Conquering the complex world of human septins: Implications for health and disease

Abstract

Septins are highly conserved filamentous proteins first characterized in budding yeast and subsequently identified in all eukaryotes. Septins can bind and hydrolyze GTP which is intrinsically related to their formation of septin hexamers and functional protein interactions. The human septin family is composed of 14 loci, SEPT1-SEPT14, which encodes dozens of different septin proteins. Their central GTPase and polybasic domain regions are highly conserved but they diverge in their N-terminus and/or C-terminus. The mechanism by which the different isoforms are generated is not yet well understood, but one can hypothesize that the use of different promoters and/or alternative splicing could give rise to these variants.

Septins perform diverse cellular functions according to tissue expression and their interacting partners. Functions identified to date include cell division, chromosome segregation, protein scaffolding, cellular polarity, motility, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, and DNA damage response. Their expression is tightly regulated to maintain proper filament assembly and normal cellular functions. Alteration of these proteins, by mutation or expression changes, has been associated with a variety of cancers and neurological diseases. The association of septins with cancer results from expression alterations in solid tumorsor translocations in leukemias (MLL). Expression changes in septins have also been associated with neurological conditions such as Alzheimer’s and Parkinson’s disease, as well as retinopathies, Hepatitis C, spermatogenesis and Listeria infection. Pathogenic mutations of SEPT9 were identified in the autosomal dominant neurological disorder Hereditary Neuralgic Amyotrophy (HNA).

Human septin research over the past decade has established their importance in cell biology and human disease. Further functional characterization of septins is crucial to our understanding of their possible diagnostic, prognostic, and therapeutic applications.

Introduction

Forty years ago, septin’s pioneer researchers may have never imagined that their discovery of four novel yeast genes would open the door to a biologically complex world of cytoskeletal GTPases that were highly relevant to human disease. Septins were first characterized in Saccharomyces cerevisiae as a group of cytoskeletal GTP-binding proteins essential for proper cell division. Genetic screening performed by Hartwell and colleagues for budding yeast mutants lead to the discovery of the first septin genes, CDC3, CDC10, CDC11 and CDC12, in which temperature sensitive mutations caused defects in budding morphology and cell cycle arrest (1). These genes were further characterized in Saccharomyces pombe in which mutations led to cytokinesis defects during mitotis. Subsequently, septins have been found to be highly conserved in all eukaryotes except plants, and many of the loci encode for different variants and/or protein isoforms (Table 1).

Table 1.

Human septins variants and isoforms per loci^**

Human septin loci	Number of recognized variants/isoforms	Variable region(s)
SEPT1	-	-
SEPT2	4 transcripts/1 isoform	5′UTR and 3′UTR
SEPT3	2 isoforms	3′UTR and C-termini
SEPT4	3 isoforms	5′UTR and 3′UTR N-and C-termini
SEPT5	-	-
SEPT6	4 isoforms	3′UTR C-termini
SEPT7	2 isoforms	alternative splicing exon2
SEPT8	4 isoforms	5′UTR and 3′UTR N-and C-termini
SEPT9	7 transcripts/6 isoforms	5′UTR and 3′UTR alternative splicing of 5′ exons
SEPT10	2 isoforms	alternative splicing exon 2
SEPT11	-	-
SEPT12	2 isoforms	alternative splicing central exon
SEPT13	-	-
SEPT14	-	-

^**Data gathered from NCBI Reference Sequence (Refseq)

Recently, phylogenetic and evolutionary analysis for septins of metazoans demonstrated that all septin proteins could be clustered into four major subgroups (Fig 1). It has been proposed that the emergence of these four subgroups occurred prior to the divergence of vertebrates and invertebrates and that septin expansion in number was due to duplication of pre-existing genes (2). Septin research has grown impressively, from four genes identified in budding yeast to 14 core loci identified in humans to date. The variety of existing septin isoforms and the diversity of their biological functions in eukaryotic cells are both intriguing and exciting. Contributions to septin biology over the last few years include the first crystal structure of septin oligomers, septin protein interactions, expression profiles, associations with well known signaling pathways and involvement in human disease. These observations have set the stage for more exciting experimental strategies to further understand the role of septins in health and disease. Many questions remain related to their multiple physiological functions in cells as related to normal development and health, and their implicated roles in the development of human diseases.

Figure 1. Phylogenetic tree of the human septin family.

A dendrogram tree illustrates the phylogenetic relationship of the human septin family members as determined by Clustal W analysis (modified from Peterson E.A., et al. (2008) Mamm Genome).

