The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment.

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

The septin modifier, forchlorfenuron, activates NLRP3 via a potassium-independent mitochondrial axis.

The Nod-like receptor protein 3 (NLRP3) inflammasome is activated by stimuli that induce perturbations in cell homeostasis, which commonly converge on cellular potassium efflux. NLRP3 has thus emerged as a sensor for ionic flux. Here, we identify forchlorfenuron (FCF) as an inflammasome activator that triggers NLRP3 signaling independently of potassium efflux. FCF triggers the rearrangement of septins, key cytoskeletal proteins that regulate mitochondrial function. We report that FCF triggered the rearrangement of SEPT2 into tubular aggregates and stimulated SEPT2-independent NLRP3 inflammasome signaling. Similar to imiquimod, FCF induced the collapse of the mitochondrial membrane potential and mitochondrial respiration. FCF thereby joins the imidazoquinolines as a structurally distinct class of molecules that triggers NLRP3 inflammasome signaling independent of potassium efflux, likely by inducing mitochondrial damage.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

Phosphorylation of the F-BAR protein Hof1 drives septin ring splitting in budding yeast.

A double septin ring accompanies cytokinesis in yeasts and mammalian cells. In budding yeast, reorganisation of the septin collar at the bud neck into a dynamic double ring is essential for actomyosin ring constriction and cytokinesis. Septin reorganisation requires the Mitotic Exit Network (MEN), a kinase cascade essential for cytokinesis. However, the effectors of MEN in this process are unknown. Here we identify the F-BAR protein Hof1 as a critical target of MEN in septin remodelling. Phospho-mimicking HOF1 mutant alleles overcome the inability of MEN mutants to undergo septin reorganisation by decreasing Hof1 binding to septins and facilitating its translocation to the actomyosin ring. Hof1-mediated septin rearrangement requires its F-BAR domain, suggesting that it may involve a local membrane remodelling that leads to septin reorganisation. In vitro Hof1 can induce the formation of intertwined septin bundles, while a phosphomimetic Hof1 protein has impaired septin-bundling activity. Altogether, our data indicate that Hof1 modulates septin architecture in distinct ways depending on its phosphorylation status.

The MoLfa1 Protein Regulates Fungal Development and Septin Ring Formation in Magnaporthe oryzae.

Septins play a key regulatory role in cell division, cytokinesis, and cell polar growth of the rice blast fungus (Magnaporthe oryzae). We found that the organization of the septin ring, which is essential for appressorium-mediated infection in M. oryzae, requires long-chain fatty acids (LCFAs), which act as mediators of septin organization at membrane interfaces. However, it is unclear how septin ring formation and LCFAs regulate the pathogenicity of the rice blast fungus. In this study, a novel protein was named MoLfa1 because of its role in LCFAs utilization. MoLfa1 affects the utilization of LCFAs, lipid metabolism, and the formation of the septin ring by binding with phosphatidylinositol phosphates (PIPs), thereby participating in the construction of penetration pegs of M. oryzae. In addition, MoLfa1 is localized in the endoplasmic reticulum (ER) and interacts with the ER-related protein MoMip11 to affect the phosphorylation level of Mps1. (Mps1 is the core protein in the MPS1-MAPK pathway.) In conclusion, MoLfa1 affects conidia morphology, appressorium formation, lipid metabolism, LCFAs utilization, septin ring formation, and the Mps1-MAPK pathway of M. oryzae, influencing pathogenicity.

Exocytic plasma membrane flows remodel endoplasmic reticulum-plasma membrane tethering for septin collar assembly.

Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons.

Palmitoylation is required for Sept8-204 and Sept5 to form vesicle-like structure and colocalize with synaptophysin.

