SEPT9: From pan-cancer to lung squamous cell carcinoma.

BACKGROUND: SEPT9 is a pivotal cytoskeletal GTPase that regulates diverse biological processes encompassing mitosis and cytokinesis. While previous studies have implicated SEPT9 in tumorigenesis and development; comprehensive pan-cancer analyses have not been performed. This study aims to systematically explore its role in cancer screening, prognosis, and treatment, addressing this critical gap. METHODS: Gene and protein expression data containing clinical information were obtained from public databases for pan-cancer analyses. Additionally, clinical samples from 90 patients with lung squamous cell carcinoma (LUSC) were used to further experimentally validate the clinical significance of SEPT9. In addition, the molecular docking tool was used to analyze the affinities between SEPT9 protein and drugs. RESULTS: SEPT9 is highly expressed in various cancers, and its aberrant expression correlates with genetic alternations and epigenetic modifications, leading to adverse clinical outcomes. Take LUSC as an example, additional dataset analyses and immunohistochemical experiments further confirm the diagnostic and prognostic values as well as the clinical relevance of the SEPT9 gene and protein. Functional enrichment, single-cell expression, and immune infiltration analyses revealed that SEPT9 promotes malignant tumor progression and modulates the immune microenvironments, enabling patients to benefit from immunotherapy. Moreover, drug sensitivity and molecular docking analyses showed that SEPT9 is associated with the sensitivity and resistance of multiple drugs and has stable binding activity with them, including Vorinostat and OTS-964. To harness its prognostic and therapeutic potential in LUSC, a mitotic spindle-associated prognostic model including SEPT9, HSF1, ARAP3, KIF20B, FAM83D, TUBB8, and several clinical characteristics, was developed. This model not only improves clinical outcome predictions but also reshapes the immune microenvironment, making immunotherapy more effective for LUSC patients. CONCLUSION: This is the first study to systematically analyze the role of SEPT9 in cancers and innovatively apply the mitotic spindle-associated model to LUSC, fully demonstrating its potential as a valuable biomarker for cancer screening and prognosis, and highlighting its application value in promoting immunotherapy and chemotherapy, particularly for LUSC.

SEPT9_i1 and Septin Dynamics in Oncogenesis and Cancer Treatment.

Despite significant advancements in the field of oncology, cancers still pose one of the greatest challenges of modern healthcare. Given the cytoskeleton's pivotal role in regulating mechanisms critical to cancer development, further studies of the cytoskeletal elements could yield new practical applications. Septins represent a group of relatively well-conserved GTP-binding proteins that constitute the fourth component of the cytoskeleton. Septin 9 (SEPT9) has been linked to a diverse spectrum of malignancies and appears to be the most notable septin member in that category. SEPT9 constitutes a biomarker of colorectal cancer (CRC) and has been positively correlated with a high clinical stage in breast cancer, cervical cancer, and head and neck squamous cell carcinoma. SEPT9_i1 represents the most extensively studied isoform of SEPT9, which substantially contributes to carcinogenesis, metastasis, and treatment resistance. Nevertheless, the mechanistic basis of SEPT9_i1 oncogenicity remains to be fully elucidated. In this review, we highlight SEPT9's and SEPT9_i1's structures and interactions with Hypoxia Inducible Factor α (HIF-1 α) and C-Jun N-Terminal Kinase (JNK), as well as discuss SEPT9_i1's contribution to aneuploidy, cell invasiveness, and taxane resistance-key phenomena in the progression of malignancies. Finally, we emphasize forchlorfenuron and other septin inhibitors as potential chemotherapeutics and migrastatics.

Predictive nomogram of the clinical outcomes of colorectal cancer based on methylated SEPT9 and intratumoral IL-10+ Tregs infiltration.

