Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

Discovery of broad-spectrum fungicides that block septin-dependent infection processes of pathogenic fungi.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.

Relationship between autophagy, apoptosis and endoplasmic reticulum stress induced by melatonin in osteoblasts by septin7 expression.

Melatonin secreted by the pineal body is associated with the occurrence and development of idiopathic scoliosis. Melatonin has a concentration­dependent dual effect on osteoblast proliferation, in which higher concentrations can inhibit osteoblast proliferation and induce apoptosis; however, the underlying mechanism remains unclear. In the present study, flow cytometry was used to demonstrate that osteoblast cells treated with melatonin exhibited significantly increased early and late stage apoptotic rates as the concentration increased. Chromatin condensation in the nucleus and apoptotic body formation could be observed using fluorescent microscopy in osteoblast cells treated with 2 mM melatonin. Western blotting results showed that there was an upregulation in the expression of apoptosis marker proteins [poly (ADP­ribose) polymerase 1 (PARP­1)], endoplasmic reticulum stress [ERS; C/EBP homologous protein (CHOP) and glucose­regulated protein, 78 kDa (GRP78)] and autophagy [microtubule­associated protein 1 light chain 3ß (LC3)­I/LC3II]. PARP­1 expression was not altered when treated with ERS inhibitor 4PBA and autophagy inhibitor 3MA, whereas 4PBA or 3MA in combination with 2 mM melatonin (or the three together) significantly increased PARP­1 expression. Furthermore, the use of septin7 small interfering RNA confirmed that increased expression of GRP78 and CHOP was related to septin7, and melatonin­â€‹mediated ERS was necessary for septin7 activation. These findings suggest that ERS and autophagy might occur in the early stage of treatment with a high concentration of melatonin, and each might play a protective role in promoting survival; in a later stage, ERS and autophagy might interact and contribute to the induction of apoptosis. Overall, the results indicated that septin7 may be a target protein of melatonin­induced ERS.

A SEPT1-based scaffold is required for Golgi integrity and function.

Compartmentalization of membrane transport and signaling processes is of pivotal importance to eukaryotic cell function. While plasma membrane compartmentalization and dynamics are well known to depend on the scaffolding function of septin GTPases, the roles of septins at intracellular membranes have remained largely elusive. Here, we show that the structural and functional integrity of the Golgi depends on its association with a septin 1 (SEPT1)-based scaffold, which promotes local microtubule nucleation and positioning of the Golgi. SEPT1 function depends on the Golgi matrix protein GM130 (also known as GOLGA2) and on centrosomal proteins, including CEP170 and components of γ-tubulin ring complex (γ-Turc), to facilitate the perinuclear concentration of Golgi membranes. Accordingly, SEPT1 depletion triggers a massive fragmentation of the Golgi ribbon, thereby compromising anterograde membrane traffic at the level of the Golgi.

A Septin Cytoskeleton-Targeting Small Molecule, Forchlorfenuron, Inhibits Epithelial Migration via Septin-Independent Perturbation of Cellular Signaling.

Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.

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.

Repression of Septin9 and Septin2 suppresses tumor growth of human glioblastoma cells.

Glioblastoma (GBM) is the most common primary malignancy of the central nervous system (CNS) with <10% 5-year survival rate. The growth and invasion of GBM cells into normal brain make the resection and treatment difficult. A better understanding of the biology of GBM cells is crucial to the targeted therapies for the disease. In this study, we identified Septin9 (SEPT9) and Septin2 (SEPT2) as GBM-related genes through integrated multi-omics analysis across independent transcriptomic and proteomic studies. Further studies revealed that expression of SEPT9 and SEPT2 was elevated in glioma tissues and cell lines (A172, U87-MG). Knockdown of SEPT9 and SEPT2 in A172/U87-MG was able to inhibit GBM cell proliferation and arrest cell cycle progression in the S phase in a synergistic mechanism. Moreover, suppression of SEPT9 and SEPT2 decreased the GBM cell invasive capability and significantly impaired the growth of glioma xenografts in nude mice. Furthermore, the decrease in GBM cell growth caused by SEPT9 and SEPT2 RNAi appears to involve two parallel signaling pathway including the p53/p21 axis and MEK/ERK activation. Together, our integration of multi-omics analysis has revealed previously unrecognized synergistic role of SEPT9 and SEPT2 in GBM, and provided novel insights into the targeted therapy of GBM.

Overexpression of septin-7 inhibits melatonin-induced cell apoptosis in human fetal osteoblastic cells via suppression of endoplasmic reticulum stress.