Sept8 is a vesicle associated protein and there are two typical transcriptional variants (Sept8-204 and Sept8-201) expressed in mice brain. Interestingly, the coexpression of Sept8-204/Sept5 induces the formation of small sized vesicle-like structure, while that of the Sept8-201/Sept5 produces large puncta. Sept8 is previously shown to be palmitoylated. Here it was further revealed that protein palmitoylation is required for Sept8-204/Sept5 to maintain small sized vesicle-like structure and colocalize with synaptophysin, since either the expression of nonpalmitoylated Sept8-204 mutant (Sept8-204-3CA) or inhibiting Sept8-204 palmitoylation by 2-BP with Sept5 produces large puncta, which barely colocalizes with synaptophysin (SYP). Moreover, it was shown that the dynamic palmitoylation of Sept8-204 is controlled by ZDHHC17 and PPT1, loss of ZDHHC17 decreases Sept8-204 palmitoylation and induces large puncta, while loss of PPT1 increases Sept8-204 palmitoylation and induces small sized vesicle-like structure. Together, these findings suggest that palmitoylation is essential for the maintenance of the small sized vesicle-like structure for Sept8-204/Sept5, and may hint their important roles in synaptic functions.

Chemical Proteomics Reveals Nε-Fatty-Acylation of Septins by Rho Inactivation Domain (RID) of the Vibrio MARTX Toxin to Alter Septin Localization and Organization.

Vibrio species, the Gram-negative bacterial pathogens causing cholera and sepsis, produce multiple secreted virulence factors for infection and pathogenesis. Among these is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin that releases several critical effector domains with distinct functions inside eukaryotic host cells. One such effector domain, the Rho inactivation domain (RID), has been discovered to catalyze long-chain Nε-fatty-acylation on lysine residues of Rho GTPases, causing inactivation of Rho GTPases and disruption of the host actin cytoskeleton. However, whether RID modifies other host proteins to exert additional functions remains to be determined. Herein, we describe the integration of bioorthogonal chemical labeling and quantitative proteomics to globally profile the target proteins modified by RID in living cells. More than 246 proteins are identified as new RID substrates, including many involved in GTPase regulation, cytoskeletal organization, and cell division. We demonstrate that RID extensively Nε-fatty-acylates septin proteins, the fourth cytoskeletal component of mammalian cells with important roles in diverse cellular processes. While affinity purification and mass spectrometry analysis show that RID-mediated Nε-fatty-acylation does not affect septin-septin interactions, this modification increases the membrane association of septins and confers localization to detergent-resistant membrane rafts. As a result, the filamentous assembly and organization of septins are disrupted by RID-mediated Nε-fatty-acylation, further contributing to cytoskeletal and mitotic defects that phenocopy the effects of septin depletion. Overall, our work greatly expands the substrate scope and function of RID and demonstrates the role of RID-mediated Nε-fatty-acylation in manipulating septin localization and organization.

Unearthing the role of septins in viral infections.

Septin proteins are a subfamily of closely related GTP-binding proteins conserved in all species except for higher plants and perform essential biological processes. Septins self-assemble into heptameric or octameric complexes and form higher-order structures such as filaments, rings, or gauzes by end-to-end binding. Their close association with cell membrane components makes them central in regulating critical cellular processes. Due to their organisation and properties, septins function as diffusion barriers and are integral in providing scaffolding to support the membrane's curvature and stability of its components. Septins are also involved in vesicle transport and exocytosis through the plasma membrane by co-localising with exocyst protein complexes. Recently, there have been emerging reports of several human and animal diseases linked to septins and abnormalities in their functions. Most of our understanding of the significance of septins during microbial diseases mainly pertains to their roles in bacterial infections but not viruses. This present review focuses on the known roles of septins in host-viral interactions as detailed by various studies.

PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension.

Convergent extension (CE) requires the coordinated action of the planar cell polarity (PCP) proteins1,2 and the actin cytoskeleton,3,4,5,6 but this relationship remains incompletely understood. For example, PCP signaling orients actomyosin contractions, yet actomyosin is also required for the polarized localization of PCP proteins.7,8 Moreover, the actin-regulating Septins play key roles in actin organization9 and are implicated in PCP and CE in frogs, mice, and fish5,6,10,11,12 but execute only a subset of PCP-dependent cell behaviors. Septin loss recapitulates the severe tissue-level CE defects seen after core PCP disruption yet leaves overt cell polarity intact.5 Together, these results highlight the general fact that cell movement requires coordinated action by distinct but integrated actin populations, such as lamella and lamellipodia in migrating cells13 or medial and junctional actin populations in cells engaged in apical constriction.14,15 In the context of Xenopus mesoderm CE, three such actin populations are important, a superficial meshwork known as the "node-and-cable" system,4,16,17,18 a contractile network at deep cell-cell junctions,6,19 and mediolaterally oriented actin-rich protrusions, which are present both superficially and deeply.4,19,20,21 Here, we exploited the amenability of the uniquely "two-dimensional" node and cable system to probe the relationship between PCP proteins, Septins, and the polarization of this actin network. We find that the PCP proteins Vangl2 and Prickle2 and Septins co-localize at nodes, and that the node and cable system displays a cryptic, PCP- and Septin-dependent anteroposterior (AP) polarity in its organization and dynamics.

