Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Registered report protocol: Quantitative analysis of septin Cdc10-associated proteome in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. C. neoformans can thrive during infections due to its three main virulence-related characteristics: the ability to grow at host temperature (37°C), formation of carbohydrate capsule, and its ability to produce melanin. C. neoformans strains lacking septin proteins Cdc3 or Cdc12 are viable at 25°C; however, they fail to proliferate at 37°C and are avirulent in the murine model of infection. The basis of septin contribution to growth at host temperature remains unknown. Septins are a family of conserved filament-forming GTPases with roles in cytokinesis and morphogenesis. In the model organism Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher order complex at the mother-bud neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother-bud neck but the septin interacting proteome in this species remains largely unknown. Moreover, it remains possible that septins play other roles important for high temperature stress that are independent of their established role in cytokinesis. Therefore, we propose to perform a global analysis of septin Cdc10 binding partners in C. neoformans, including those that are specific to high temperature stress. This analysis will shed light on the underlying mechanism of survival of this pathogenic yeast during infection and can potentially lead to the discovery of novel drug targets.

Expression and localization of Septin 14 gene and protein in infertile men testis.

An increasing body of data implicates the Septin family in the pathology of several diseases, including male fertility. The objective of this study was to evaluate the gene and protein expression pattern of Septin 14 in the testis tissue of azoospermic men. In addition, Septin 14 localization was also assessed in the sperm. Testicular tissues were obtained from biopsies of non-obstrutive azoospermic men who underwent diagnostic testicular biopsy in Royan institute and were divided into two groups: TESE + with positive result in testicular sperm extraction (with hypospermatogenesis pathology) and TESE- with negative result (included patients with Sertoli cell only syndrome and maturation arrest pathologies). Total RNA and protein was extracted using trizol reagent. Septin 14 gene and protein expression level were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, respectively. The localization of Septin 14 protein was also studied by Immunocytochemistry. The expression of Septin 14 was significantly lower (p < 0. 05) in TESE- group than TESE + in both mRNA and protein levels. The localization of Septin 14 protein was detected in the head to tail of normal sperms with high localization in front of the acrosome and the neck. This is a novel localization report on Septin 14 in sperm. Regarding the presence of this protein in the sperm acrosome and neck, it can be concluded that decreasing of Septin 14 protein expression may be associated with the pathogenesis of male infertility and therefore Septin 14 expression level maybe critical for human spermatogenesis.

Utility of the methylated SEPT9 test for the early detection of colorectal cancer: a systematic review and meta-analysis of diagnostic test accuracy.

Background: Circulating tumour DNA from colorectal cancer (CRC) is a biomarker for early detection of the disease and therefore potentially useful for screening. One such biomarker is the methylated SEPT9 (mSEPT9) gene, which occurs during CRC tumourigenesis. This systematic review and meta-analysis aims to establish the sensitivity, specificity and accuracy of mSEPT9 tests for the early diagnosis of CRC. Methods: A systematic search of the relevant literature was conducted using Medline and Embase databases. Data were extracted from the eligible studies and analysed to estimate pooled sensitivity, specificity and diagnostic test accuracy. Results: Based on 19 studies, the pooled estimates (and 95% CIs) for mSEPT9 to detect CRC were: sensitivity 69% (62-75); specificity 92% (89-95); positive likelihood ratio 9.1 (6.1-13.8); negative likelihood ratio 0.34 (0.27-0.42); diagnostic OR 27 (15-48) and area under the curve 0.89 (0.86-0.91). The test has a positive predictive value of 2.6% and negative predictive value of 99.9% in an average risk population (0.3% CRC prevalence), and 9.5% (positive predictive value) and 99.6% (negative predictive value) in a high-risk population (1.2% CRC prevalence). Conclusion: The mSEPT9 test has high specificity and moderate sensitivity for CRC and is therefore a potential alternative screening method for those declining faecal immunochemical test for occult blood (FIT) or other screening modalities. However, it is limited by its poor diagnostic performance for precancerous lesions (advanced adenomas and polyps) and its relatively high costs, and little is known about its acceptability to those declining to use the FIT.

