Biology subjects

Spiliotis, E. T.

Publications and source records attributed to Spiliotis, E. T..

9 recordsLinked to original sources

bioRxiv2026

Microtubule-templated actin assembly by septin9 drives apical expansion of epithelial cells

Cells dynamically regulate the size of their apical domain during epithelial morphogenesis. Multiciliated cells (MCCs) represent an extreme case, undergoing massive apical expansion to accommodate hundreds of basal bodies that generate an array of motile cilia. While actin-generated forces drive this expansion, the molecular mechanisms that govern actin assembly and organization remain poorly understood. We show that in Xenopus, depletion of Septin9 results in failure of filamentous actin assembly, apical domain expansion, and ciliogenesis. Notably, human Septin9 (SEPT9) localizes to cortical microtubules rather than actin filaments. Live-cell imaging and pharmacological studies reveal that SEPT9 and microtubules form a mutually stabilizing scaffold that directs actin assembly. Using isoform-specific depletion and rescue experiments that genetically separate the microtubule from its actin-bound functions of SEPT9, we demonstrate that SEPT9 association with microtubules is required for actin assembly. These results provide the first in vivo demonstration that microtubule-templated actin assembly via Septin9 drives apical surface expansion during epithelial morphogenesis.

cell biology↗

bioRxiv2026

Septin crosstalk with microtubules and actin is regulated by a GSK3-dependent phosphoswitch

Septins are cytoskeletal filaments that associate with the actin and microtubule cytoskeleton, but the mechanisms that govern septin crosstalk and function with these networks are largely unknown. Here, we show that glycogen synthase kinase 3 (GSK3) directly phosphorylates septin-9 (SEPT9), acting as a molecular switch that bidirectionally controls septin distribution between actin and microtubules. We show that GSK3 inhibition redistributes endogenous SEPT9 toward microtubules in multiple cell types. Phosphomimetic mutations at serines 82 and 85 reduce microtubule binding and enhance actin association in cells and in vitro, while phosphonull mutations promote microtubule binding and growth. In primary hippocampal neurons, GSK3{beta} inactivation promotes SEPT9-microtubule association, and phosphomimetic mutations impair asymmetric neurite growth during neuronal polarization. These findings reveal a phosphorylation-dependent mechanism of septin partitioning between actin and microtubules, placing the cytoskeletal functions of septins under the control of GSK3 - a kinase linked to multiple signaling pathways of cell physiology and metabolism. HighlightsO_LIGSK3{beta} phosphorylates SEPT9, and its activity gates septin-cytoskeleton association C_LIO_LIS82/S85 phosphorylation reduce microtubule binding and increase actin localization C_LIO_LIUnphosphorylated SEPT9 binds preferentially to microtubules, promoting their growth C_LIO_LIGSK3{beta} inactivation drives SEPT9 to microtubules to establish neuronal polarity C_LI

cell biology↗

bioRxiv2026

Septins buffer actomyosin forces to protect the nucleus from genotoxic mechanical stress

Invasively migrating cells thread their nucleus through confined interstitial spaces. How cells protect the nucleus from intracellular forces is poorly understood. Here, we show that the septin cytoskeleton buffers the actomyosin forces that power nuclear movement. Septin filaments comprising SEPT9, a septin amplified in breast cancer, align with perinuclear actomyosin cables which exhibit higher tensile stress during 3D confined migration through narrower pores. SEPT9 depletion amplifies actin stress during confined migration and after myosin II hyper-activation in non-migrating cells causing actin and nuclear envelope ruptures. Following confined migration, DNA breaks, nuclear blebs, micronuclei and cell death increase in SEPT9-depleted cells, phenotypes rescued by the oncogenic SEPT9 isoform 1. Clinicogenomic data reveal that SEPT9 amplification associates with lower genomic alteration in aggressive breast tumors and higher patient mortality. We propose that septins are a mechanoprotective element of the cytoskeleton, and SEPT9 amplification enhances tumor cell survival by preventing nuclear damage.

