Proper phosphorylation of septin 12 regulates septin 4 and soluble adenylyl cyclase expression to induce sperm capacitation.

Septin-based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12- and Sept4-null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4-null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation-deficient (S196A), and SEPT4-depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E- and Sept12 S196A-knock-in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E- and S196A-mutant mouse sperm. In the sperm of the SEPT4-knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation.

Impaired Platelet Function in Sept8-Deficient Mice In Vitro.

Septins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbß3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbß3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

The pro-apoptotic ARTS protein induces neutrophil apoptosis, efferocytosis, and macrophage reprogramming to promote resolution of inflammation.

ARTS (Sept4_i2) is a pro-apoptotic protein and a product of the Sept4 gene. ARTS acts upstream of mitochondria to initiate caspase activation. ARTS induces apoptosis by specifically binding XIAP and allowing de-repression of active caspases required for Mitochondrial Outer Membrane Permeabilzation (MOMP). Moreover, ARTS promotes apoptosis by inducing ubiquitin-mediated degradation of both major anti-apoptotic proteins XIAP and Bcl-2. In the resolution phase of inflammation, the infiltrating leukocytes, which execute the acute innate response, undergo apoptosis and are subsequently cleared by phagocytic macrophages (i.e. efferocytosis). In this course, macrophages undergo reprogramming from inflammatory, to anti-inflammatory, and eventually to resolving macrophages that leave the injury sites. Since engulfment of apoptotic leukocytes is a key signaling step in macrophage reprogramming and resolution of inflammation, we hypothesized that a failed apoptosis in leukocytes in vivo would result in an impaired resolution process. To test this hypothesis, we utilized the Sept4/ARTS-/- mice, which exhibit resistance to apoptosis in many cell types. During zymosan A-induced peritonitis, Sept4/ARTS-/- mice exhibited impaired resolution of inflammation, characterized by reduced neutrophil apoptosis, macrophage efferocytosis and expression of pro-resolving mediators. This was associated with increased pro-inflammatory cytokines and reduced anti-inflammatory cytokines, secreted by resolution-phase macrophages. Moreover, ARTS overexpression in leukocytes in vitro promoted an anti-inflammatory behavior. Overall, our results suggest that ARTS is a key master-regulator necessary for neutrophil apoptosis, macrophage efferocytosis and reprogramming to the pro-resolving phenotype during the resolution of inflammation.

SEPT7 Interacts with KIF20A and Regulates the Proliferative State of Neural Progenitor Cells During Cortical Development.

Balanced proliferation and differentiation of neural progenitor cells (NPCs) are critical for brain development, but how the process is regulated and what components of the cell division machinery is involved are not well understood. Here we report that SEPT7, a cell division regulator originally identified in Saccharomyces cerevisiae, interacts with KIF20A in the intercellular bridge of dividing NPCs and plays an essential role in maintaining the proliferative state of NPCs during cortical development. Knockdown of SEPT7 in NPCs results in displacement of KIF20A from the midbody and early neuronal differentiation. NPC-specific inducible knockout of Sept7 causes early cell cycle exit, precocious neuronal differentiation, and ventriculomegaly in the cortex, but surprisingly does not lead to noticeable cytokinesis defect. Our data uncover an interaction of SEPT7 and KIF20A during NPC divisions and demonstrate a crucial role of SEPT7 in cell fate determination. In addition, this study presents a functional approach for identifying additional cell fate regulators of the mammalian brain.

Septins are critical regulators of osteoclastic bone resorption.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

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.

MicroRNA-127 post-transcriptionally downregulates Sept7 and suppresses cell growth in hepatocellular carcinoma cells.

BACKGROUND/AIMS: Hepatocellular carcinoma is one of the most common cancers worldwide. It has been suggested that microRNAs, a class of small regulatory RNAs, are associated with tumorigenesis by targeting the mRNAs of hundreds of genes that modulate a variety of biological processes, including cellular differentiation, apoptosis, metabolism, and proliferation. METHODS/RESULTS: we analyzed the expression levels of mir-127 in 33 HCC and non-cancerous tissues using qRT-PCR. MiR-127 is downregulated in 69.7% of HCC tissues compared with adjacent normal tissues, but its expression level is not correlated with the TNM stage, AFP level, or age. In vitro, miR-127 can arrest Huh7 at the G2/M phase and inhibit Huh7 cell proliferation. In an in vivo xenograft model, the overexpression of miR-127 can inhibit Huh7 cell tumorigenicity. The luciferase reporter and western blot results confirm that miR-127 downregulates Sept7 expression by targeting its 3'UTR. Furthermore, the knockdown of Sept7 has the same effect on cell proliferation as the overexpression of miR-127 in Huh7 cells. CONCLUSION: miR-127 plays a tumor-suppressor role and can serve as a potential diagnostic biomarker for HCC.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry.

