REVIEW

Spatial effects − site-specific regulation of actin and microtubuleorganization by septin GTPasesElias T. Spiliotis

ABSTRACTThe actin and microtubule cytoskeletons comprise a variety ofnetworks with distinct architectures, dynamics and proteincomposition. A fundamental question in eukaryotic cell biology ishow these networks are spatially and temporally controlled, so theyare positioned in the right intracellular places at the right time. Whilesignificant progress has been made in understanding the self-assembly of actin andmicrotubule networks, less is known about howthey are patterned and regulated in a site-specific manner. Inmammalian systems, septins are a large family of GTP-bindingproteins that multimerize into higher-order structures, whichassociate with distinct subsets of actin filaments and microtubules,as well as membranes of specific curvature and lipid composition.Recent studies have shedmore light on how septins interact with actinand microtubules, and raised the possibility that the cytoskeletaltopology of septins is determined by their membrane specificity.Importantly, new functions have emerged for septins regarding thegeneration, maintenance and positioning of cytoskeletal networkswith distinct organization and biochemical makeup. This Reviewpresents new and past findings, and discusses septins as a uniqueregulatory module that instructs the local differentiation andpositioning of distinct actin and microtubule networks.

KEYWORDS: Septins, Actin, Microtubules, Spatial organization andregulation, Actin microtubule patterning, Membrane-cytoskeletoncrosstalk, Rho signaling

IntroductionIn eukaryotic organisms, sub-cellular compartmentalization enablesnumerous metabolic processes to occur optimally with high efficiencyand fidelity (Diekmann and Pereira-Leal, 2013; Gabaldón and Pittis,2015). Inherent to the principle of compartmentalization is a networkof cytoskeletal fibers that positions and coordinates cellular organelles(Bonifacino and Neefjes, 2017; Gurel et al., 2014). Owing to theirstructural polarity, actin filaments and microtubules (MTs) provide aframework that is well-suited for the asymmetric positioning anddirectional movement of membrane compartments (Li and Gundersen,2008). However, actin and MTs are remarkably heterogeneous,consisting of sub-networks that vary in architecture and propertiesdepending on their intracellular location (Sanchez and Feldman, 2017;Subramanian and Kapoor, 2012; Vignaud et al., 2012).Much work has been devoted to deciphering the mechanisms that

control actin and MT organization and dynamics, but less isunderstood about how these cytoskeletal networks differ in theirpattern and region-specificity. Actin filament and MT organization

is regulated by effectors of the Ras, Rho and Ran families of smallGTPases, but additional mechanisms are now thought to maintain thehomeostatic balance of different sub-networks and control their size,density and pattern (Heasman and Ridley, 2008; Malumbres andBarbacid, 2003). New studies suggest that spatial differentiationinvolves the sorting of actin-binding proteins (ABPs) andmicrotubule-associated proteins (MAPs) through competitive andcooperative interactions with each other and their underlyingpolymers (Christensen et al., 2017; Winkelman et al., 2016; Yadavet al., 2014). In parallel, competition for non-polymerized actin and/or tubulinmay limit the size and density of different networks (Suarezand Kovar, 2016). Spatial boundaries and geometrical constrains,which can bias protein diffusion and polymer arrangements, are alsoof crucial importance in the generation and positioning of actinfilaments and MTs with varying architecture (Vignaud et al., 2012).Here, I review and discuss how actin and MTs are regulated byseptins, a family of GTP-binding proteins that assemble into higher-order fibrillar structures, and function as spatial barriers and scaffolds(Caudron and Barral, 2009; Mostowy and Cossart, 2012).

Septins demarcate andmaintain spatially distinct subsets ofactin filamentsOriginally discovered in the budding yeast Saccharomyces cerevisiaeas membrane-associated filaments that affect cell polarity anddivision (Byers and Goetsch, 1976; Hartwell, 1971), septins areGTP-binding proteins that are evolutionarily and structurally relatedto the Ras superfamily of P-loop GTPases (Leipe et al., 2002;Mostowy and Cossart, 2012). In contrast to the Ras GTPases, whichare functionally active as GTP-bound monomers, septins belong to abroader class of G proteins, whose biological functions depend onGTP-dependent dimerization (Gasper et al., 2009). By dimerizing intandem through two alternative interfaces that utilize uniquestructural elements of their GTP-binding pockets, septins assembleinto non-polar oligomeric complexes and filamentous polymers(Sirajuddin et al., 2007). With a slow GTP turnover, septins areinherently more stable than actin filaments and MTs (Bridges et al.,2014; Hagiwara et al., 2011; Hu et al., 2008; Schmidt and Nichols,2004). Thus, septins are suited to form scaffold-like structures thatpersist spatially and temporally, thereby marking distinct cytoskeletaland membrane domains.