Human septin family conserved domains

The four human septin subgroups are characterized by sequence homology and domain composition (Fig 1 and Fig 2). All human septins share a highly conserved central region that contains a polybasic domain and the GTP-binding domain. The N- and C-terminal regions vary in length and amino acid composition. They contain a proline-rich domain and an α-helical coiled-coil domain, respectively. The relevance of the differences in the N- and C- termini of some septins and their isoforms is not completely understood, but these variations might play a role in their unique cellular localizations, protein interactions, and biological functions. While the specific functions of these domains remain to be fully elucidated, recent studies have begun to reveal information about the possible roles of these domains, as described later on. Several members of the human septin family also contain the less well studied proline-rich domain near the N terminal region. Even though there are no specific studies dedicated to understanding the functional role of the proline-rich domain of septins, one can hypothesize that this domain may be essential for protein-protein interactions, including interaction with proteins containing SH3 domains (3, 4). Preceding the GTPase domain, all mammalian septins, including human septins, contain a highly conserved polybasic domain responsible for the association of septins with cellular membranes. Studies of the polybasic domain of SEPT4 have shown that this domain binds to phosphoinositol phosphates (5). The polybasic domain might mediate the targeting of septins to membrane domains relevant for their role as diffusion barriers in yeast and mammalian cells. The Wittinghofer team studied the human septin complex composed of SEPT2, SEPT6 and SEPT7 and found that these three septins form a hexameric complex of two copies of each septin through a GTP/GDP bound interface, which is also crucial for the GTPase activity (6, 7). Future studies of these domains are necessary for comprehensive insight into septins’ intricate functional roles in mammalian cells.

Figure 2. General structure of septins.

N- and C- termini vary between Septin family members and their isoforms. Some septins contain a proline rich domain in their amino terminus that is involved in protein interaction. The C- terminus of some septins consists of a coiled-coil domain predicted to contain a α-helix. The polybasic domain is responsible for the association of septins with the plasma membrane. The central GTP-binding domain contains conserved motifs G1 (GxxxxGK[S/T]), G3 (DxxG) and G4 (xKxD) and is implicated in the formation of septin heteroligomers and protein interaction.

Septins are widely subjected to functionally significant post-translational modifications. For example, sumoylation of yeast septins in G2/M-arrested cells is well described, and the ubiquitin-protein ligase (E3) Siz1p is required for yeast septin sumoylation (8). Sumoylation of mammalian septins has not yet been reported. However, mammalian septins contain multiple phosphorylation sites and they can be phosphorylated by Ser/Thr kinases in post-mitotic neurons (9–11) and by Aurora-B kinase in mitotic cells (12). This suggests that sumoylation and/or phosphorylation of septins might modulate conformational changes that can alter their functional role.

Functional role of septins

Despite the mechanistic differences between budding yeast and dividing animal cells (13), several orthologs of yeast septins were first identified in Caenorhabditis elegans (unc-59 and unc-61), Drosophila melanogaster (pnut) and mammals (SEPT2 (Nedd5) and SEPT4(H5)) (14–17). Similar to yeast septins, animal septins can form polymeric actin-associated filaments, hydrolyze GTP and produce multinucleated cells when mutated (18, 19). Septins can form microfilaments by interacting with each other or with cytoskeletal and filamentous proteins such as actin, myosin and tubulin, indicative of their functional roles in cytokinesis during contractile ring formation, cell morphology changes and dynamic scaffolds (14, 20–24). Several mammalian septins have many interacting partners (Table 2) and form complexes between family members to create filaments important in different cellular functions. One can speculate that some of the diverse functional roles of mammalian septins are intrinsically related to their physical interactions, which might explain how one septin can have multiple functions in different tissues and at different times during cell cycle and development.

Table 2.