BACKGROUND: Gene methylation and the immune-related tumor microenvironment (TME) are highly correlated in tumor progression and therapeutic efficacy. Although both of them can be used to predict the clinical outcomes of colorectal cancer (CRC) patients, their predictive value is still unsatisfactory. Whether a combination risk model comprising these two prediction parameters performs better predictive effectiveness than independent factor is still unclear. Methylated Septin9 (mSEPT9) is an early diagnosis biomarker of CRC, in this study, we aimed to investigate mSEPT9-related biomarkers of immunosuppressive TME and identify the value of the combination risk model in predicting the clinical outcomes of CRC. METHODS: Immunofluorescence staining was performed to clarify the correlation between intratumoral IL-10+ Treg infiltration and mSEPT9 in peripheral blood. Survival time, response to 5-fluorouracil (5-FU)-based chemotherapy and PD-1 blockade, and the probability of recurrence or metastasis were analyzed in study (197 CRC samples) and validation (195 CRC samples) sets to evaluate the efficacy of combination risk model. Potential mechanisms were explored by mRNA sequencing. RESULTS: Hypermethylated SEPT9 in the peripheral blood of patients with CRC (stage I-III, and stage IV with resectable M1) before radical resection was positively correlated with high intratumoral IL-10+ Treg infiltration. The high-risk model revealed poor overall survival and progression-free survival, inferior therapeutic response to 5-FU-based chemotherapy and PD-1 blockade, and high probability of recurrence or metastasis. The underlying mechanisms may be associated with the increase in mSEPT9 and mediation of IL-10 via methionine metabolic reprogramming-induced infiltration of IL-10+ Tregs in the TME, which promotes tumor progression and resistance to 5-FU-based chemotherapy and PD-1 blockade. CONCLUSIONS: The combination risk model of peripheral mSETP9 and intratumoral IL-10+ Treg infiltration in CRC can effectively predict prognosis, responsiveness to 5-FU-based chemotherapy and PD-1 blockade, and the probability of recurrence or metastasis. Therefore, this model can be used for precision treatment of CRC.

Septins promote macrophage pyroptosis by regulating gasdermin D cleavage and ninjurin-1-mediated plasma membrane rupture.

The septin cytoskeleton is primarily known for roles in cell division and host defense against bacterial infection. Despite recent insights, the full breadth of roles for septins in host defense is poorly understood. In macrophages, Shigella induces pyroptosis, a pro-inflammatory form of cell death dependent upon gasdermin D (GSDMD) pores at the plasma membrane and cell surface protein ninjurin-1 (NINJ1) for membrane rupture. Here, we discover that septins promote macrophage pyroptosis induced by lipopolysaccharide (LPS)/nigericin and Shigella infection, but do not affect cytokine expression or release. We observe that septin filaments assemble at the plasma membrane, and cleavage of GSDMD is impaired in septin-depleted cells. We found that septins regulate mitochondrial dynamics and the expression of NINJ1. Using a Shigella-zebrafish infection model, we show that septin-mediated pyroptosis is an in vivo mechanism of infection control. The discovery of septins as a mediator of pyroptosis may inspire innovative anti-bacterial and anti-inflammatory treatments.

Proteomic landscape profiling of primary prostate cancer reveals a 16-protein panel for prognosis prediction.

Prostate cancer (PCa) is the most common malignant tumor in men. Currently, there are few prognosis indicators for predicting PCa outcomes and guiding treatments. Here, we perform comprehensive proteomic profiling of 918 tissue specimens from 306 Chinese patients with PCa using data-independent acquisition mass spectrometry (DIA-MS). We identify over 10,000 proteins and define three molecular subtypes of PCa with significant clinical and proteomic differences. We develop a 16-protein panel that effectively predicts biochemical recurrence (BCR) for patients with PCa, which is validated in six published datasets and one additional 99-biopsy-sample cohort by targeted proteomics. Interestingly, this 16-protein panel effectively predicts BCR across different International Society of Urological Pathology (ISUP) grades and pathological stages and outperforms the D'Amico risk classification system in BCR prediction. Furthermore, double knockout of NUDT5 and SEPTIN8, two components from the 16-protein panel, significantly suppresses the PCa cells to proliferate, invade, and migrate, suggesting the combination of NUDT5 and SEPTIN8 may provide new approaches for PCa treatment.

Septin 9 expression regulates 'don't eat me' signals and identifies an immune-epithelial class of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.