cell biology↗

bioRxiv2023

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 invadopodia formation and the clustering of invadopodia 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. HighlightsO_LIThe oncogenic SEPT9_i1 is enriched in breast cancer invadopodia in 2D and 3D ECM C_LIO_LISEPT9_i1 promotes invadopodia precursor clustering and invadopodia elongation C_LIO_LISEPT9_i1 localizes to the nuclear envelope and reduces nuclear deformability C_LIO_LISEPT9_i1 is required for EGF-induced amplification of juxtanuclear invadopodia C_LI eTOC BlurbInvadopodia promote the invasion of metastatic cancers. The nucleus is a mechanosensory organelle that determines migratory strategies, but how it crosstalks with invadopodia is unknown. Okletey et al show that the oncogenic isoform SEPT9_i1 promotes nuclear envelope stability and the formation of invadopodia at juxtanuclear areas of the plasma membrane.

cell biology↗

bioRxiv2022

Pyramidal neuron morphogenesis requires a septin network that stabilizes filopodia and suppresses lamellipodia during neurite initiation

Pyramidal neurons are the major cell type of the forebrain, consisting of a pyramidally shaped soma with axonal and apicobasal dendritic processes. It is poorly understood how the neuronal soma morphs from a sphere to pyramid, while generating neurites of the proper shape and orientation. Here, we discovered that the spherical somata of immature neurite-less neurons possess a circumferential wreath-like network of septin filaments, which promotes myosin II localization and suppresses Arp2/3 activity at the base of filopodial actin bundles. The septin network facilitates neurite formation by stabilizing nascent filopodia, which mature to neurites, and concomitantly maintains a consolidated soma by suppressing the extension of lamellipodia. We show that this septin function is critical for the morphogenesis and spatial orientation of pyramidal somata and their neurites in vitro and in vivo. Therefore, the somatic septin cytoskeleton provides a key morphogenetic mechanism for neuritogenesis and the development of pyramidal neurons. Highlights- A septin wreath-like network controls the shape of neuronal somata and nascent neurites - Septins promote and suppress filopodial and lamellipodial protrusions, respectively - Septins scaffold myosin II and exclude Arp2/3 at the base of filopodial actin - Development of pyramidally shaped neurons requires septins in vitro and in vivo eTOC SummaryRadler et al report a new morphogenetic mechanism in the development of pyramidal neurons, which is mediated by a septin wreath-like cytoskeleton in the soma of immature spherical neurons. The septin network stabilizes somatic filopodia and suppresses lamellipodia by differentially controlling the localization of myosin II and Arp2/3.

cell biology↗

bioRxiv2022

Septins mediate a microtubule-actin crosstalk that enables actin growth on microtubules

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons (1, 2). Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin (1, 2). Septins are filamentous GTP-binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin and intermediate filaments (3, 4). Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Super-resolution imaging shows that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We show that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings provide the first evidence of septins directly mediating microtubule interactions with actin filaments, and reveal a new mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis. Significance StatementCellular morphogenesis and processes such as cell division and cell migration require the coordination of the actin and microtubule cytoskeletons. Despite its broader physiological significance, actin-microtubule crosstalk is molecularly and mechanistically poorly understood. Actin-microtubule crosstalk has been viewed primarily as the regulation or guidance of microtubule dynamics by actin filaments. Here, we have discovered a new mechanism by which actin filament growth is guided by microtubules. We report that septins, a poorly understood component of the cytoskeleton, can capture and link the polymerizing ends of actin filaments to microtubule polymers. We present evidence that this new septin-mediated mechanism is critical for the morphology of growth cones, which are structures that direct the growth of neuronal axons through actin-microtubule crosstalk.

cell biology↗

bioRxiv2020

Production and analysis of a mammalian septin hetero-octamer complex

The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

biochemistry↗

bioRxiv2019

Septin 2/6/7 complexes tune microtubule plus end growth and EB1 binding in a concentration-dependent manner

Septins are filamentous GTP-binding proteins, which affect microtubule (MT) dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus end growth and elongation, while higher and intermediate concentrations inhibit and pause plus end growth, respectively. We show that SEPT2/6/7 has a 1.5-fold preference for GTP-over GDP-bound MT lattice, and competes with EB1 for binding to GTP{gamma}S-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus end growth and the tracking of plus end-binding proteins, and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment.\n\nHighlight Summary for eTOCKnowledge of septin roles in MT dynamics is poor and confounded by knockdown studies. Here, in vitro reconstitution assays show concentration-dependent effects of SEPT2/6/7 on MT plus end growth, pausing and EB1 tracking. We found that SEPT2/6/7 filaments are potent than actin in pausing MT growth and dissociating EB1 from intersecting plus ends.

cell biology↗

bioRxiv2019

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 signaling and ubiquitination. Notably, 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, signaling 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.

cell biology↗