Representative interacting partners and functional roles of mammalian septins

Complexes	Putative Functions	Implicated Septin Domain	Reference
SEPT1/Aurora B	Chromosome segregation, Cytokinesis	not determined	Qi et al., 2005
SEPT2/F-Actin	Cytokinesis	GTP binding	Kinoshita et al., 1997
SEPT2/5/6/Anillin	Cytokinesis	not determine	Kinoshita et al., 2002
SEPT2/6/7	Filament formation	C- & N- termini	Low et al., 2006
SEPT2/GLAST	Neurotransmitter release	not determined	Kinoshita et al., 2004
SEPT2/myosin II	Cytokinesis	not determined	Joo et al., 2007
SEPT2/6/CENP-E	Chromosome segregation	not determined	Spiliotis et at., 2005
SEPT2/6/7/MAP4	Microtubule stability	not determined	Kremer et al., 2005
SEPT2/4/7/Sec6/8	Vesicle trafficking	not determined	Hsu et al., 1998
SEPT3/5/7	Neuronal biology	not determined	Fujishuma et al., 2007
SEPT4/5/8	Vesicle Targeting/Exocytosis	N- & C- termini	Martinez et al., 2006
SEPT4/8	Platelet biology	not determined	Blaser et al., 2004
SEPT5/parkin	Parkinson’s pathogenesis	not determined	Choi et al., 2003
SEPT5/11	Exocytosis in endothelial cells	GTP binding & C- termini	Blaser et al., 2006
SEPT5/SNARE complex	Vesicle Targeting/Exocytosis	GTP binding & C- terminal	Beites et al., 2005
SEPT6/12	Filament formation	not determined	Ding et al., 2007
SEPT7/9/11/Actin	Filament formation	N-terminal	Nagata et al., 2004
SEPT7/CENP-E	Chromosome segregation	not determined	Zhu et al., 2008
SEPT8/Vamp2	Snare complex formation Neurotransmitter release	not determined	Ito et al., 2009
SEPT9/Actin	Stress Fiber	not determined	Nagata et al., 2005 González et al., 2007
SEPT9/SA-RhoGEF/Actin	Rho Signaling	N-terminal	Nagata et al., 2005
SEPT9/HIF-1	Cell proliferation, angiogenesis, prostate Cancer	N-terminus GTP binding	Amir et al., 2006 Amir et al., 2009
SEPT9/JNK	Cell proliferation, breast cancer	GTP binding	Gonzalez et al., 2009
SEPT9/Tubulins	Filament formation, microtubules, spindle formation,	GTP binding	Surka et al., 2002 Nagata et al., 2003 González et al., 2007
SEPT11/12	Filament formation	not determined	Ding et al., 2008
SEPT14/SEPT 1-7/9/11-12	Testicular biology	not determined	Peterson et al., 2008

In addition to their roles in cytokinesis, mammalian septins have been associated with other distinct cellular processes including vessicle trafficking, cell polarity, cytoskeletal dynamics, apoptosis, neurodegeneration and oncogenesis. The diversity and complexity of these septin-associated functions is not well understood, but it is believed that a wide range of hetero-oligomeric septin complexes are major players in these cellular processes. For example, SEPT4/ARTS, an alternative transcript of SEPT4, is translocated from the mitochondrion to the nucleus upon exposure to the apoptotic agent TGF-β, possibly providing a mechanistic link between cell division and cell death (25–27). Recent data indicate that several septins, including SEPT2, 4, 6 and 7, co-precipitate with the mammalian sec6/8 complex, suggesting an association of septins with membrane dynamics and vesicle trafficking (28–31). In addition, septins have been implicated in platelet function (SEPT4, 5 and 8) (32–35), cardiac myocyte development (36), chromosome dynamics (SEPT2) (37), cell polarity, motility and microtubule dynamics (SEPT9 and others) (20, 22–24, 38).

In a very elegant study, Kremer et al. showed that a complex of septin proteins (SEPT2, 6 and 7) interacts with SOCS7, which is necessary to retain SOCS7 and NCK in the cytoplasm at a steady state to regulate actin organization and the DNA damage response. The SOCS7/NCK complex accumulates in the nucleus to activate CHK2 and p53 mediated cell cycle arrest upon DNA damage. This action is potentiated by the depletion of the septin complex by siRNA. The spatial distribution of NCK as mediated by the septin complex is important in the reorganization of the actin cytoskeleton, cell polarity and the DNA damage response. One can predict that in vivo the distribution of septins in the cytoplasm is an important mechanism that regulates the localization of many proteins and their site of action in the cell (39).

SEPT2 and SEPT11 are required for phagosome formation which is important for cell membrane dynamics (31). SEPT2 is also required for epithelial cell polarity by associating with tubulin networks and facilitating vesicle transport by preventing polyGlu microtubule tracts from binding to MAP4 (38). SEPT12 has been implicated in mammalian spermatogenesis (40, 41). In addition, SEPT4 and SEPT7 serve as diagnostic markers for human male asthonozoospermia, since healthy individuals show septin expression in the annuli of spermatozoa while infertile patients do not (42). Ihara et al. demonstrated that expression of murine Sept4 is important for the cortical organization required for morphology and motility of the sperm flagellum (43). SEPT2 and SEPT11 are modulators of InlB-mediated invasion by Listeria monocytogenes. SEPT2 is essential for the entry of Listeria to the cells, but SEPT11 expression restricts the efficacy of Listeria invasion. Overall, these findings demonstrate the diverse roles of septins despite their high structural conservation (44–46).

Most recently SEPT14, a novel testis-specific septin was identified and characterized as the newest member of the human septin family. This septin was identified in an effort to find novel interactors of SEPT9 isoforms using a yeast-two hybrid system. SEPT14 maps to 7p11.2 in humans and includes a conserved GTPase domain and a carboxy-terminus coil-coiled domain characteristic of other septins. SEPT14 was found to interact with 10 other members of the human septins and localized with actin stress fibers, a general feature of septin filaments. Expression analyses in many fetal, adult and cancer tissues showed that SEPT14 expression is limited to the normal testes by Northern blot and RT-PCR. Interestingly, SEPT14 expression was negative when tested in cancer cell lines isolated from testis (47).