Early in the identification and characterization of mammalianseptins (Box 1), septins such as SEPT2, SEPT4, SEPT6, SEPT7 andSEPT9 were reported to colocalize with actin (Joberty et al., 2001;Kinoshita et al., 1997; Surka et al., 2002; Xie et al., 1999). In non-dividing cells, septins localize most prominently to actin stressfibers (SFs), which consist of bundles of linear actin filaments(Calvo et al., 2015; Dolat et al., 2014b; Hanai et al., 2004; Joo et al.,2007; Kinoshita et al., 1997; Kremer et al., 2007; Xie et al., 1999)(Fig. 1A). Although less reported, septins have been observed atlamellipodia, pseudopodia and phagocytic cups, which containbranched actin filaments (Huang et al., 2008; Mizutani et al., 2013;

Drexel University, Department of Biology, Drexel University, Philadelphia,PA 19104, USA.

*Author for correspondence (ets33@drexel.edu)

E.T.S., 0000-0003-1082-9763

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Shankar et al., 2010; Xie et al., 1999). Some septins, however, areclearly excluded from the actin network of ruffling lamellipodia(e.g. SEPT2) while they colocalize with actin SFs (Schmidt andNichols, 2004). In mitotic cells, septins concentrate at the cleavagefurrow and the underlying contractile ring, a network of circularactomyosin bundles (Estey et al., 2010; Joo et al., 2007; Kinoshitaet al., 1997; Kinoshita and Noda, 2001). A growing number ofstudies have shown that septins accumulate at sites of actinformation and/or reorganization induced by bacterial pathogens(Mostowy and Cossart, 2011). Although septins and actin filamentsare structurally inter-dependent – i.e. septin disruption alters actinorganization and vice versa – spatial overlap between septins andactin filaments is mostly only partial (Fig. 1A,B); septins localize tosubsets of actin filaments and/or to discrete domains of individualfilaments (Joo et al., 2007; Kinoshita et al., 2002; Kremer et al.,2007; Schmidt and Nichols, 2004).

The SF network comprises a diversity of actin filaments, whosepositioning is critical to harness and balance the mechanical forcesof adherent and migrating cells (Burridge and Wittchen, 2013).Depending on their intracellular position, actin SFs vary in theirmembrane contacts, contractility and composition of ABPs. Thereare three types of SF: dorsal (also termed radial), transverse arc andventral (Geiger and Yamada, 2011; Tojkander et al., 2012). Dorsal(radial) SFs are non-contractile linear bundles of actin with one endanchored to peripheral focal adhesions (FAs) and the other growingfreely toward the cell center. Transverse arcs are contractile curvedacto-myosin filaments that are oriented parallel to the cell edge andflow retrogradely toward the cell center, without being anchored toFAs. By contrast, ventral SFs transverse cells longitudinally acrossthe nucleus or along the cell edges and both of their ends areanchored to FAs. Formed by annealed actin filaments of the dorsal(radial) and transverse arc networks, the ventral SFs are contractile,transducing forces to and from the extracellular matrix.

