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.

Septins and a formin have distinct functions in anaphase chiral cortical rotation in the Caenorhabditis elegans zygote.

Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the Caenorhabditis elegans zygote, actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and cross-link the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior-posterior axis and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.

Revised subunit order of mammalian septin complexes explains their in vitro polymerization properties.

Septins are conserved GTP-binding cytoskeletal proteins that polymerize into filaments by end-to-end joining of hetero-oligomeric complexes. In human cells, both hexamers and octamers exist, and crystallography studies predicted the order of the hexamers to be SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7, while octamers are thought to have the same core, but with SEPT9 at the ends. However, based on this septin organization, octamers and hexamers would not be expected to copolymerize due to incompatible ends. Here we isolated hexamers and octamers of specific composition from human cells and show that hexamers and octamers polymerize individually and, surprisingly, with each other. Binding of the Borg homology domain 3 (BD3) domain of Borg3 results in distinctive clustering of each filament type. Moreover, we show that the organization of hexameric and octameric complexes is inverted compared with its original prediction. This revised septin organization is congruent with the organization and behavior of yeast septins suggesting that their properties are more conserved than was previously thought.

The state of the septin cytoskeleton from assembly to function.

Septins are conserved guanine nucleotide-binding proteins that polymerize into filaments at the cell cortex or in association with other cytoskeletal proteins, such as actin or microtubules. As integral players in many morphogenic and signaling events, septins form scaffolds important for the recruitment of the cytokinetic machinery, organization of the plasma membrane, and orientation of cell polarity. Mutations in septins or their misregulation are associated with numerous diseases. Despite growing appreciation for the importance of septins in different aspects of cell biology and disease, septins remain relatively poorly understood compared with other cytoskeletal proteins. Here in this review, we highlight some of the recent developments of the last two years in the field of septin cell biology.

Masters of asymmetry - lessons and perspectives from 50 years of septins.

Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.

Septin4 regulates endoplasmic reticulum stress and apoptosis in melatonin­induced osteoblasts.

Idiopathic scoliosis (IS) is a spinal 3­dimensional deformity with an unknown cause. Melatonin is secreted by the pineal body and contributes to the occurrence and progression of IS. In our previous preliminary study, it was reported that high concentrations of melatonin can induce osteoblast apoptosis, thus acting as an IS treatment, but the mechanism of action is unknown. Therefore, the present study was performed to further investigate the possible mechanism underlying the efficacy of melatonin as a treatment for IS. The present results indicated that high concentrations of melatonin mediate endoplasmic reticulum stress (ERS)­induced apoptosis in hFOB 1.19 cells, and this resulted in a significant and dose­dependent increase in the expression of Septin4, as well as the expression levels of glucose­regulated protein (GRP)78, GRP94 and cleaved caspase­3. Furthermore, osteoblasts were overexpressed with Septin4 and the mechanism via which melatonin induces osteoblast ERS was demonstrated to be via the regulation of Septin4. In addition, it was indicated that cytoskeleton destruction, cell morphology changes and the decrease in the number of cells were aggravated after osteoblasts were overexpressed with Septin4, as indicated by phalloidin and DAPI staining. Collectively, the present results suggest that the Septin4 protein may be a target of ERS in melatonin­induced osteoblast apoptosis, which is involved in bone metabolism diseases, thus providing novel evidence for clinical melatonin treatment of IS.

Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration.

Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration.

Septin 9 isoforms promote tumorigenesis in mammary epithelial cells by increasing migration and ECM degradation through metalloproteinase secretion at focal adhesions.

The cytoskeletal interacting protein Septin 9 (SEPT9), a member of the septin gene family, has been proposed to have oncogenic functions. It is a known hot spot of retroviral tagging insertion and a fusion partner of both de novo and therapy-induced mixed lineage leukemia (MLL). Of all septins, SEPT9 holds the strongest link to cancer, especially breast cancer. Murine models of breast cancer frequently exhibit SEPT9 amplification in the form of double minute chromosomes, and about 20% of human breast cancer display genomic amplification and protein over expression at the SEPT9 locus. Yet, a clear mechanism by which SEPT9 elicits tumor-promoting functions is lacking. To obtain unbiased insights on molecular signatures of SEPT9 upregulation in breast tumors, we overexpressed several of its isoforms in breast cancer cell lines. Global transcriptomic profiling supports a role of SEPT9 in invasion. Functional studies reveal that SEPT9 upregulation is sufficient to increase degradation of the extracellular matrix, while SEPT9 downregulation inhibits this process. The degradation pattern is peripheral and associated with focal adhesions (FAs), where it is coupled with increased expression of matrix metalloproteinases (MMPs). SEPT9 overexpression induces MMP upregulation in human tumors and in culture models and promotes MMP3 secretion to the media at FAs. Downregulation of SEPT9 or chemical inhibition of septin filament assembly impairs recruitment of MMP3 to FAs. Our results indicate that SEPT9 promotes upregulation and both trafficking and secretion of MMPs near FAs, thus enhancing migration and invasion of breast cancer cells.