Biochemical and morphological characterization of SEPT1 in mouse brain.

Septins are a highly conserved family of GTPases which are identified in diverse organisms ranging from yeast to humans. In mammals, nervous tissues abundantly contain septins and associations of septins with neurological disorders such as Alzheimer's disease and Parkinson's disease have been reported. However, roles of septins in the brain development have not been fully understood. In this study, we produced a specific antibody against mouse SEPT1 and carried out biochemical and morphological characterization of SEPT1. When the expression profile of SEPT1 during mouse brain development was analyzed by western blotting, we found that SEPT1 expression began to increase after birth and the increase continued until postnatal day 22. Subcellular fractionation of mouse brain and subsequent western blot analysis revealed the distribution of SEPT1 in synaptic fractions. Immunofluorescent analyses showed the localization of SEPT1 at synapses in primary cultured mouse hippocampal neurons. We also found the distribution of SEPT1 at synapses in mouse brain by immunohistochemistry. These results suggest that SEPT1 participates in various synaptic events such as the signaling, the neurotransmitter release, and the synapse formation/maintenance.

iTRAQ-based proteomic analysis of DMH-induced colorectal cancer in mice reveals the expressions of ß-catenin, decorin, septin-7, and S100A10 expression in 53 cases of human hereditary polyposis colorectal cancer.

PURPOSE: The aim of this study is to explore the roles of ß-catenin, decorin, septin-7, and S100A10 expression in colorectal cancer development. METHODS: Twenty-five BALB/c mice were divided into five groups; four groups were administrated N,N-dimethylhydrazine for 0, 10, 15, and 20 weeks, and one group was administrated normal saline for 20 weeks. The colons were collected for histopathological analysis. Protein samples prepared from the frozen colon tissues of mice treated with N,N-dimethylhydrazine for the different time points were evaluated using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled with the 2D liquid chromatography-tandem mass spectrometry analysis. Based on the proteomic analysis results, immunohistochemical staining of ß-catenin, decorin, septin-7, and S100A10 was performed in paraffin-embedded mice colorectal tissue, and 53 cases of human hereditary polyposis colorectal cancer samples. RESULTS: Colorectal cancer was observed in mice treated with N,N-dimethylhydrazine for 20 weeks, and adenomas were observed in mice subjected to the 10-, and 15-week treatments. Seventy-two differentially expressed proteins were involved in the development of cancer as per the iTRAQ and spectrometry analysis. In normal epithelium, adenoma, and cancer from human hereditary polyposis colorectal cancer, S100A10 expression (c2 = 100.989, P = 0.000) was highest in cancer, whereas decorin (c2 = 12.852, P = 0.002) and septin-7 (c2 = 66.519, P = 0.002) expressions were highest in the normal epithelium, which was confirmed via immunohistochemical staining. CONCLUSIONS: The subcellular localization of ß-catenin and decorin, septin-7, and S100A10 expressions are associated with the development of colorectal cancer in mice after N,N-dimethylhydrazine treatment and in human hereditary polyposis colorectal cancers.

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.

SEPT12-NDC1 Complexes Are Required for Mammalian Spermiogenesis.