Most septins decorate transverse arc and ventral SFs in adiscontinuous manner (Calvo et al., 2015; Dolat et al., 2014b; Hanaiet al., 2004; Joo et al., 2007; Kinoshita et al., 2002; Kinoshita et al.,1997; Nagata et al., 2004; Schmidt and Nichols, 2004; Xie et al.,1999). In migrating epithelia that have undergone partial epithelial-to-mesenchymal transition (EMT), septins (SEPT2, SEPT6, SEPT7and SEPT9) localize at the interface between dorsal and transversearcs (Dolat et al., 2014b) (Fig. 1B). After FA formation, theseseptins accumulate at the non-anchored ends of dorsal (radial) SFs,which contain anti-parallel actin filaments, but are absent from theFA-proximal ends, where actin polymerization generates parallelunipolar actin filaments (Cramer et al., 1997; Dolat et al., 2014b;Geiger and Yamada, 2011). It is unclear whether septins specificallyassociate with anti-parallel actin filaments or whether they areexcluded from ABPs (e.g. fascin) that occupy bundles of parallelactin filaments (Elkhatib et al., 2014). At the flared ends of dorsal(radial) SFs, septins interweave with actin, thereby formingfibrillary cross-brace-like structures that overlap with transversearc filaments, which are more densely coated with septins (Dolatet al., 2014b). Septin cross-braces have also been observed betweenthe ventral SFs of cancer-associated fibroblasts (Calvo et al., 2015).

Septins are crucial for the spatial organization and function of theactin SF network. Septin knockdown results in loss of SFs anddiminished mechanotransduction − as revealed by reduced FAmaturation, and ECM remodeling and contraction (Calvo et al.,2015). In the absence of septins, dorsal (radial) SFs are shorter,sparser and less stable, dissipating quickly after formation, whilethere is a dramatic loss of transverse arc and ventral SFs (Dolat et al.,2014b). On the basis of how transverse arc and ventral SFs areformed, septins appear to be strategically positioned for thecrosslinking of converging actin filaments (Fig. 1C). At the edgeof peripheral lamellae, septins may encounter not only the ends ofdorsal (radial) SFs as they merge with actin filaments of transversearc, but also the dorsal SFs from two opposing focal adhesions asthey connect with transverse arc segments forming a ventral SF(Hotulainen and Lappalainen, 2006; Tojkander et al., 2015).Interestingly, septin depletion or their dissociation from actinaffects the ventral SFs that transverse the cytoplasm, but notperipheral SFs and/or actin filaments that are aligned parallel to thenon-protrusive cell edges (Calvo et al., 2015; Dolat et al., 2014b;Joo et al., 2007; Kinoshita et al., 2002; Kremer et al., 2007). Hence,septins are crucial for the formation and maintenance of spatiallydistinct populations of SFs.

Recent work suggests that septins are also involved in themaintenance of a perinuclear actin network (Verdier-Pinard et al.,

Box 1. The Septin GTPasesMammalian septins comprise a family of thirteen paralogous genes(Kinoshita, 2003b; Pan et al., 2007; Russell and Hall, 2011). On the basisof sequence similarity, septins are assigned into four main groups, i.e.SEPT2 (SEPT1, SEPT2, SEPT4, SEPT5), SEPT3 (SEPT3, SEPT9,SEPT12), SEPT6 (SEPT6, SEPT8, SEPT10, SEPT11, SEPT14) andSEPT7 (SEPT7) (see figure). Driven by the propensity of their GTP-binding domains to homo- and hetero-dimerize, septins assemble in acombinatorial fashion, forming the palindromic apolar octamer SEPT9−SEPT7−SEPT6−SEPT2−SEPT2−SEPT6−SEPT7−SEPT9 that isthought of as the basic unit of septin oligomers and polymers (Kimet al., 2011; Kinoshita, 2003a; Sellin et al., 2011; Sheffield et al., 2003;Sirajuddin et al., 2007). In this octameric complex, septins of the samegroup can substitute one another, increasing compositional andfunctional diversity (Kinoshita, 2003a; Nakahira et al., 2010; Sandrocket al., 2011). Depending on tissue or cell type, levels of expression andpost-translational modifications, septins may follow alternative modes ofassembly forming hetero-hexamers (e.g. SEPT7−SEPT6−SEPT2−SEPT2−SEPT6−SEPT7) that lack the terminal member of theSEPT3 group or complexes with multiple septins of the same group(Dolat et al., 2014a; Sellin et al., 2014). How GTP binding and hydrolysisregulate septin assembly is not fully understood. GTP hydrolysisstabilizes the dimeric interfaces of some septins (Sirajuddin et al.,2009; Zent et al., 2011; Zent and Wittinghofer, 2014). However, septinsof the SEPT6 group are constitutive bound to GTP. New evidencesuggests that the dimeric interface between a GDP- and a GTP-boundseptin is more stable than between twoGDP-bound septins (Weems andMcMurray, 2017). Thus, the nucleotide-bound state of a monomericseptin can determine its septin partner (Weems and McMurray, 2017).Once assembled into oligomers and polymers, it is unclear how septindynamics can be influenced by GTP hydrolysis and/or exchange. Giventhat septin subunits turn over within a filamentous polymer, post-translational modifications and other unknown factors might control thedynamics of mammalian septins (Akhmetova et al., 2015; Alonso et al.,2015; Bridges et al., 2014; Hagiwara et al., 2011; Hernández-Rodríguezand Momany, 2012; Hu et al., 2010; Schmidt and Nichols, 2004).