Our previous study demonstrated that melatonin could induce apoptosis in the human fetal osteoblastic (hFOB) 1.19 cell line via induction of endoplasmic reticulum stress (ERS), and recent studies have demonstrated that the expression of septin­7 (SEPT7) exhibits a positive correlation with the concentration of melatonin. Western blotting demonstrated the expression level of SEPT7 was significantly upregulated in a dose­dependent manner following treatment with differing concentrations of melatonin compared with the control groups, which did not receive any treatment. The expression of proteins associated with cell apoptosis and endoplasmic reticulum stress (ERS; pro-caspase­3, cleaved caspase­3, C/EBP­homologous protein, 78 kDa glucose­regulated protein and phosphorylated­eukaryotic translation initiation factor 2α) were decreased following transfection with SEPT7 overexpression plasmid and increased following transfection with SEPT7 small interfering RNA compared with the control groups. The results of the present study suggest that SEPT7 inhibits melatonin­induced cell apoptosis via suppression of ERS.

The requirement of SEPT2 and SEPT7 for migration and invasion in human breast cancer via MEK/ERK activation.

Septins are a novel class of GTP-binding cytoskeletal proteins evolutionarily conserved from yeast to mammals and have now been found to play a contributing role in a broad range of tumor types. However, their functional importance in breast cancer remains largely unclear. Here, we demonstrated that pharmaceutical inhibition of global septin dynamics would greatly suppress proliferation, migration and invasiveness in breast cancer cell lines. We then examined the expression and subcellular distribution of the selected septins SEPT2 and SEPT7 in breast cancer cells, revealing a rather variable localization of the two proteins with cell cycle progression. To determine the role of both septins in mediating malignant behavior of cancer cells, we used RNA silencing to specifically deplete endogenous SEPT2 or SEPT7 in highly invasive breast cancer cell line MDA-MB-231. Our findings showed that SEPT2/7 depletion had virtually identical inhibitory effects on cellular proliferation, apoptosis, migration and invasion. Moreover, the opposite performance in migration and invasion was observed after enforced expression of SEPT2/7 in the same cell line. We further demonstrated MEK/ERK activation, but not other MAPKs and AKT, was positively correlated with the protein levels of SEPT2 and SEPT7. Additionally, in SEPT2/7-overexpressing cells, the MEK specific inhibitor U0126 was able to correct the high active status of MEK/ERK while normalizing the increased invasive behaviors of these cells. Taken together, these results strongly suggest that SEPT2 and SEPT7 are involved in breast carcinogenesis and may serve as valuable therapeutic targets for breast cancer.

Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells.

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies.

Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes.

The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199-206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4(+)) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.

Functional analysis of the SEPT9-ABL1 chimeric fusion gene derived from T-prolymphocytic leukemia.

We analyzed the function of a SEPT9-ABL1 fusion identified in a case of T-prolymphocytic leukemia with tyrosine kinase inhibitor (TKI) resistance. Five isoforms with different N-termini, including SEPT9a-ABL1, SEPT9b-ABL1, SEPT9d-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1, were detected in the leukemic cells. All isoforms except SEPT9d-ABL1 are localized in the cytoplasm, undergo autophosphorylation and phosphorylate the downstream targets, STAT-5 and Crkl, and provided IL-3-independence and in vivo invasiveness to 32D cells. Additionally, these SEPT9-ABL1 isoforms were resistant to TKIs in vitro and in vivo, in comparison to BCR-ABL1. These findings demonstrated that SEPT9-ABL1 had oncogenic activity and conferred resistance to TKIs.

Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins.

Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodelling and scaffolds, and with diverse binding partners. Precise roles for these structural proteins in most processes often remain elusive. Identification of small molecules that inhibit septins could aid in elucidating the functions of septins and has become increasingly important, including the description of roles for septins in pathogenic phenomena such as tumorigenesis. The plant growth regulator forchlorfenuron, a synthetic cytokinin known to inhibit septin dynamics, likely represents an informative probe for septin function. This report deals with septins of the human blood fluke Schistosoma mansoni and their interactions with forchlorfenuron. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with forchlorfenuron, leading to rapid polymerization of filaments. Culturing developmental stages (miracidia, cercariae, adult males) of schistosomes in FCF at 50-500 µM rapidly led to paralysis, which was reversible upon removal of the cytokinin. The reversible paralysis was concentration-, time- and developmental stage-dependent. Effects of forchlorfenuron on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of forchlorfenuron on fluke motility. The findings implicated a mechanism targeting a molecular system controlling movement in these developmental stages: a direct effect on muscle contraction due to septin stabilization might be responsible for the reversible paralysis, since enrichment of septins has been described within the muscles of schistosomes. This study revealed the reversible effect of forchlorfenuron on both schistosome motility and its striking impact in hastening polymerization of septins. These novel findings suggested routes to elucidate roles for septins in this pathogen, and exploitation of derivatives of forchlorfenuron for anti-schistosomal drugs.