The S-Nitrosylation of Septin2 (SNO-Septin2) axis: A novel potential therapeutic target for treating aneurysms and dissection.

Aortic aneurysm and aortic dissection (AAD) are severe life-threatening cardiovascular disorders for which no approved pharmaceutical therapies are currently available. Protein S-nitrosylation (SNO) is a typical redox-dependent posttranslational modification whose role in AAD has yet to be described. Recently, Zhang et al. revealed for the first time that SNO modification of macrophage cytoskeletal protein septin2 promotes vascular inflammation and extracellular matrix degradation in aortic aneurysm. Mechanically, the TIAM1-RAC1(T lymphoma invasion and metastasis-inducing protein 1-Ras-related C3 botulinum toxin substrate 1) axis participates in the progression of AAD induced with S-nitrosylated septin2. More importantly, developing R-ketorolac and NSC23766 compounds that specifically target the TIAM1-RAC1 pathway may be new a potential strategy for alleviating AAD.

The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF.

Septin-coated microtubules promote maturation of multivesicular bodies by inhibiting their motility.

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.

S-Nitrosylation of Septin2 Exacerbates Aortic Aneurysm and Dissection by Coupling the TIAM1-RAC1 Axis in Macrophages.

BACKGROUND: S-Nitrosylation (SNO), a prototypic redox-based posttranslational modification, is involved in cardiovascular disease. Aortic aneurysm and dissection are high-risk cardiovascular diseases without an effective cure. The aim of this study was to determine the role of SNO of Septin2 in macrophages in aortic aneurysm and dissection. METHODS: Biotin-switch assay combined with liquid chromatography-tandem mass spectrometry was performed to identify the S-nitrosylated proteins in aortic tissue from both patients undergoing surgery for aortic dissection and Apoe-/- mice infused with angiotensin II. Angiotensin II-induced aortic aneurysm model and ß-aminopropionitrile-induced aortic aneurysm and dissection model were used to determine the role of SNO of Septin2 (SNO-Septin2) in aortic aneurysm and dissection development. RNA-sequencing analysis was performed to recapitulate possible changes in the transcriptome profile of SNO-Septin2 in macrophages in aortic aneurysm and dissection. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation were used to uncover the TIAM1-RAC1 (Ras-related C3 botulinum toxin substrate 1) axis as the downstream target of SNO-Septin2. Both R-Ketorolac and NSC23766 treatments were used to inhibit the TIAM1-RAC1 axis. RESULTS: Septin2 was identified S-nitrosylated at cysteine 111 (Cys111) in both aortic tissue from patients undergoing surgery for aortic dissection and Apoe-/- mice infused with Angiotensin II. SNO-Septin2 was demonstrated driving the development of aortic aneurysm and dissection. By RNA-sequencing, SNO-Septin2 in macrophages was demonstrated to exacerbate vascular inflammation and extracellular matrix degradation in aortic aneurysm. Next, TIAM1 (T lymphoma invasion and metastasis-inducing protein 1) was identified as a SNO-Septin2 target protein. Mechanistically, compared with unmodified Septin2, SNO-Septin2 reduced its interaction with TIAM1 and activated the TIAM1-RAC1 axis and consequent nuclear factor-κB signaling pathway, resulting in stronger inflammation and extracellular matrix degradation mediated by macrophages. Consistently, both R-Ketorolac and NSC23766 treatments protected against aortic aneurysm and dissection by inhibiting the TIAM1-RAC1 axis. CONCLUSIONS: SNO-Septin2 drives aortic aneurysm and dissection through coupling the TIAM1-RAC1 axis in macrophages and activating the nuclear factor-κB signaling pathway-dependent inflammation and extracellular matrix degradation. Pharmacological blockade of RAC1 by R-Ketorolac or NSC23766 may therefore represent a potential treatment against aortic aneurysm and dissection.

External quality assessment for detection of colorectal cancer by Septin9 DNA methylation in clinical laboratories.