Reorganization of Septins Modulates Synaptic Transmission at Neuromuscular Junctions.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

Transfection of choanoflagellates illuminates their cell biology and the ancestry of animal septins.

As the closest living relatives of animals, choanoflagellates offer unique insights into animal origins and core mechanisms underlying animal cell biology. However, unlike traditional model organisms, such as yeast, flies, and worms, choanoflagellates have been refractory to DNA delivery methods for expressing foreign genes. Here we report a robust method for expressing transgenes in the choanoflagellate Salpingoeca rosetta, overcoming barriers that have previously hampered DNA delivery and expression. To demonstrate how this method accelerates the study of S. rosetta cell biology, we engineered a panel of fluorescent protein markers that illuminate key features of choanoflagellate cells. We then investigated the localization of choanoflagellate septins, a family of GTP-binding cytoskeletal proteins that are hypothesized to regulate multicellular rosette development in S. rosetta. Fluorescently tagged septins localized to the basal poles of S. rosetta single cells and rosettes in a pattern resembling septin localization in animal epithelia. The establishment of transfection in S. rosetta and its application to the study of septins represent critical advances in the use of S. rosetta as an experimental model for investigating choanoflagellate cell biology, core mechanisms underlying animal cell biology, and the origin of animals.

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin.

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that, during entry into dendrites, axonally destined cargos move with a retrograde bias toward the cell body, while dendritically destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysine-rich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axons-dendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

Septins suppress the release of vaccinia virus from infected cells.

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.

The role of septin 7 in physiology and pathological disease: A systematic review of current status.

Septins are a conserved family of cytoskeletal GTPases present in different organisms, including yeast, drosophila, Caenorhabditis elegans and humans. In humans, septins are involved in various cellular processes, including exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Septin 7 is unique out of 13 human septins. Mammalian septin 6, septin 7, septin 2 and septin 9 coisolate together in complexes to form the core unit for the generation of the septin filaments. Physiological septin filaments are hetero-oligomeric complexes consisting of core septin hexamers and octamers. Furthermore, septin 7 plays a crucial role in cytokinesis and mitosis. Septin 7 is localized to the filopodia and branches of developing hippocampal neurons, and is the most abundant septin in the adult rat forebrain as well as a structural component of the human and mouse sperm annuli. Septin 7 is crucial to the spine morphogenesis and dendrite growth in neurons, and is also a structural constituent of the annulus in human and mouse sperm. It can suppress growth of some tumours such as glioma and papillary thyroid carcinoma. However, the molecular mechanisms of involvement of septin 7 in human disease, especially in the development of cancer, remain unclear. This review focuses on the structure, function and mechanism of septin 7 in vivo, and summarizes the role of septin 7 in cell proliferation, cytokinesis, nervous and reproductive systems, as well as the underlying molecular events linking septin 7 to various diseases, such as Alzheimer's disease, schizophrenia, neuropsychiatric systemic lupus erythematosus, tumour and so on.

Septin7 regulates inner ear formation at an early developmental stage.

Septins are guanosine triphosphate-binding proteins that are evolutionally conserved in all eukaryotes other than plants. They function as multimeric complexes that interact with membrane lipids, actomyosin, and microtubules. Based on these interactions, septins play essential roles in the morphogenesis and physiological functions of many mammalian cell types including the regulation of microtubule stability, vesicle trafficking, cortical rigidity, planar cell polarity, and apoptosis. The inner ear, which perceives auditory and equilibrium sensation with highly differentiated hair cells, has a complicated gross morphology. Furthermore, its development including morphogenesis is dependent on various molecular mechanisms, such as apoptosis, convergent extension, and cell fate determination. To determine the roles of septins in the development of the inner ear, we specifically deleted Septin7 (Sept7), the non-redundant subunit in the canonical septin complex, in the inner ear at different times during development. Foxg1Cre-mediated deletion of Sept7, which achieved the complete knockout of Sept7 within the inner ear at E9.5, caused cystic malformation of inner ears and a reduced numbers of sensory epithelial cells despite the existence of mature hair cells. Excessive apoptosis was observed at E10.5,E11.5 and E12.5 in all inner ear epithelial cells and at E10.5 and E11.5 in prosensory epithelial cells of the inner ears of Foxg1Cre;Septin7floxed/floxed mice. In contrast with apoptosis, cell proliferation in the inner ear did not significantly change between control and mutant mice. Deletion of Sept7 within the cochlea at a later stage (around E15.5) with Emx2Cre did not result in any apparent morphological anomalies observed in Foxg1Cre;Septin7floxed/floxed mice. These results suggest that SEPT7 regulates gross morphogenesis of the inner ear and maintains the size of the inner ear sensory epithelial area and exerts its effects at an early developmental stage of the inner ear.