Septins as microtubule associated proteins

Several studies suggest that septins might be involved in microtubule dynamics and chromosome segregation. Two groups showed that mammalian SEPT2 and SEPT7 may form a mitotic scaffold for CENP-E and other effectors to coordinate chromosome segregation and cytokinesis (37, 48). Kremer et al. proposed a novel molecular function for septins in mammalian cells through the modulation of microtubule dynamics via an interaction with MAP4 (22). Looking specifically at SEPT9 isoforms, SEPT9_v1 (MSFA) was found to localize specifically with microtubules. This localization was required for the completion of cytokinesis and it could be disrupted by nocodazole treatment (24). Interaction of SEPT9_v1 with microtubule networks was found to be essential for septin filament formation (23). We propose that there is a tight regulation between tubulin filaments, microtubules and septins filaments. How this regulation is mediated, and what other proteins are involved remains unclear. Fluorescence microscopy and further characterization of protein interactions might give better insight into septin-tubulin interaction and its regulation of chromosome segregation during mitosis.

Septins and neurological disorders

Possible roles for septins in neurological disorders have emerged based on the brain-specific expression of some septins (Table 3). SEPT2/NEDD5, SEPT1/DIFF6 and SEPT4/H5 have been found to associate with Alzheimer-specific neurofibrillary tangles (5, 49). The SEPT5/CDCREL-1 interaction with Parkin, a pathogenic protein in Parkinson’s disease (50, 51), provides evidence for the involvement of another subset of septins in neuronal development and disease. SEPT4 has also been implicated in brain pathogenesis by its association with cytoplasmic inclusions in Parkinson’s disease and other synocleinopathies (52). The SEPT3/5/7 complex was identified in the mammalian brain and SEPT3 specifically is developmentally regulated and enriched in presynaptic nerve terminals, suggesting a role for these septins in neuronal biology (53, 54). SEPT2 and SEPT8 are associated with neurotransmitter release due to their interaction with the glutamate transporter (Glast) and the synaptic vesicle protein synaptobrevin 2 (Vamp2), respectively (55, 56). Other septin complexes have been associated with myelin formation in the peripheral nervous system (57). Recently, SEPT9 point mutations and a duplication within the gene have been identified in patients with the autosomal dominant neuropathy Hereditary Neuralgic Amyotrophy (HNA) (58–60), further supporting a role for septins in neurological disease. The mechanism by which these mutations are pathogenic in HNA is still not fully elucidated, but McDade et al. showed that isoforms v4 and v4* have distinct 5′ ends encoded by exons where germline mutations of HNA are found. The two mRNAs are translated with different efficiencies and cellular stress can alter this pattern (61). These mutations dramatically enhance the translational efficiency of the v4 5′-UTR, leading to elevated SEPT9_v4 protein under hypoxic conditions (61). These data provide mechanistic insight into the effect of HNA mutations on fine control of SEPT9_v4 protein and its regulation under physiologically relevant conditions. The data are consistent with the episodic and stress-induced nature of the clinical features of HNA (61).

Table 3.

Characteristics of human septin loci and disease associations

Human Septin	Alternative Names	Human Chromosome Location	Expression	Disease Associations
SEPT1	DIFF6	16p11.1	Brain, Lymphocytes, Others	Alzheimer’s disease, Leukemia, Lymphoma
SEPT2	NEDD5, DIFF6	2q37	Brain, Lymphocytes, Others	Brain, Liver and Renal cancer, Von Hippel-Lindau syndrome, Alzheimer’s disease Shigella and Listeria infections
SEPT3	SEP3, G-SEPTIN	22q13.2	Brain-specific	Brain cancer, Alzheimer’s disease
SEPT4	ARTS, H5, PNUTL2, BRADEION, SEP4	17q22	Brain, Testes, Eye, Lymphocytes	Alzheimer’s disease, Skin, Urogenital and Colon cancer, Leukemia, Male infertility (spermatogenesis defect)
SEPT5	CDCREL-1, PNUTL1	22q11.21	Brain, Eye, Platelets	Leukemia, Parkinson’s disease, Schizophrenia, Pancreatic Cancer
SEPT6	SEP2, KIAA0128	Xq24	Ubiquitous	Schizophrenia, Leukemia, Hepatitis C
SEPT7	CDC10	7q36.1	Brain, Testes	Nervous system cancers, Male infertility (spermatogenesis defect)
SEPT8	KIAA0202	5q31	Brain, Retina and others	Retinal Degeneration
SEPT9	MSF, PNUTL4 AF17Q25, OvBrSEPT, SINT1, KIAA0991	17q25	Ubiquitous	Hereditary Neuralgic Amyotrophy, Leukemia, Breast, Ovarian and Colorectal cancers, Head and Neck cancers, classical Hodgkin lymphoma, Shigella and Listeria infections
SEPT10	SEPT1-like	2q13	Ubiquitous	-
SEPT11	FLJ10849	4q21.1	Ubiquitous	Schizophrenia, Bipolar Disorder, Leukemia, Shigella and Listeria infections
SEPT12	FLJ25410	16p13.3	Lymphocytes, Testes	Male Infertility (spermatogenesis defect)
SEPT13	-	7p13	Ubiquitous	-
SEPT14	-	7q11	Testes	-