BACKGROUND AND AIMS: The incidence and mortality rate of colorectal cancer (CRC) are increasing worldwide. Septin9 methylated (mSEPT9) DNA in circulation can be used as a non-invasive detection method to assist in the early diagnosis of CRC; however, the detection methods and procedures are complicated. This study aimed to evaluate the ability of clinical laboratories to detect Septin9 methylation in plasma cell-free DNA (cfDNA). MATERIALS AND METHODS: We prepared a sample panel consisting of positive and negative Septin9 methylation cells and CRC cells. Three positive samples with different methylation levels, one negative sample and one duplicate sample, two samples containing interference, three different CRC cell samples, and a fictitious case report were included. The panel was distributed to 59 laboratories for mSEPT9 analysis, result comparison, and scoring. RESULTS: The sample panel, validated by National Medical Products Administration (NMPA)-approved tests and targeted bisulfite sequencing, met expectations and could be used for external quality assessment (EQA). Among the 59 laboratories, 55 (93.22%) correctly reported the mSEPT9 results for all samples, while four (6.79%) reported 15 false negatives and were considered improvable. All false negatives originated from four laboratories using laboratory-developed tests (LDTs), with three failing to detect weakly positive samples, samples containing interference, and samples from different CRC cells, and one reported erroneous results on all positive samples. CONCLUSION: Our results illustrated that the detection of mSEPT9 in cfDNA is satisfactory in China. EQA is indispensable because it can help improve the diagnostic capability and quality management of the laboratories, and provide suggestions for the problems existing in mSEPT9 detection.

The value of serum methylated septin 9 and carcinoembryonic antigen in efficacy evaluation and follow-up monitoring of colorectal cancer.

This study explored the value of the detection of serum methylated septin 9 (mSEPT9) and carcinoembryonic antigen (CEA) in the auxiliary diagnosis, curative effect evaluation, and follow-up monitoring of colorectal cancer (CRC). The diagnosis and treatment data of 208 CRC patients in the First Affiliated Hospital of Xinjiang Medical University (China) were collected from March 2019 to December 2019, and these patients were followed up. The correlation between serum CEA, mSEPT9 levels, and tumor location and size were analyzed. Serum mSEPT9 and CEA were detected before and after surgery and during follow-up after treatment to analyze the value of mSEPT9 in efficacy evaluation and follow-up monitoring. In 87 patients with CRC patients who underwent surgery, the average size of poorly differentiated tumors was the largest (25.01±14.08 cm2), which was significantly different from that of moderately differentiated tumors (P =0.039). There was a statistically significant difference in serum CEA level among different degrees of differentiation (P=0.018). The level of CEA was relatively low when tumors occurred in the transverse and ascending colon. When the level of CEA was high, negative mSEPT9 suggested that the probability of a tumor occurring in the cecum was high; positive mSEPT9 indicated that the tumor was highly likely to occur in the descending or sigmoid colon. Detection before and after surgery revealed that the level of mSEPT9 may be related to the tumor-bearing state of patients. A Follow-up study also showed that the sensitivity and specificity of mSEPT9 for recurrence and metastasis were 83.3% and 97.7%, respectively, and the sensitivity and specificity of CEA were 61.1% and 89.5%, respectively. The combined detection of mSEPT9 and CEA can indicate the location and size of colorectal cancer, while the detection of serum mSEPT9 may have clinical significance in the efficacy evaluation and follow-up monitoring of colorectal cancer. KEY WORDS: Colorectal Cancer, mSEPT9, Recurrence, Metastasis, CEA.

Reconstituted in vitro systems to reveal the roles and functions of septins.

Septins are essential cytoskeletal proteins involved in key cellular processes and have also been implicated in diseases from cancers to neurodegenerative pathologies. However, they have not been as thoroughly studied as other cytoskeletal proteins. In vivo, septins interact with other cytoskeletal proteins and with the inner plasma membrane. Hence, bottom-up in vitro cell-free assays are well suited to dissect the roles and behavior of septins in a controlled environment. Specifically, in vitro studies have been invaluable in describing the self-assembly of septins into a large diversity of ultrastructures. Given that septins interact specifically with membrane, the details of these septin-membrane interactions have been analyzed using reconstituted lipid systems. In particular, at a membrane, septins are often localized at curvatures of micrometer scale. In that context, in vitro assays have been performed with substrates of varying curvatures (spheres, cylinders or undulated substrates) to probe the sensitivity of septins to membrane curvature. This Review will first present the structural properties of septins in solution and describe the interplay of septins with cytoskeletal partners. We will then discuss how septins interact with biomimetic membranes and induce their reshaping. Finally, we will highlight the curvature sensitivity of septins and how they alter the mechanical properties of membranes.

