Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells.

Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary significantly in their N-terminal extensions. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (e.g., nuclei, centrosomes, cilia) and functions such as cell signalling and ubiquitination. The interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear, signalling, and ubiquitinating proteins. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.

A Septin Double Ring Controls the Spatiotemporal Organization of the ESCRT Machinery in Cytokinetic Abscission.

Abscission is the terminal step of mitosis that physically separates two daughter cells [1, 2]. Abscission requires the endocytic sorting complex required for transport (ESCRT), a molecular machinery of multiple subcomplexes (ESCRT-I/II/III) that promotes membrane remodeling and scission [3-5]. Recruitment of ESCRT-I/II complexes to the midbody of telophase cells initiates ESCRT-III assembly into two rings, which subsequently expand into helices and spirals that narrow down to the incipient site of abscission [6-8]. ESCRT-III assembly is highly dynamic and spatiotemporally ordered, but the underlying mechanisms are poorly understood. Here, we report that, after cleavage furrow closure, septins form a membrane-bound double ring that controls the organization and function of ESCRT-III. The septin double ring demarcates the sites of ESCRT-III assembly into rings and disassembles before ESCRT-III rings expand into helices and spirals. We show that septin 9 (SEPT9) depletion, which abrogates abscission, impairs recruitment of VPS25 (ESCRT-II) and CHMP6 (ESCRT-III). Strikingly, ESCRT-III subunits (CHMP4B and CHMP2A/B) accumulate to the midbody, but they are highly disorganized, failing to form symmetric rings and to expand laterally into the cone-shaped helices and spirals of abscission. We found that SEPT9 interacts directly with the ubiquitin E2 variant (UEV) domain of ESCRT-I protein TSG101 through two N-terminal PTAP motifs, which are required for the recruitment of VPS25 and CHMP6, and the spatial organization of ESCRT-III (CHMP4B and CHMP2B) into functional rings. These results reveal that septins function in the ESCRT-I-ESCRT-II-CHMP6 pathway of ESCRT-III assembly and provide a framework for the spatiotemporal control of the ESCRT machinery of cytokinetic abscission.

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.

Novel septin 9 repeat motifs altered in neuralgic amyotrophy bind and bundle microtubules.

Septin 9 (SEPT9) interacts with microtubules (MTs) and is mutated in hereditary neuralgic amyotrophy (HNA), an autosomal-dominant neuropathy. The mechanism of SEPT9 interaction with MTs and the molecular basis of HNA are unknown. Here, we show that the N-terminal domain of SEPT9 contains the novel repeat motifs K/R-x-x-E/D and R/K-R-x-E, which bind and bundle MTs by interacting with the acidic C-terminal tails of ß-tubulin. Alanine scanning mutagenesis revealed that the K/R-R/x-x-E/D motifs pair electrostatically with one another and the tails of ß-tubulin, enabling septin­septin interactions that link MTs together. SEPT9 isoforms lacking repeat motifs or containing the HNA-linked mutation R88W, which maps to the R/K-R-x-E motif, diminished intracellular MT bundling and impaired asymmetric neurite growth in PC-12 cells. Thus, the SEPT9 repeat motifs bind and bundle MTs, and thereby promote asymmetric neurite growth. These results provide the first insight into the mechanism of septin interaction with MTs and the molecular and cellular basis of HNA.

Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons.

Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.

Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia.

Establishment of epithelial polarity requires the reorganization of the microtubule (MT) cytoskeleton from a radial array into a network positioned along the apicobasal axis of the cell. Little is known about the mechanisms that spatially guide the remodeling of MTs during epithelial polarization. Septins are filamentous guanine triphosphatases (GTPases) that associate with MTs, but the function of septins in MT organization and dynamics is poorly understood. In this paper, we show that in polarizing epithelia, septins guide the directionality of MT plus end movement by suppressing MT catastrophe. By enabling persistent MT growth, two spatially distinct populations of septins, perinuclear and peripheral filaments, steer the growth and capture of MT plus ends. This navigation mechanism is essential for the maintenance of perinuclear MT bundles and for the orientation of peripheral MTs as well as for the apicobasal positioning of MTs. Our results suggest that septins provide the directional guidance cues necessary for polarizing the epithelial MT network.