Septins in cancer

The first evidence that septins may contribute to neoplasia occurred with the discovery of septin SEPT5/CDCREL-1 as a carboxy-terminus fusion partner with mixed lineage leukemia (MLL) in an acute myeloid leukemia (AML) patient with a t(11;22)(q23;q11.2) translocation (62). Subsequently, SEPT9/MSF, SEPT6, SEPT2, and SEPT11 were also identified as fusion partners with MLL in human leukemic cells (63–66). MLL, the human homolog of Drosophila trithorax, is a common translocation partner in leukemias with more than 80 rearrangements with 50 fusion partners identified (67). The well-characterized MLL fusion products produce in-frame translated chimeric proteins associated with phenotypic disease variability such as leukemia-type and prognostic outcome. These carboxy partners, in addition to MLL, appear to be essential contributors to the pathogenesis of leukemia (68).

Five human septins have also been implicated in other types of cancer including SEPT2, SEPT3, SEPT4, SEPT5 and SEPT9 (25, 69, 70). In fact, SEPT4 acts as a tumor suppressor in leukemias and solid tumors. The SEPT4 gene promotes apoptosis, and is lost in 70% of leukemia patients (26), in addition to being associated with inhibition of colorectal cancer tumorigenesis (71). SEPT9 seems to be important not only in leukemias but also in solid tumor cancers (72–77). SEPT2 and SEPT3 were abundantly expressed in several brain tumors and brain tumor cell lines, suggesting that these family members are potential oncogenes (70). SEPT2 phosphorylation by casein kinase 2 was found to be important for hepatoma carcinoma cell proliferation (78). In addition, SEPT2 is the only human septin that has been associated with a specific human cancer predisposition syndrome, Von Hippel-Lindau (VHL). VHL is anautosomal dominant genetic condition characterized by hemangioblastomas in different tissues and renal cell carcinoma. Increased SEPT2 expression is a common event in renal cell carcinomas (78, 79). A proteomic study by Craven et al. showed that SEPT2 was downregulated upon expression of VHL in the UMRC2 renal cell carcinoma line. This suggests that SEPT2 is subject to either direct or indirect VHL regulation(78, 79).

Overall, these studies show that human septins are important in many cellular and molecular functions, as summarized in Fig 3. It seems that their expression needs to be highly regulated and the dynamics with their interaction partners are crucial for their function as either scaffolds for cytoskeletal proteins or signaling GTPases. Mutations or altered expression of these genes can arise by multiple mechanisms that are not well understood. Many septins are strikingly associated with multiple human diseases, primarily cancers and neurological disorders (Table 3 and Fig 3).

Figure 3. Human septins in health and disease.

Human septins are involved in multiple tissue and cellular phenotypes. Alterations in their expression or gene mutations affect these phenotypes, which are mechanistically important in the pathogenesis of many diseases such neurological diseases and cancer.

Human SEPT9 locus

SEPT9, with demonstrated roles in both cancer and neurological disease, provides an illustration of the diversity of human septin loci. Human SEPT9 became of great interest to the septin scientific community after it was found to be an MLL-translocation partner in leukemia (66). Subsequently, SEPT9 was mapped to chromosome 17q25, a region linked to breast and ovarian cancer, by positional cloning to a region of allelic imbalance in breast and ovarian cancers (74, 80–82). This suggested that alterations of this novel septin gene may be important not only leukemia, but also in breast and ovarian cancer. Afterward, it was shown that the SEPT9 locus gives rise to multiple alternative transcripts encoding at least seven annotated isoforms (SEPT9_v1-SEPT9_v7) (74). The SEPT9 locus contains 13 exons and shows high variability at the 5′ and 3′ ends by either alternative splicing of exons or different transcription start sties. The variable exons 1–3 and 12–13 encode alternative translational start and stop sequences and are spliced onto a core of 8 coding exons (Fig 4) (74). These isoforms maintain the general structure of septins with a highly conserved central region containing the polybasic domain and GTP-binding domain (Fig 2). They vary in the 5′-and 3′-untranslated regions (UTR) and at the N- and C- terminus of the protein. Interestingly, the SEPT9_v4 and SEPT9_v4* isoforms encode for the exact same protein, but they differ at their 5′ UTRs, suggesting that these transcripts might be differentially regulated in time or tissue of expression (73, 76, 77). SEPT9_v1 and SEPT9_v3, the longest transcripts, are highly similar except for 25 distinct amino acids at the N- terminus of SEPT9_v1. The biological reason for the presence of multiple SEPT9 transcripts is still not elucidated, but many studies, including expression analyses, show that these isoforms are differentially expressed and may have distinct functional roles in mammalian cells.