Methylated Septin9 identified patients with colorectal carcinoma and showed higher sensitivity than conventional biomarkers in detecting tumor.

INTRODUCTION: It is worth noting the limitations in sensitivity of the existing biomarkers carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) in detection of colorectal cancer (CRC). In our study, we address the performance of the liquid biopsy biomarker "methylated septin 9" (mSEPT9) in the detection and disease surveillance of CRC. MATERIALS AND METHODS: The monocentric prospective survey encompassed 120 patients diagnosed with CRC who underwent planned curative resection between December 2018 and December 2020. Blood samples were collected from the participants preoperatively as well as at 7 days, 6 weeks, and 3 months postoperatively. The presence of mSEPT9, CEA, and CA 19-9 was detected using the pro Epi Colon® 2.0 CE test, Elecsys® CEA, and Elecsys® CA19-9 electrochemiluminescence immunoassay, respectively. RESULTS: In the preoperative setting, mSEPT9 demonstrated superior capability in identifying patients with CRC compared to CEA and CA 19-9, with detection rates of 57%, 32%, and 18% respectively. Combining all three biomarkers increased the overall sensitivity to 66% preoperatively. In considering UICC stage and T-status, mSEPT9 exhibited higher sensitivity across all stages in comparison with conventional tumor markers, and 65% of patients with metastases were identified postoperatively through mSEPT9. Tumor recognition after surgery was achieved with the sensitivity of 72% and specificity of 91%. CONCLUSIONS: We recommend using mSEPT9 as a non-invasive diagnostic tool for the ongoing monitoring of patients with CRC. The sensitivity and specificity exhibited by mSEPT9 in recognition of tumor after surgery, highlights its particular potential for monitoring of CRC patients.

Mechanistic model for nuclear migration in hyphae during mitosis.

Saccharomyces cerevisiae and Candida albicans, the two well-known human pathogens, can be found in all three morphologies, i.e., yeast, pseudohyphae, and true hyphae. The cylindrical daughter-bud (germ tube) grows very long for true hyphae, and the cell cycle is delayed compared to the other two morphologies. The place of the nuclear division is specific for true hyphae determined by the position of the septin ring. However, the septin ring can localize anywhere inside the germ tube, unlike the mother-bud junction in budding yeast. Since the nucleus often migrates a long path in the hyphae, the underlying mechanism must be robust for executing mitosis in a timely manner. We explore the mechanism of nuclear migration through hyphae in light of mechanical interactions between astral microtubules and the cell cortex. We report that proper migration through constricted hyphae requires a large dynein pull applied on the astral microtubules from the hyphal cortex. This is achieved when the microtubules frequently slide along the hyphal cortex so that a large population of dyneins actively participate, pulling on them. Simulation shows timely migration when the dyneins from the mother cortex do not participate in pulling on the microtubules. These findings are robust for long migration and positioning of the nucleus in the germ tube at the septin ring.

CFTR and PC2, partners in the primary cilia in autosomal dominant polycystic kidney disease.

Defects in the primary cilium are associated with autosomal dominant polycystic kidney disease (ADPKD). We used a combination of animal models, Western blotting, and confocal microscopy and discovered that CFTR and polycystin 2 (PC2) are both colocalized to the cilium in normal kidneys, with the levels of both being decreased in cystic epithelia. Cilia were longer in CFTR-null mice and in cystic cells in our ADPKD animal models. We examined septin 2, known to play a role in cilia length, to act as a diffusion barrier and to serve as an enhancer of proliferation. We found that septin 2 protein levels were upregulated and colocalized strongly with CFTR in cystic cells. Application of VX-809, the CFTR corrector, restored CFTR and PC2 toward normal in the cilia, decreased the protein levels of septin 2, and drastically reduced septin 2 colocalization with CFTR. Our data suggest that CFTR is present in the cilia and plays a role there, perhaps through its conductance of Cl-. We also postulate that septin 2 is important for localizing CFTR to the apical membrane in cystic epithelia.NEW & NOTEWORTHY CFTR is present in the primary cilia together with polycystin 2 (PC2). Ablation of CFTR makes cilia longer suggesting that CFTR plays a role there, perhaps through its conductance of Cl.