Anillin-dependent organization of septin filaments promotes intercellular bridge elongation and Chmp4B targeting to the abscission site.

The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.

Sept6 is required for ciliogenesis in Kupffer's vesicle, the pronephros, and the neural tube during early embryonic development.

Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer's vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated α-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pronephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.

Borg5 is required for angiogenesis by regulating persistent directional migration of the cardiac microvascular endothelial cells.

The microvasculature is important for vertebrate organ development and homeostasis. However, the molecular mechanism of microvascular angiogenesis remains incompletely understood. Through studying Borg5 (Binder of the Rho GTPase 5), which belongs to a family of poorly understood effector proteins of the Cdc42 GTPase, we uncover a role for Borg5 in microvascular angiogenesis. Deletion of Borg5 in mice results in defects in retinal and cardiac microvasculature as well as heart development. Borg5 promotes angiogenesis by regulating persistent directional migration of the endothelial cells (ECs). In primary mouse cardiac ECs (MCECs), Borg5 associates with septins in the perinuclear region and colocalizes with actomyosin fibers. Both Borg5 deletion and septin 7 knockdown lead to a disruption of the perinuclear actomyosin and persistent directional migration. Our findings suggest that Borg5 and septin cytoskeleton spatially control actomyosin activity to ensure persistent directional migration of MCECs and efficient microvascular angiogenesis. Our studies reported here should offer a new avenue to further investigate the functions of Borg5, septin, and actomyosin in the microvasculature in the context of development and disease.

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.

SEPT4 is regulated by the Notch signaling pathway.

Notch receptor-mediated signaling is an evolutionarily conserved pathway that regulates diverse developmental processes and its dysregulation has been implicated in a variety of developmental disorders and cancers. Notch functions in these processes by activating expression of its target genes. Septin 4 (SEPT4) is a polymerizing GTP-binding protein that serves as scaffold for diverse molecules and is involved in cell proliferation and apoptosis. After activation of the Notch signal, the expression of SEPT4 is up-regulated and cell proliferation is inhibited. When the Notch signal is inhibited by the CSL (CBF1/Su(H)/Lag-1)-binding-domain-negative Mastermind-like protein 1, the expression of SEPT4 is down-regulated, proliferation and colony formation of cells are promoted, but cell adhesion ability is decreased. Nevertheless, the SEPT4 expression is not affected after knock-down of CSL. Meanwhile, if SEPT4 activity is inhibited through RNA interference, the protein level and activity of NOTCH1 remains unchanged, but cell proliferation is dysregulated. This indicates that SEPT4 is a Notch target gene. This relationship between Notch signaling pathway and SEPT4 offers a potential basis for further study of developmental control and carcinogenesis.

ARTS-based anticancer therapy: taking aim at cancer stem cells.

Apoptosis related protein in TGF-ß signaling pathway (ARTS/septin 4 isoform 2) hereforth referred to as ARTS, was originally found to promote apoptosis induced by TGF-ß, but later was shown to promote apoptosis induced by a wide variety of apoptotic stimuli. In vivo and in vitro studies revealed that ARTS-induced apoptosis is mainly executed through direct binding and antagonizing XIAP. High levels of XIAP are found in many types of cancers and often correlate with poor prognosis. ARTS was shown to function as a tumor-suppressor protein in human patients and mouse-tumor models. In particular, Septin 4/ARTS-deficient mice have increased tumor susceptibility and contain increased numbers of stem cells (SCs) and progenitor cells, apparently owing to their resistance towards apoptosis. Based on these results we propose that loss of proapoptotic ARTS may act as the 'first hit' initiating tumorigenesis in two distinct ways. First, loss of ARTS-mediated apoptosis leads to increased numbers of normal SCs. Elevated numbers of normal SCs may lead to increased cancer risk due to higher numbers of cellular targets available for transforming mutations. Second, after these SCs acquire additional transforming mutations and become cancer SC (CSCs), they are more likely to survive in the absence of ARTS owing to increased resistance toward apoptosis. A combination of these two mechanisms, over time, is expected to significantly increase tumor risk. Because CSCs appear to share phenotypic markers with normal SCs, targeting the signaling pathways that affect normal SC development and maintenance can serve as a useful approach towards true eradication of cancer. In this article we describe the role of ARTS in apoptosis and cancer, with focus on its potential role as a CSC marker and as a potential target for anticancer and anti-CSC therapy.