Investigation of the role of four mitotic septins and chitin synthase 2 for cytokinesis in Kluyveromyces lactis.

Septins are key components of the cell division machinery from yeast to humans. The model yeast Saccharomyces cerevisiae has five mitotic septins, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1. Here we characterized the five orthologs from the genetically less-redundant milk yeast Kluyveromyces lactis. We found that except for KlSHS1 all septin genes are essential. Klshs1 deletions displayed temperature-sensitive growth and morphological defects. Heterologous complementation analyses revealed that all five K. lactis genes encode functional orthologs of their S. cerevisiae counterparts. Fluorophore-tagged versions of the K. lactis septins localized to a ring at the incipient bud site and split into two separate rings at the bud neck later in cytokinesis. One of the key proteins recruited to the bud neck by septins in S. cerevisiae is the chitin synthase Chs2, which synthesizes the primary septum. KlCHS2 was found to be essential and deletions showed cytokinetic defects upon spore germination. KlChs2-GFP also localized to the bud neck and to punctate structures in K. lactis. We conclude that cytokinesis in K. lactis is similar to S. cerevisiae and chimeric septin complexes are fully functional in both yeasts. In contrast to some S. cerevisiae strains, KlChs2 and KlCdc10 were found to be essential.

Septins are involved in nuclear division, morphogenesis and pathogenicity in Fusarium graminearum.

Septins are GTP-binding proteins that regulate cell polarity, cytokinesis and cell morphogenesis. Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating diseases worldwide. In this study, we have functionally characterized the core septins, Cdc3, Cdc10, Cdc11 and Cdc12 in F. graminearum. The loss of FgCdc3, FgCdc11, FgCdc12, but not FgCdc10, mutants showed significant reduction in growth, conidiation and virulence. Microscopic analyses revealed that all of them were involved in septum formation and nuclear division. Moreover, disruption of septin genes resulted in morphological defects in ascospores and conidia. Interestingly, conidia produced by ΔFgcdc3, ΔFgcdc11 and ΔFgcdc12 mutants exhibited deformation with interconnecting conidia in contrast to their parent wild-type strain PH-1 and the ΔFgcdc10 mutant that produced normal conidia. Using yeast two-hybrid assays, we determined the interactions among FgCdc3, FgCdc10, FgCdc11 and FgCdc12. Taken together, our results indicate that septins play important roles in the nuclear division, morphogenesis and pathogenicity in F. graminearum.

Roles of septins in prospore membrane morphogenesis and spore wall assembly in Saccharomyces cerevisiae.

The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D-associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis.

Septins promote stress fiber-mediated maturation of focal adhesions and renal epithelial motility.

Organogenesis and tumor metastasis involve the transformation of epithelia to highly motile mesenchymal-like cells. Septins are filamentous G proteins, which are overexpressed in metastatic carcinomas, but their functions in epithelial motility are unknown. Here, we show that a novel network of septin filaments underlies the organization of the transverse arc and radial (dorsal) stress fibers at the leading lamella of migrating renal epithelia. Surprisingly, septin depletion resulted in smaller and more transient and peripheral focal adhesions. This phenotype was accompanied by a highly disorganized lamellar actin network and rescued by the actin bundling protein α-actinin-1. We show that preassembled actin filaments are cross-linked directly by Septin 9 (SEPT9), whose expression is increased after induction of renal epithelial motility with the hepatocyte growth factor. Significantly, SEPT9 overexpression enhanced renal cell migration in 2D and 3D matrices, whereas SEPT9 knockdown decreased migration. These results suggest that septins promote epithelial motility by reinforcing the cross-linking of lamellar stress fibers and the stability of nascent focal adhesions.