Identification of a novel alternatively spliced septin.

Septins are a family of cytoskeletal proteins involved in cytokinesis, targeting of proteins to specific sites on the plasma membrane, and cellular morphogenesis. While many aspects of their function in cytokinesis in yeast cells have been investigated, the function of septins in mammalian cells is less well understood. For example, septins are present in post-mitotic neurons, suggesting they have other roles in, for example, establishing cell polarity. The full extent of the septin gene family is not known in mammalian cells. To better understand the septin gene family, we have cloned and characterized a novel mammalian septin.     

A PI3K activity-independent function of p85 regulatory subunit in control of mammalian cytokinesis

Cytosolic division in mitotic cells involves the function of a number of cytoskeletal proteins, whose coordination in the spatio-temporal control of cytokinesis is poorly defined. We studied the role of p85/p110 phosphoinositide kinase (PI3K) in mammalian cytokinesis. Deletion of the p85alpha regulatory subunit induced cell accumulation in telophase and appearance of binucleated cells, whereas inhibition of PI3K activity did not affect cytokinesis. Moreover, reconstitution of p85alpha-deficient cells with a Deltap85alpha mutant, which does not bind the catalytic subunit, corrected the cytokinesis defects of p85alpha(-/-) cells. We analyzed the mechanism by which p85alpha regulates cytokinesis; p85alpha deletion reduced Cdc42 activation in the cleavage furrow and septin 2 accumulation at this site. As Cdc42 deletion also triggered septin 2 and cytokinesis defects, a mechanism by which p85 controls cytokinesis is by regulating the local activation of Cdc42 in the cleavage furrow and in turn septin 2 localization. We show that p85 acts as a scaffold to bind Cdc42 and septin 2 simultaneously. p85 is thus involved in the spatial control of cytosolic division through regulation of Cdc42 and septin 2, in a PI3K-activity independent manner.     

SEPT9 occupies the terminal positions in septin octamers and mediates polymerization-dependent functions in abscission

Septins are filamentous guanosine triphosphatase-binding proteins that are required for cytokinesis in a wide range of organisms from yeast to man. Several septins, including SEPT9, have been found to be altered in cancers, but their roles in malignancy and cytokinesis remain unclear. It is known that they assemble into rod-shaped oligomeric complexes that join end-on-end to form filaments, but whether SEPT9 incorporates into these complexes and how it does so are unanswered questions. We used tandem affinity purification of mammalian septin complexes to show that SEPT9 occupies a terminal position in an octameric septin complex. A mutant SEPT9, which cannot self-associate, disrupted septin filament formation and resulted in late abscission defects during cytokinesis but did not affect septin-dependent steps earlier in mitosis. These data suggest that mammalian SEPT9 holds a terminal position in the septin octamers, mediating abscission-specific polymerization during cytokinesis.     

septin基因家族的研究进展

septin是一个广泛存在于除植物以外所有真核生物中的基因家族。最初认为septin家族是与酵母细胞胞质分裂相关的基因家族。然而随着研究的深入, 人们发现这类基因编码的蛋白质在许多生物体内出现了较大的功能分化, 尤其在哺乳动物细胞中, 他们不仅成员众多, 且参与了细胞分裂、细胞极化、囊泡运输及胞膜重构等多个过程。更引起研究人员重视的是: 最近有大量数据表明, 这一家族的一些成员与肿瘤发生、神经功能障碍和病原微生物感染的过程直接相关。因此, 近年来septin家族的功能研究正逐步成为细胞生物学及病理学研究的新热点。文章将试图从septin基因家族的种类、结构特点、生物学功能及其与人类疾病的关系等方面的研究进展进行综述。     

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.     

The mammalian septin MSF localizes with microtubules and is required for completion of cytokinesis

Cytokinesis in animal cells involves the contraction of an actomyosin ring formed at the cleavage furrow. Nuclear division, or karyokinesis, must be precisely timed to occur before cytokinesis in order to prevent genetic anomalies that would result in either cell death or uncontrolled cell division. The septin family of GTPase proteins has been shown to be important for cytokinesis although little is known about their role during this process. Here we investigate the distribution and function of the mammalian septin MSF. We show that during interphase, MSF colocalizes with actin, microtubules, and another mammalian septin, Nedd5, and coprecipitates with six septin proteins. In addition, transfections of various MSF isoforms reveal that MSF-A specifically localizes with microtubules and that this localization is disrupted by nocodazole treatment. Furthermore, MSF isoforms localize primarily with tubulin at the central spindle during mitosis, whereas Nedd5 is mainly associated with actin. Microinjection of affinity-purified anti-MSF antibodies into synchronized cells, or depletion of MSF by small interfering RNAs, results in the accumulation of binucleated cells and in cells that have arrested during cytokinesis. These results reveal that MSF is required for the completion of cytokinesis and suggest a role that is distinct from that of Nedd5.     