Figure 4. SEPT9 genomic structure.

Genomic structure of SEPT9 alternatively spliced transcripts and variants. SEPT9 exhibits 5′ and 3′ alternative spliced and variable exons and is composed of 13 exons in total. Gray boxes indicate common exons and colored boxes indicate variant-specific exons, all drawn to scale. Transcriptional start sites are indicated by (*) and the end of each transcript by (**). The variable exons (colored boxes) encode different translational start (arrow) and stop (⊗) and are spliced into a core of common coding exons shared between variants (gray boxes).

SEPT9 is widely expressed based on ubiquitous adult and fetal transcript expression, although individual isoforms may have tissue specific expression (74). The cellular localization of SEPT9 isoforms is largely cytoplasmic in interphase cells, but the SEPT9_v1 isoform has a bipartite nuclear localization signal that may direct the shuttling of SEPT9_v1 between the nucleus and the cytoplasm. In mitotic cells SEPT9 exhibits a punctate staining pattern located between the separating chromosomes and at the cleavage furrow during telophase (24). SEPT9_v1 is localized to microtubule networks in interphase cells and in mitotic cells it is localized at the mitotic spindle and the bundle of microtubules at the midzone (23). Both studies showed that when SEPT9 is ablated, cells become binucleated, suggesting a role for SEPT9 in cell division via interaction with components of the cytoskeleton such as tubulins.

Since they were discovered, septins had been characterized as cytoskeletal GTPases important in cell division. Mutations of this family of genes in yeast lead to budding defects, cell cycle arrest and cytokinesis defects. In mammals, several septins(eg. SEPT2, SEPT5, SEPT7) also have been implicated in mitosis, specifically in chromosome segregation dynamics and cytokinesis (Table 2). SEPT9 is not an exception. Its localization to microtubules, the cleavage furrow and midbody at different stages in mitosis suggests a possible specific role of SEPT9 isoforms in chromosome segregation and/or completion of cell division by affecting the mitotic spindle assembly, disassembly or dynamics and the cytokinesis process (Fig 5).

Figure 5. SEPT9 functional role in mitosis.

Diagram showing the phases in mitosis in which SEPT9 expression is present. SEPT9 is localized to the microtubules at the midzone during anaphase and at the cleavage furrow in telophase. Its particular localization during mitosis suggests a role in chromosome segregation and cytokinesis.

Intricate human SEPT9 nomenclature

Human septin nomenclature has evolved on the past decade as illustrated by many alternative names as shown in Table 3, making the septin literature difficult to reconcile. SEPT9 is no exception, as it was originally called MLL septin-like fusion (MSF) due to the fact that it was identified as a fusion partner of MLL in leukemias (66). In 2002, a committee of septin researchers developed an official nomenclature system for SEPT9 transcripts (83). This new name was derived from the functional role of these genes in septae formation in yeast. Some research groups published with the old nomenclature while others used their own versions, making progress in SEPT9 research hard to reconcile. New nomenclature was established by the National Center for Biotechnology Information (NCBI) and HUGO Gene Nomenclature Committee (HGNC), but it has yet to be universally accepted by the septin community. These nomenclature changes are depicted in Table 4 by group and year. Throughout this review the nomenclature utilized was established in 2005 by Scott et al. and endorsed at the 2009 International Septin Meeting(76).

Table 4.