Septins provide microenvironment sensing and cortical actomyosin partitioning in motile amoeboid T lymphocytes.

The all-terrain motility of lymphocytes in tissues and tissue-like gels is best described as amoeboid motility. For amoeboid motility, lymphocytes do not require specific biochemical or structural modifications to the surrounding extracellular matrix. Instead, they rely on changing shape and steric interactions with the microenvironment. However, the exact mechanism of amoeboid motility remains elusive. Here, we report that septins participate in amoeboid motility of T cells, enabling the formation of F-actin and α-actinin-rich cortical rings at the sites of cell cortex-indenting collisions with the extracellular matrix. Cortical rings compartmentalize cells into chains of spherical segments that are spatially conformed to the available lumens, forming transient "hourglass"-shaped steric locks onto the surrounding collagen fibers. The steric lock facilitates pressure-driven peristaltic propulsion of cytosolic content by individually contracting cell segments. Our results suggest that septins provide microenvironment-guided partitioning of actomyosin contractility and steric pivots required for amoeboid motility of T cells in tissue-like microenvironments.

External quality assessment for detection of colorectal cancer by Septin9 DNA methylation in clinical laboratories.

BACKGROUND AND AIMS: The incidence and mortality rate of colorectal cancer (CRC) are increasing worldwide. Septin9 methylated (mSEPT9) DNA in circulation can be used as a non-invasive detection method to assist in the early diagnosis of CRC; however, the detection methods and procedures are complicated. This study aimed to evaluate the ability of clinical laboratories to detect Septin9 methylation in plasma cell-free DNA (cfDNA). MATERIALS AND METHODS: We prepared a sample panel consisting of positive and negative Septin9 methylation cells and CRC cells. Three positive samples with different methylation levels, one negative sample and one duplicate sample, two samples containing interference, three different CRC cell samples, and a fictitious case report were included. The panel was distributed to 59 laboratories for mSEPT9 analysis, result comparison, and scoring. RESULTS: The sample panel, validated by National Medical Products Administration (NMPA)-approved tests and targeted bisulfite sequencing, met expectations and could be used for external quality assessment (EQA). Among the 59 laboratories, 55 (93.22%) correctly reported the mSEPT9 results for all samples, while four (6.79%) reported 15 false negatives and were considered improvable. All false negatives originated from four laboratories using laboratory-developed tests (LDTs), with three failing to detect weakly positive samples, samples containing interference, and samples from different CRC cells, and one reported erroneous results on all positive samples. CONCLUSION: Our results illustrated that the detection of mSEPT9 in cfDNA is satisfactory in China. EQA is indispensable because it can help improve the diagnostic capability and quality management of the laboratories, and provide suggestions for the problems existing in mSEPT9 detection.

The cell-end protein Tea4 spatially regulates hyphal branch initiation and appressorium remodeling in the blast fungus Magnaporthe oryzae.

The differentiation of specialized infection cells, called appressoria, from polarized germ tubes of the blast fungus Magnaporthe oryzae, requires remarkable remodeling of cell polarity and architecture, yet our understanding of this process remains incomplete. Here we investigate the behavior and role of cell-end marker proteins in appressorium remodeling and hyphal branch emergence. We show that the SH3 domain-containing protein Tea4 is required for the normal formation of an F-actin ring at Tea1-GFP-labeled polarity nodes, which contributes to the remodeling of septin structures and repolarization of the appressorium. Further, we show that Tea1 localizes to a cortical structure during hyphal septation which, unlike contractile septin rings, persists after septum formation, and, in combination with other polarity determinants, likely spatially regulates branch emergence. Genetic loss of Tea4 leads to mislocalization of Tea1 at the hyphal apex and with it, impaired growth directionality. In contrast, Tea1 is largely depleted from septation events in Δtea4 mutants and branching and septation are significantly reduced. Together, our data provide new insight into polarity remodeling during infection-related and vegetative growth by the blast fungus.