Male factor infertility accounts for approximately 50 percent of infertile couples. The male factor-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile sperm, immature sperm, abnormally structured sperm, and sperm with nuclear damage. Our knockout and knock-in mice models demonstrated that SEPTIN12 (SEPT12) is vital for the formation of sperm morphological characteristics during spermiogenesis. In the clinical aspect, mutated SEPT12 in men results in oligozoospermia or teratozoospermia or both. Sperm with mutated SEPT12 revealed abnormal head and tail structures, decreased chromosomal condensation, and nuclear damage. Furthermore, several nuclear or nuclear membrane-related proteins have been identified as SEPT12 interactors through the yeast 2-hybrid system, including NDC1 transmembrane nucleoporin (NDC1). NDC1 is a major nuclear pore protein, and is critical for nuclear pore complex assembly and nuclear morphology maintenance in mammalian cells. Mutated NDC1 cause gametogenesis defects and skeletal malformations in mice, which were detected spontaneously in the A/J strain. In this study, we characterized the functional effects of SEPT12-NDC1 complexes during mammalian spermiogenesis. In mature human spermatozoa, SEPT12 and NDC1 are majorly colocalized in the centrosome regions; however, NDC1 is only slightly co-expressed with SEPT12 at the annulus of the sperm tail. In addition, SEPT12 interacts with NDC1 in the male germ cell line through coimmunoprecipitation. During murine spermiogenesis, we observed that NDC1 was located at the nuclear membrane of spermatids and at the necks of mature spermatozoa. In male germ cell lines, NDC1 overexpression restricted the localization of SEPT12 to the nucleus and repressed the filament formation of SEPT12. In mice sperm with mutated SEPT12, NDC1 dispersed around the manchette region of the sperm head and annulus, compared with concentrating at the sperm neck of wild-type sperm. These results indicate that SEPT12-NDC1 complexes are involved in mammalian spermiogenesis.

Screening for Colorectal Cancer: US Preventive Services Task Force Recommendation Statement.

IMPORTANCE: Colorectal cancer is the second leading cause of cancer death in the United States. In 2016, an estimated 134,000 persons will be diagnosed with the disease, and about 49,000 will die from it. Colorectal cancer is most frequently diagnosed among adults aged 65 to 74 years; the median age at death from colorectal cancer is 68 years. OBJECTIVE: To update the 2008 US Preventive Services Task Force (USPSTF) recommendation on screening for colorectal cancer. EVIDENCE REVIEW: The USPSTF reviewed the evidence on the effectiveness of screening with colonoscopy, flexible sigmoidoscopy, computed tomography colonography, the guaiac-based fecal occult blood test, the fecal immunochemical test, the multitargeted stool DNA test, and the methylated SEPT9 DNA test in reducing the incidence of and mortality from colorectal cancer or all-cause mortality; the harms of these screening tests; and the test performance characteristics of these tests for detecting adenomatous polyps, advanced adenomas based on size, or both, as well as colorectal cancer. The USPSTF also commissioned a comparative modeling study to provide information on optimal starting and stopping ages and screening intervals across the different available screening methods. FINDINGS: The USPSTF concludes with high certainty that screening for colorectal cancer in average-risk, asymptomatic adults aged 50 to 75 years is of substantial net benefit. Multiple screening strategies are available to choose from, with different levels of evidence to support their effectiveness, as well as unique advantages and limitations, although there are no empirical data to demonstrate that any of the reviewed strategies provide a greater net benefit. Screening for colorectal cancer is a substantially underused preventive health strategy in the United States. CONCLUSIONS AND RECOMMENDATIONS: The USPSTF recommends screening for colorectal cancer starting at age 50 years and continuing until age 75 years (A recommendation). The decision to screen for colorectal cancer in adults aged 76 to 85 years should be an individual one, taking into account the patient's overall health and prior screening history (C recommendation).

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases, Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall.

MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains-TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.

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.

Septin 7 immunoexpression in papillary thyroid carcinoma: a preliminary study.