Human SEPT9 nomenclature

Kalikin et al.	McIlhatton et al.	Macara et al.	Inagaki et al.	Scott et al.	NCBI	HGNC	NCBI Accession Numbers
2000	2001	2002	2004	2005β	2008	2008
					variant/isoform	isoform	transcript/protein
MSF-A	epsilon	SEPT9_v1	SEPT9a	SEPT9_v1a	SEPT9_v1/SEPT9a	SEPT9_i1	NM_001113491/NP_001106963
-	gamma	-	-	SEPT9_v2a	SEPT9_v2/SEPT9b	SEPT9_i2	NM_001113493/NP_001106965
MSF	alpha	SEPT9	SEPT9b	SEPT9_v3a	SEPT9_v3/SEPT9c	SEPT9_i3	NM_006640/NP_006631
-	-	-	-	-	SEPT9_v4/SEPT9d	SEPT9_i4	NM_001113495/NP_001106967
MSF-B	zeta	SEPT9_v2	SEPT9c	SEPT9_v4*a	SEPT9_v5/SEPT9ea	SEPT9_i5	NM_001113492/NP_001106966
MSF-C	beta	SEPT9_v3	SEPT9c	SEPT9_v4a	SEPT9_v6/SEPT9ea	SEPT9_i6	NM_001113494/NP_001106966
-	delta	SEPT9_v4	-	SEPT9_v5a	SEPT9_v7/SEPT9f	SEPT9_i7	NM_001113496/NP_001106968
-	-	SEPT9_v5	-	SEPT9_v1-5b-c*	-	-	-

^aSEPT9 variant 5 and 6 differ in their 5′ UTR but encode the same protein isoform.

^*SEPT9_v1-5b-c variants differ at the 3′ UTR. No well characterized.

^βCurrent nomenclature in peer reviewed publications

SEPT9 as a translocation partner of MLL in leukemia

Originally, SEPT9 was identified as part of a fusion protein with the myeloid/lymphoid leukemia gene (MLL) in a therapy-induced acute myeloid leukemia (t-AML) patient with a t(11;17)(q23;q25) rearrangement and was named MSF (MLL septin-like fusion; (66)). The translocation codes for an in-frame transcript joining the 5′ of MLL through exon 5 to SEPT9 at the start of exon 3 through the 3′ terminus. A reciprocal transcript was amplified in one patient joining the 5′ of MSF through exon 1 to exon 7 of MLL but was out of frame (66, 84). The fusion protein was composed of the amino protein terminus of MLL, including the nuclear localization signal, the A-T hook DNA binding domain, and the DNA methyltransferase-like DNA binding domain, and a carboxy terminus of SEPT9_v1, including the proline rich domain, the polybasic and GTPase domains. Lost from the carboxy terminus of MLL is the PHD zinc finger protein-protein interaction domain and the SET domain thought to regulate gene expression through chromatin remodeling (66, 84). It has been suggested that MLL is the sole clinical cause in leukemias with 11q23 rearrangements as it is fused to a wide variety of other genes. However, all of the well-characterized MLL fusion products produce in-frame translated chimeric proteins associated with phenotypic disease variability such as leukemia type and prognostic outcome which provide further evidence that proteins at the varying reciprocally translocated chromosomes are essential contributors to the pathogenesis of leukemia (67, 68, 85). Thus, speculation on the contributions of MLL-SEPT9 fusion protein expression to haematopoetic cellular transformation would include potential mislocalization of MSF from the cytoplasm to the nucleus, aberrant expression of MLL target proteins and altered activation of MSF GTPase signaling pathways. This suggests that these carboxy partners, in addition to MLL, are essential contributors to the pathogenesis of leukemia (68) and implicates septin family members in oncogenesis(85).

SEPT9 model of oncogenesis in solid tumors

A compelling example of the role of septins in the oncogenesis of solid tumors was the identification of the MSF/SEPT9 murine ortholog Sint1/Sept9 at a provirus insertion site in SL3-3 MLV-induced lymphomas (86), which demonstrated that this septin locus is a site for oncogenic integration. SEPT9 also maps to a region of allelic imbalance at chromosome 17q25.3 in breast cancer (74) and sporadic ovarian cancer (82). Additonally, there is differential expression of SEPT9 isoforms in tumors of various tissues (21, 77, 87, 88). SEPT9 has been characterized as a candidate gene in head and neck squamous cell carcinomas by a genome-wide screen for methylated genes (89, 90). SEPT9 was also identified as a possible candidate gene for classical Hodgkin lymhoma (cHL) (91). Circulating methylated SEPT9 DNA in plasma was recently identified as a valuable biomarker for minimally invasive detection of colorectal cancer. This led to the development of a new methylation (m)SEPT9 assay which may prove useful in clinical research for the detection of invasive colorectal cancer with a simple blood test from patients (92–94).

Finally, an isoform of SEPT9, SEPT9_v1, might be important as a biomarker for therapeutic resistance of many cancers to microtubule disrupting agents. SEPT9_v1 expression was strongly correlated with susceptibility of a wide range of cancer cells to drugs such as paclitaxel (95). In general, cancer cells with high SEPT9_v1 expression were more resistant to these drugs (95).