Cyclophilin A Isomerisation of Septin 2 Mediates Abscission during Cytokinesis.

The isomerase activity of Cyclophilin A is important for midbody abscission during cell division, however, to date, midbody substrates remain unknown. In this study, we report that the GTP-binding protein Septin 2 interacts with Cyclophilin A. We highlight a dynamic series of Septin 2 phenotypes at the midbody, previously undescribed in human cells. Furthermore, Cyclophilin A depletion or loss of isomerase activity is sufficient to induce phenotypic Septin 2 defects at the midbody. Structural and molecular analysis reveals that Septin 2 proline 259 is important for interaction with Cyclophilin A. Moreover, an isomerisation-deficient EGFP-Septin 2 proline 259 mutant displays defective midbody localisation and undergoes impaired abscission, which is consistent with data from cells with loss of Cyclophilin A expression or activity. Collectively, these data reveal Septin 2 as a novel interacting partner and isomerase substrate of Cyclophilin A at the midbody that is required for abscission during cytokinesis in cancer cells.

Septin 9 Orients the Apico-Basal Polarity Axis and Controls Plasticity Signals.

The cytoskeleton is a master organizer of the cellular cortex and membrane trafficking and therefore plays a crucial role in apico-basal polarity. Septins form a family of GTPases that assemble into non-polar filaments, which bind to membranes and recruit cytoskeletal elements such as microtubules and actin using their polybasic (PB) domains, to perform their broad biological functions. Nevertheless, the role of septins and the significance of their membrane-binding ability in apico-basal polarity remains under-investigated. Here, using 3D cultures, we demonstrated that septin 9 localizes to the basolateral membrane (BM). Its depletion induces an inverted polarity phenotype, decreasing ß-catenin at BM and increasing transforming growth factor (TGFß) and Epithelial-Mesenchymal Transition (EMT) markers. Similar effects were observed after deleting its two PB domains. The mutant became cytoplasmic and apical. The cysts with an inverted polarity phenotype displayed an invasive phenotype, with src and cortactin accumulating at the peripheral membrane. The inhibition of TGFß-receptor and RhoA rescued the polarized phenotype, although the cysts from overexpressed septin 9 overgrew and presented a filled lumen. Both phenotypes corresponded to tumor features. This suggests that septin 9 expression, along with its assembly through the two PB domains, is essential for establishing and maintaining apico-basal polarity against tumor development.

An oncogenic isoform of septin 9 promotes the formation of juxtanuclear invadopodia by reducing nuclear deformability.

Invadopodia are extracellular matrix (ECM) degrading structures, which promote cancer cell invasion. The nucleus is increasingly viewed as a mechanosensory organelle that determines migratory strategies. However, how the nucleus crosstalks with invadopodia is little known. Here, we report that the oncogenic septin 9 isoform 1 (SEPT9_i1) is a component of breast cancer invadopodia. SEPT9_i1 depletion diminishes invadopodium formation and the clustering of the invadopodium precursor components TKS5 and cortactin. This phenotype is characterized by deformed nuclei and nuclear envelopes with folds and grooves. We show that SEPT9_i1 localizes to the nuclear envelope and juxtanuclear invadopodia. Moreover, exogenous lamin A rescues nuclear morphology and juxtanuclear TKS5 clusters. Importantly, SEPT9_i1 is required for the amplification of juxtanuclear invadopodia, which is induced by the epidermal growth factor. We posit that nuclei of low deformability favor the formation of juxtanuclear invadopodia in a SEPT9_i1-dependent manner, which functions as a tunable mechanism for overcoming ECM impenetrability.