GTPase PartnerGroup

SEPT2

SEPT3

SEPT6

SEPT7

PB CTENTE

SEPT1,2,4,5

SEPT3,9,12

SEPT6,8,10,11,14

SEPT7

NTE, N-terminal extension; SUE, septin-unique element;CTE, C-terminal extension; PB, polybasic (membrane-binding domain);GTP binding, GTP-binding domain

SUEGTP binding

SEPT6

SEPT7,6

SEPT2,7,3

SEPT6,3

Yes

Yes

Yes

No

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2017). On the ventral side of nuclei, septins form a dense network offilaments, which colocalizes with actin. Surprisingly, overexpressionof a specific isoform of SEPT9 (SEPT9_i2), which suppresses themigration of breast cancer cells, disrupts the integrity of the septinmeshwork and its colocalization with actin (Verdier-Pinard et al.,2017). Overexpression of SEPT9_i2 phenocopies in part thedepletion of the formin FMN2, which nucleates a subnuclear actinnetwork, thereby maintaining nuclear shape, position and integrityduring cell migration (Skau et al., 2016; Verdier-Pinard et al., 2017).Although more studies are needed to determine whether septins areinvolved in the generation and maintenance of this actin network, itappears that specific septin paralogs and/or isoforms are linked tospatially and functionally distinct actin networks.During cell division, septins are involved in the organization of

the actomyosin contractile ring of the cytokinetic cleavage furrow.In adherent mammalian cells, knockdown of some septins (e.g.SEPT2, SEPT7, SEPT11) disrupts the ingression of cleavagefurrows, which then fail to close completely, leading to theformation of binucleated cells (Estey et al., 2010; Joo et al., 2007;Kinoshita et al., 1997; Menon et al., 2014; Spiliotis et al., 2005).However, cells of hematopoietic origin complete cytokinesisindependently of septins, and certain septin isoforms (e.g.SEPT9_i3) do not affect cleavage furrow ingression but functionin cytokinetic abscission (Estey et al., 2010; Menon and Gaestel,2015; Menon et al., 2014). Thus, depending on cell type, septinsmay have different localizations and functions that range frommembrane-coupling and positioning of the actomyosin ring toactivation of myosin II and actin turnover. From studies inDrosophilia melanogaster, it is nevertheless apparent that septinsare crucial for the correct organization of actin within the contractilering (Mavrakis et al., 2014).

Our knowledge of septin localization and function in branchedactin networks is limited. There is scant evidence of septinenrichment in lamellipodia. In squamous carcinoma and melanomacells, SEPT1 and SEPT5 are found in lamellipodia, and required forcell spreading (Mizutani et al., 2013). In dorsal root ganglia neurons,SEPT6 also exhibits a paralog-specific enrichment in patches ofbranched actin that gives rise to axonal filopodia (Hu et al., 2012).Moreover, SEPT6 overexpression enhances lamellipodial actin inMadin-Darby canine kidney (MDCK) epithelia cells (Hu et al.,2012). Furthermore, several septins (e.g. SEPT2, SEPT11) have beenimplicated in the formation of phagocytic cups in macrophages, butwithout affecting actin accumulation (Huang et al., 2008). Inmetastatic human cancer cells, proteomic analysis of pseudopodia,which are enriched with branched actin, yielded SEPT9 as apseudopod-specific protein (Shankar et al., 2010). These data suggestthat specific septin paralogs and complexes are involved in theorganization of branched actin networks, but more work is needed toascertain their exact localization and function.