Mammalian septin function in hemostasis and beyond

Interest in the biology of mammalian septin proteins has undergone a birth in recent years. Originally identified as critical for yeast budding throughout the 1970s, the septin family is now recognized to extend from yeast to humans and is associated with a variety of events ranging from cytokinesis to vesicle trafficking. An emerging theme for septins is their presence at sites where active membrane or cytoplasmic partitioning is occurring. Here, we briefly review the mammalian septin protein family and focus on a prototypic human and mouse septin, termed SEPT5, that is expressed in the brain, heart, and megakaryocytes. Work from neurobiology laboratories has linked SEPT5 to the exocytic complex of neurons, with implications that SEPT5 regulates neurotransmitter release. Striking similarities exist between neurotransmitter release and the platelet-release reaction, which is a critical step in platelet response to vascular injury. Work from our laboratory has characterized the platelet phenotype from mice containing a targeted deletion of SEPT5. Most strikingly, platelets from SEPT5(null) animals aggregate and release granular contents in response to subthreshold levels of agonists. Thus, the characterization of a SEPT5-deficient mouse has linked SEPT5 to the platelet exocytic process and, as such, illustrates it as an important protein for regulating platelet function. Recent data suggest that platelets contain a wide repertoire of different septin proteins and assemble to form macromolecular septin complexes. The mouse platelet provides an experimental framework to define septin function in hemostasis, with implications for neurobiology and beyond.     

Regulation of septin organization and function in yeast

Septins are a conserved eukaryotic family of GTP-binding filament-forming proteins with functions in cytokinesis and other processes. In the budding yeast Saccharomyces cerevisiae, septins initially localize to the presumptive bud site and then to the cortex of the mother-bud neck as an hourglass structure. During cytokinesis, the septin hourglass splits and single septin rings partition with each of the resulting cells. Septins are thought to function in diverse processes in S. cerevisiae, mainly by acting as a scaffold to direct the neck localization of septin-associated proteins.     

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.     

Genetic Deletion of SEPT7 Reveals a Cell Type-Specific Role of Septins in Microtubule Destabilization for the Completion of Cytokinesis

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.     

Septin structure and function in yeast and beyond

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

Phosphorylation-dependent septin interaction of Bni5 is important for cytokinesis

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Delta. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.     

Localization and evolution of septins in algae

Septins are a group of GTP‐binding proteins that are multi‐functional, with a well‐known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog‐possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.     

The actin-binding and bundling protein,EPLIN, is required for cytokinesis

Cytokinesis involves two phases: 1) membrane ingression followed by 2) membrane abscission. The ingression phase generates a cleavage furrow and this requires co-operative function of the actin-myosin II contractile ring and septin filaments. We demonstrate that the actin-binding protein, EPLIN, locates to the cleavage furrow during cytokinesis and this is possibly via association with the contractile ring components, myosin II, and the septin, Sept2. Depletion of EPLIN results in formation of multinucleated cells and this is associated with inefficient accumulation of active myosin II (MRLCS19) and Sept2 and their regulatory small GTPases, RhoA and Cdc42, respectively, to the cleavage furrow during the final stages of cytokinesis. We suggest that EPLIN may function during cytokinesis to maintain local accumulation of key cytokinesis proteins at the furrow.     

Self- and actin-templated assembly of Mammalian septins

Septins are polymerizing GTPases required for cytokinesis and cortical organization. The principles by which they are targeted to, and assemble at, specific cell regions are unknown. We show that septins in mammalian cells switch between a linear organization along actin bundles and cytoplasmic rings, approximately 0.6 microm in diameter. A recombinant septin complex self-assembles into rings resembling those in cells. Linear organization along actin bundles was reconstituted by adding an adaptor protein, anillin. Perturbation of septin organization in cells by expression of a septin-interacting fragment of anillin or by septin depletion via siRNA causes loss of actin bundles. We conclude that septins alone self-assemble into rings, that adaptor proteins recruit septins to actin bundles, and that septins help organize these bundles.     

Sept12 is a component of the mammalian sperm tail annulus

Since their essential role in cytokinesis was first shown in yeast, the septins have been described to function in diverse cellular contexts. The members of this unique class of GTPases are capable of binding and hydrolyzing GTP, associating with membranes and oligomerizing into higher order structures. Here we describe Sept12, a novel septin, identified in a yeast two hybrid screen using Sept5 as the bait. Sept12 contains the primary sequence elements of a septin and is capable of interacting with other septins. In addition, Sept12 purifies with bound nucleotide and binds to phosphoinositides, confirming its identity as a septin. RT-PCR and Northern blots reveal that Sept12 mRNA is expressed predominantly in testis, and this is supported by tissue Western blots. In rats, Sept12 protein levels rise upon sexual maturity and the Sept12 protein colocalizes with the annulus in isolated mature spermatozoa. Further, coexpression of Sept12 with Sept4, an essential annulus component, results in complete colocalization of both proteins into robust and highly curved filaments in CHO cells. This study suggests Sept12 may be involved in mammalian fertility.     

Cortical organization by the septin cytoskeleton is essential for structural and mechanical integrity of mammalian spermatozoa

Septins are polymerizing GTP binding proteins required for cortical organization during cytokinesis and other cellular processes. A mammalian septin gene Sept4 is expressed mainly in postmitotic neural cells and postmeiotic male germ cells. In mouse and human spermatozoa, SEPT4 and other septins are found in the annulus, a cortical ring which separates the middle and principal pieces. Sept4-/- male mice are sterile due to defective morphology and motility of the sperm flagellum. In Sept4 null spermatozoa, the annulus is replaced by a fragile segment lacking cortical material, beneath which kinesin-mediated intraflagellar transport stalls. The sterility is rescued by injection of sperm into oocytes, demonstrating that each Sept4 null spermatozoon carries an intact haploid genome. The annulus/septin ring is also disorganized in spermatozoa from a subset of human patients with asthenospermia syndrome. Thus, cortical organization based on circular assembly of the septin cytoskeleton is essential for the structural and mechanical integrity of mammalian spermatozoa.     

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.     

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.     

Borg/septin interactions and the assembly of mammalian septin heterodimers,trimers, and filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.