Papillary thyroid carcinoma (PTC) is the most common type among thyroid cancers. The diagnosis of PTC may be challenging when follicular variant (FVPTC) of this disease is present due to the resemblance of nuclear properties of the classical type (CVPTC). However, making use of ancillary molecular markers in the diagnosis of PTC may help. In our study, we aimed to evaluate the SEPT7 protein expression in PTC. A total of 55 paraffin block tissue samples comprising encapsulated FVPTC (FVPTC(e), n=25), and CVPTC (n=15), and benign hyperfunctioning thyroid nodules (HypN, n=15) were used in this study. Nuclear, cytoplasmic, and overall (total) SEPT7 protein expression levels were determined by using immunohistochemistry. Nuclear, cytoplasmic, and overall SEPT7 expressions (p=0.02, p=0.001, p=0.002, respectively) were significantly lower in FVPTC(e) tissues when compared to HypN. In CVPTC group, nuclear expression was significantly lower (p=0.004) while overall and cytoplasmic expressions were not changed (p>0.05). In HypN group, highest nuclear (mean=2.73), cytoplasmic (mean=2.86), and overall (mean=2.86) expression scores were detected. Significantly lower SEPT7 expression in all expressional categories in FVPTC(e) group may be a sign of different molecular signature in this type of tissue.

Mucosal expression of aquaporin 5 and epithelial barrier proteins in chronic rhinosinusitis with and without nasal polyps.

OBJECTIVES: The purpose of this study is to characterize the association between altered epithelial barrier function, represented by changes in histology and differential expression of the mucosal water membrane permeability protein aquaporin 5 (AQP5), and the pathophysiology of chronic refractory sinusitis (CRS) in patients with and without nasal polyposis. STUDY DESIGN: Prospective clinical study. SETTING: Tertiary rhinology referral center. PARTICIPANTS: Sinonasal samples were obtained from seven CRS subjects with nasal polyps (CRSwNP), seven CRS without nasal polyposis (CRSsNP), and five control healthy patients. METHODS: Mucosal membrane changes were evaluated through hematoxylin and eosin staining of the membrane barrier and immunohistochemical staining of AQP5 expression, a membrane channel protein that affects trans-epithelial water permeability and tissue edema. AQP5 expression was confirmed by real-time PCR (rt-PCR) and western blot. Levels of other membrane proteins, including E-cadherin and Septin-2, were also assessed. RESULTS: CRSwNP patients showed substantial histologic evidence of membrane remodeling with increased edema and glandular hyperplasia. The epithelial expression of AQP5 was significantly lower in CRSwNP as compared to CRSsNP or control. There was no significant difference in the expression of E-cadherin and Septin-2. CONCLUSIONS: Collectively, these data suggest that the mucosal epithelial barrier is compromised in the context of CRS (predominantly in CRSwNP) when compared to control and that AQP5 acts as a key tight junction protein in the maintenance of mucosal water homeostasis. We hypothesize that AQP5 plays a possible role in the pathophysiology of mucosal edema and polyp formation.

Septin localization in the dimorphic fungus Paracoccidioides brasiliensis.

Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control.

Modeling vesicle traffic reveals unexpected consequences for Cdc42p-mediated polarity establishment.

BACKGROUND: Polarization in yeast has been proposed to involve a positive feedback loop whereby the polarity regulator Cdc42p orients actin cables, which deliver vesicles carrying Cdc42p to the polarization site. Previous mathematical models treating Cdc42p traffic as a membrane-free flux suggested that directed traffic would polarize Cdc42p, but it remained unclear whether Cdc42p would become polarized without the membrane-free simplifying assumption. RESULTS: We present mathematical models that explicitly consider stochastic vesicle traffic via exocytosis and endocytosis, providing several new insights. Our findings suggest that endocytic cargo influences the timing of vesicle internalization in yeast. Moreover, our models provide quantitative support for the view that integral membrane cargo proteins would become polarized by directed vesicle traffic given the experimentally determined rates of vesicle traffic and diffusion. However, such traffic cannot effectively polarize the more rapidly diffusing Cdc42p in the model without making additional assumptions that seem implausible and lack experimental support. CONCLUSIONS: Our findings suggest that actin-directed vesicle traffic would perturb, rather than reinforce, polarization in yeast.