Septin-based mechanisms of actin organizationChallenging a decade-long posit that septins associate with actinfilaments indirectly, new evidence from in vitro reconstitutionexperiments indicates that septins interact with actin (Dolat et al.,2014b; Mavrakis et al., 2014; Smith et al., 2015). Polymerization ofactin together with recombinant Drosophila Sep1−Sep2−Pnut orhuman SEPT2−SEPT6−SEPT7 complexes results in long, highlycurved and looped actin filaments that are, in patches, coated withseptins (Mavrakis et al., 2014). Interestingly, the linear or curvedmorphology of actin depends on the filamentous state of septins.Actin polymerization in the presence of septin filaments yieldsstraighter SF-like actin bundles that are heavily enriched with septins

A

Actin SEPT7

Actin Septins

Focal adhesion

Dorsal SF

Transversearc SF

Ventral SF

CB

Actin SEPT2 Paxillin

MergedMerged

Key

Fig. 1. The roles of septin in actin SF organization. (A) Confocal images showing a fibroblast cell (NIH 3T3) stained with phalloidin (actin; black and white) andanti-SEPT7 (green). SEPT7 decorates partially a subset of SFs and is largely absent from lammelipodia. Scale bar: 5 µm. (B) Structured illumination super-resolution image shows a peripheral lamellar region from a motile MDCK cell stained with phalloidin (actin; red), anti-SEPT2 (green) and anti-paxillin (white).Arrows point to SEPT2 localization at transverse arc SFs and their junctions with dorsal (radial) SFs. Scale bar: 5 µm. (C) Model of how septins may coordinateactin SF interactions at the peripheral lamellae of motile cells. Septins associate with the distal ends of FA-anchored dorsal (radial) SFs and may mediate theiranchoring to transverse arc SFs (left). In parallel, septins may promote the generation of ventral SFs by annealing transverse arc SFs with the free ends of dorsal(radial) SFs from two opposing FAs (right).

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(Mavrakis et al., 2014). In contrast, circular actin bundles, whichresemble the contractile ring, coincide with the presence of punctateseptin complexes (Mavrakis et al., 2014). Consistent with these invitro data, septin-null mutants alter the organization of the contractilering in cellularizingDrosophila embryos, and contractile rings have apolygonal appearancewith actin filaments that are loosely packed andless parallel to the membrane (Mavrakis et al., 2014). Consequently,the fast phase of membrane ingression is slower (Mavrakis et al.,2014). Taken together, these data indicate that (i) septin complexescross-link and bend actin into curved and circular filaments, and(ii) higher-order septin filaments provide a template for the linearpolymerization of actin.Despite the now-confirmed direct interaction of SEPT2−SEPT6

−SEPT7 complexes with polymerizing actin, initial attempts toreconstitute pre-polymerized phalloidin-stabilized actin filaments withrecombinant SEPT2−SEPT6−SEPT7 were successful only whenactin filaments had been first decorated with anillin, a mitotic proteinthat links the contractile ring to the membrane of the cleavage furrow(Kinoshita et al., 2002; Piekny and Maddox, 2010). As neither fascinnor filamin enables actin coating with SEPT2−SEPT6−SEPT7,anillin emerged as a specific adaptor for septin-actin binding duringmitosis (Kinoshita et al., 2002). Subsequent work indicated that,during interphase, non-muscle myosin IIA functions similarly as anactin-binding adaptor, by interacting directly with SEPT2 through thecoiled-coil domain of its heavy chain (Joo et al., 2007). Notably,overexpression of this myosin IIA domain disrupts septin localizationto cytoplasmic actin SFs, which are significantly reduced (Joo et al.,2007).In contrast to SEPT2−SEPT6−SEPT7, whose binding to actin