The STIM1-ORAI1 pathway of store-operated Ca(2+) entry is an essential component of cellular Ca(2+) signalling. STIM1 senses depletion of intracellular Ca(2+) stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca(2+) channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca(2+) entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum-plasma membrane junctions before and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.

MiR-30a-5p antisense oligonucleotide suppresses glioma cell growth by targeting SEPT7.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting the mRNAs of hundreds of human genes. Variations in miRNA expression levels were shown to be associated with glioma. We have previously found miR-30a-5p overexpression in glioma cell lines and specimens. Bioinformatics analyses predict that several miRNAs, including miR-30a-5p, are involved in the post-transcriptional regulation of SEPT7. SEPT7 is a member of the septin family, which is a highly conserved subfamily of GTPases implicated in exocytosis, apoptosis, synaptogenesis, neurodegeneration and tumorigenesis. Our previous study has also demonstrated that SEPT7 expression is decreased in astrocytic gliomas with different grades and plays a tumor suppressor role. In the present study, we knocked down miR-30a-5p with antisense oligonucleotide (miR-30a-5p AS) in LN229 and SNB19 glioblastoma(GBM) cells, and found that cell growth and invasion were inhibited, while apoptosis was induced. miR-30a-5p AS treated cells showed upregulation of SEPT7 and downregulation of PCNA, cyclin D1, Bcl2, MMP2 and MMP9. In contrast, when miR-30a-5p mimics were transfected into LN229 and SNB19 GBM cells, cell growth and invasion were promoted and the expression of relevant proteins increased. Meanwhile, the effect of miR-30a-5p mimics on glioma cells can be reversed by transfection of SEPT7 construct. Additionaly, miR-30a-5p directly targeting SEPT7 was identified by the reporter gene assay. Our study demonstrates,for the first time, that miR-30a-5p is a bona fide negative regulator of SEPT7 and the oncogenic activity of miR-30a-5p in human gliomas is at least in part through the repression of SEPT7.

Localization of septin proteins in the mouse cochlea.

Septins are a family of GTP binding proteins that are well conserved in eukaryotic species except plants. Septins contribute to the lateral compartmentalization of membranes, cortical rigidity, and the regulation of membrane trafficking by associating with membrane lipids, actin, and microtubules. The organ of Corti in the cochlea has pivotal roles in auditory perception and includes two kinds of highly polarized cells, hair and supporting cells, both of which are rich in actin and microtubules. To identify the roles of septins in the cochlea, we analyzed the localization of three septin proteins, septin 4 (SEPT4), septin 5 (SEPT5), and septin 7 (SEPT7) that are abundantly expressed in brain tissues, and also examined auditory functions of Sept4 and Sept5 null mice. SEPT4, SEPT5, and SEPT7 were expressed in inner and outer pillar cells and Deiters' cells but the distribution patterns of each protein in Deiters' cells were different. SEPT4 and SEPT7 were expressed in the phalangeal process where SEPT5 was not detected. In addition to these cells SEPT5 and SEPT7 were co-localized with presynaptic vesicles of efferent nerve terminals. Only SEPT7 was expressed in the cochlea at embryonic stages. Although expression patterns of septin proteins suggested their important roles in the function of the cochlea, both Sept4 and Sept5 null mice had similar auditory functions to their wild type littermates. Immunohistochemical analysis of Sept4 null mice showed that compensatory expression of SEPT5 in the phalangeal process of Deiters' cells may have caused functional compensation of hearing ability in Sept4 null mice.

A role for septins in the interaction between the Listeria monocytogenes INVASION PROTEIN InlB and the Met receptor.

Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InlB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InlB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InlB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InlB-Met interaction and the viscosity of membrane tethers at locations where the InlB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function.

SEPT12 deficiency causes sperm nucleus damage and developmental arrest of preimplantation embryos.

Oocytes fertilized with spermatozoa obtained from Septin 12+/- chimeric mice failed to develop beyond the morula stage after IVF and intracytoplasmic sperm injection because of significant DNA defects in the spermatozoa. Given that SEPT12 is expressed at the edge of the sperm nucleus in both humans and mice, we hypothesized the vital roles of Septin 12 in sperm head shaping, nuclear DNA condensation, and early embryonic development.