flat supported lipid bilayers and thereafter induce the remodeling of membranes. Short septin rods (octamers subunits) can indeed destabilize supported lipid bilayers and reshape the membrane to form 3D structures such as rings and tubes, demonstrating that long filaments are not necessary for septin-induced membrane buckling.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton. Summary Human septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

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.

Protein enrichment at specific membrane locations in cells is crucial for many cellular functions. It is well-recognized that the ability of some proteins to sense membrane curvature contributes partly to their enrichment in highly curved cellular membranes. In the past, different theoretical models have been developed to reveal the physical mechanisms underlying curvature-driven protein sorting. This review aims to provide a detailed discussion of the two continuous models that are based on the Helfrich elasticity energy, (1) the spontaneous curvature model and (2) the curvature mismatch model. These two models are commonly applied to describe experimental observations of protein sorting. We discuss how they can be used to explain the curvature-induced sorting data of two BAR proteins, amphiphysin and centaurin. We further discuss how membrane rigidity, and consequently the membrane curvature generated by BAR proteins, could influence protein organization on the curved membranes. Finally, we address future directions in extending these models to describe some cellular phenomena involving protein sorting.