SEPT9 isoforms have been associated with signaling pathways relevant to oncogenesis. For example, SEPT9_v1 interacts with HIF1-α preventing its ubiquitination and degradation, thus activating HIF downstream survival genes to promote tumor progression and angiogenesis in prostate cancer cells (72). Also, a new study showed that SEPT9 up-regulates HIF-1 by preventing Rack1-mediated degradation in an oxygen independent manner (96). In addition, a gene expression profile of prostate cancer showed that SEPT9 was over-expressed in tumors with chromosomal fusion between androgen-regulated transmembrane protease serine 2 (TMPRSS2) and v-ets erythroblastocis virus E26 oncogne (ERG). This fusion is associated with a more aggressive clinical phenotype and tumors with TMPRSS2-ERG are regulated by a estrogen-dependent signaling and are considered a distinct molecular subclass (97). The SEPT9_v3 isoform binds SA-RhoGEF, functioning as a scaffold to keep it in an inactive state, thereby inhibiting SA-RhoGEF mediated Rho activation (98, 99). This finding was crucial for the study of septins in cancer because it provided a direct link between septins and signaling proteins involved in cellular functions such as actin cytoskeletal organization, transcriptional activation, tumor invasion, cell morphology, cell motility and cytokinesis.

SEPT9 expression is also altered in many types of cancers including head and neck tumors, ovarian, prostate, and breast cancers and it is over-expressed in a MMTV mouse model of mammary tumorigenesis (20, 72–76, 86, 92). Most recently, Gonzalez et al., showed that preferential expression of SEPT9_v1 in human mammary epithelial cells promotes pro-oncogenic phenotypes. Briefly, ectopic expression of SEPT9_v1 using retroviral constructs in two immortalized human mammary epithelial cells, MCF10A and HPV 4-12, increases cell proliferation, decreases apoptotic response and enhances motility and invasiveness, in addition to present a reminiscent of epithelial to mesenchymal transition (EMT), all hallmarks of oncogenic transformation in epithelial tissues. In addition, knockdown of SEPT9, and specifically SEPT9_v1, via RNA interference assays in two cancer cell lines wth high endogenous levels of SEPT9_v1 (MDA-MB-231 and BT-549) complemented the observations made in the expression model by rescuing tumorigenic phenotypes. Another striking phenotype found was that SEPT9_v1 high expression disrupts proper formation of tubulin microfilaments in interphase cells and increases aneuploidy. This study provides compelling evidence that SEPT9_v1 could drive malignant progression in mammary epithelial cells (20). Another study also by Gonzalez et al., demonstrated that up-regulation of SEPT9_v1 might increases proliferation rates of mammary epithelial cells by stabilizing c-Jun-N-terminal kinase (JNK) proteins, which are involved in cell proliferation and cell cycle progression. High SEPT9_v1 expression also showed to increase JNK kinase activiy and the transcriptional activation of JNK target genes important in cell cycle progression, including cyclin D1(100).

As a result of these studies, SEPT9 is now accepted as a novel cancer associated protein and is emerging as a potential biomarker for diagnosis and chemotherapeutic response in some epithelial cancers.

Conclusion

Since septins were discovered almost 40 years ago, septin research has grown impressively, from four genes identified in budding yeast in the mid 1970’s to 14 members identified in humans today. In yeast, septins were primarily described as filamentous proteins necessary for budding morphology and cell cycle progression by possibly interacting with cytoskeleton components. The variety of septin isoforms and the significant diversity of their functions in eukaryotic cells have proven to be intriguing and exciting. Studies of mammalian septins have been primarily focused on their association with cancer and neurological diseases. However, many other interesting functions are coming to light such as the role of certain septins in bacterial infection (44–46), angiogenesis (72), spermatogenesis (40–43, 101), retinal degeneration (102, 103), platelet-release reaction (32–35, 104), and beyond. Still, questions related to their normal function in cells and to their implication in human diseases are still puzzling. Major fundamental questions still need to be studied including but not limited to: which factors regulate the tissue-specific and temporal-specific expression of these proteins?, why do human cells needs the presence of 14 distinct septins?, or which molecular and/or cellular alterations dictate the involvement of each specific septin in human diseases? Still, many of the contributions in septin biology in the last years including the first crystal structure of septin oligomers, additional protein interactions, expression profiles and associations with signaling pathways and human diseases have set the path to more exciting experimental strategies to understand further their role in sickness and in health.

Abbreviations

MLL

myeloid/lymphoid leukemia gene

HNA

Hereditary Neuralgic Amyotrophy

MSF

MLL septin-like fusion

VHL

von Hippel Lindau

NCBI

National Center for Biotechnology

HGNC

HUGO Gene Nomenclature Committee

MMTV

mouse mammary tumor virus

SEPT

septin

NEDD5

Neural precursor cell expressed, developmentally down regulated 5

CDCREL

cell division control-related protein

DIFF

differentiation protein

PNUTL

peanut-like protein