Septins modulate the autophagy response after nutrient starvation.

The pathways that induce macroautophagy (referred to as autophagy hereafter) in response to the stress of starvation are well conserved and essential under nutrient-limiting conditions. However, less is understood about the mechanisms that modulate the autophagy response. Here we present evidence that after induction of autophagy in budding yeast septin filaments rapidly assemble into discrete patches distributed along the cell cortex. These patches gradually mature over 12 h of nutrient deprivation to form extended structures around Atg9 membranes tethered at the cortical endoplasmic reticulum, a class of membranes that are limiting for autophagosome biogenesis. Loss of cortical septin structures alters the kinetics of autophagy activation and most dramatically extends the duration of the autophagy response. In wild-type cells, diffusion of Atg9 membranes at the cell cortex undergoes transient pauses that are dependent on septins, and septins at the bud neck block the diffusion of Atg9 membranes between mother and daughter cells. We conclude that septins reorganize at the cell cortex during autophagy to locally limit access of Atg9 membranes to autophagosome assembly sites, and thus modulate the autophagy response during nutrient deprivation.

Palmitoylated Sept8-204 modulates learning and anxiety by regulating filopodia arborization and actin dynamics.

Septin proteins are involved in diverse physiological functions, including the formation of specialized cytoskeletal structures. Septin 8 (Sept8) is implicated in spine morphogenesis and dendritic branching through palmitoylation. We explored the role and regulation of a Sept8 variant in human neural-like cells and in the mouse brain. We identified Sept8-204 as a brain-specific variant of Sept8 that was abundant in neurons and modified by palmitoylation, specifically at Cys469, Cys470, and Cys472. Sept8-204 palmitoylation was mediated by the palmitoyltransferase ZDHHC7 and was removed by the depalmitoylase PPT1. Palmitoylation of Sept8-204 bound to F-actin and induced cytoskeletal dynamics to promote the outgrowth of filopodia in N2a cells and the arborization of neurites in hippocampal neurons. In contrast, a Sept8-204 variant that could not be palmitoylated because of mutation of all three Cys residues (Sept8-204-3CA) lost its ability to bind F-actin, and expression of this mutant did not promote morphological changes. Genetic deletion of Sept8, Sept8-204, or Zdhhc7 caused deficits in learning and memory and promoted anxiety-like behaviors in mice. Our findings provide greater insight into the regulation of Sept8-204 by palmitoylation and its role in neuronal morphology and function in relation to cognition.

The Septin Gene StSep4 Contributes to the Pathogenicity of Setosphaeria turcica by Regulating the Morphology, Cell Wall Integrity, and Pathogenic Factor Biosynthesis.

Septins are a conserved group of GTP-binding proteins found in all eukaryotes and are the fourth-most abundant cytoskeletal proteins. Septins of some pathogenic fungi are involved in morphological changes related to infection. Our previous studies have identified four core septins (StSep1-4) in Setosphaeria turcica, the causal agent of northern corn leaf blight, while only StSep4 is significantly upregulated during the invasive process. We therefore used forchlorfenuron (FCF), the specific inhibitor of septin, and ΔStSep4 knockout mutants to further clarify the role of septins in S. turcica pathogenicity. FCF treatment caused a dose-dependent reduction in S. turcica colony growth, delayed the formation of infection structures, and reduced the penetration ability. ΔStSep4 knockout mutants displayed abnormal mycelium morphology, slow mycelial growth, conidiation deficiency, delayed appressorium development, and weakened pathogenicity. StSep4 deletion also broke cell wall integrity, altered chitin distribution, decreased the melanin content, and disrupted normal nuclear localization. A transcriptomic comparison revealed that genes differentially expressed between ΔStSep4 and WT were enriched in terms of ribosomes, protein translation, membrane components, and transmembrane transport activities. Our results demonstrate that StSep4 is required for morphology and pathogenicity in S. turcica, making it a promising target for the development of novel fungicides.

Reconstituted in vitro systems to reveal the roles and functions of septins.

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.