requires co-polymerization, SEPT9 can crosslink and bundlepreformed actin filaments (Dolat et al., 2014b; Smith et al., 2015).Moreover, the basic N-terminal domain of SEPT9_i1, a uniquesequence within the septin family, bundles actin on its own (Smithet al., 2015). Electron microscopy and 3D helical reconstruction ofactin filaments in complex with the N-terminal domain of SEPT9revealed three different and mutually exclusive modes of binding(Smith et al., 2015). On individual actin segments, SEPT9 utilizesonly one mode at a time, which is indicative of a highly cooperativeassociation (Smith et al., 2015). Switching between these threemodesof binding may enable SEPT9 to access actin in the presence of otherABPs. However, SEPT9 is predicted to compete with ABPs that havesimilar affinities and modes of binding (Smith et al., 2015). Indeed,SEPT9 competes with the motor domain of myosin V while in itsweaker ATP-bound state and inhibits actin depolymerization bycofilin, which has similar modes of actin-binding as SEPT9 (Smithet al., 2015). Given that SFs are subject to disassembly by myosin IIcontractility and cofilin (Tojkander et al., 2015; Wilson et al., 2010),SEPT9 could protect actin from depolymerization in a site-specificmanner. For example, at the interface of transverse arc and dorsal(radial) SFs, SEPT9 might ensure the integrity of actin filaments asthey adjoin to form ventral SFs (Fig. 1C). It is unclear whether theseroles of SEPT9 antagonize the binding of SEPT2−SEPT6−SEPT7complexes to ABPs, such as myosin II and anillin. But if so, it mightprovide a mechanism to regulate the actin- and ABP-bindingproperties of septin complexes – i.e. the presence of specific septinparalogs or isoforms within a septin complexmight modulate bindingto actin and ABPs.Crosslinking of linear actin filaments by septins points to similar

functions in the organization of branched actin networks that arenucleated by the Arp2/Arp3 complex. Formation of branches thatextend from linear actin occurs preferentially at sites of filamentcurvature and bending (Risca et al., 2012), but it is unclear whether

septins promote or inhibit such branching. However, in vitro bindingexperiments have shown that SEPT6 binds to Arp2/Arp3-nucleatedbranched filamentsmore favorably than to linear actin and localizes totheir branch points (Hu et al., 2012). By contrast, SEPT7 decoratesthe linear segments of branched actin and its binding is not enhancedby Arp2/Arp3 (Hu et al., 2012). In agreement with theseobservations, Arp2 has been identified as a SEPT6-specificinteractor (Nakahira et al., 2010). Moreover, SEPT6 overexpressionincreases the recruitment of cortactin to actin patches andlamellipodia (Hu et al., 2012). Because SEPT6 was found toenhance the transition of actin patches to filopodia, SEPT6 andcortactin appear to synergize in promoting the polymerization and/orstability of branched actin (Hu et al., 2012). Although more work isneeded to test directly whether septins affect Arp2/Arp3-mediatedactin dynamics, septins have been reported to interact directly withthe yeast F-BAR protein Hof1, whose mammalian homolog Pro-Ser-Thr phosphatase-interacting protein 1 (PSTPIP1) scaffolds therecruitment of the Arp2/Arp3 activator Wiskott-Aldrich syndromeprotein (WASP) to the immunological synapse (Badour et al., 2003;Finnigan et al., 2016). Hence, septins might interact with scaffoldsand regulators of the Arp2/Arp3 complex during the formation ofmembrane-bound networks of branched actin.

While increasing evidence suggests that septins influence actinorganization by interacting directly with actin filaments and/orABPs, septins have also been implicated in the regulation of actin bythe Rho-signaling GTPases Cdc42 and RhoA. Septin localizationand function in actin SFs is dependent on Cdc42 activity, and themammalian Borg (binding of Rho GTPases) family of Cdc42effector proteins (Cdc42EPs), which regulates the formation ofhigher-order septin filaments in a Cdc42-dependent manner (Calvoet al., 2015; Farrugia and Calvo, 2017; Joberty et al., 1999, 2001).Experiments with mutants of Cdc42EP3 that abrograte the bindingof septin or actin suggest that Cdc42EP3 functions like a molecularglue, reinforcing the connections between septin and actin filaments(Calvo et al., 2015). Interestingly, Cdc42EP1 is required for themaintenance of a spatially distinct network of parallel actin bundlesthat colocalize with septins on the dorsal side of the nuclei of mousecardiac endothelial cells (Liu et al., 2014).

In addition to this interplay with effectors of Cdc42, SEPT9 hasbeen shown to interact with the Rho guanine nucleotide exchangefactor (GEF) 18 termed SA-RhoGEF (also known as ARHGEF18),and rhotekin (RTKN), a downstream effector of RhoA/C (Ito et al.,2005; Nagata and Inagaki, 2005). SEPT9_i3 binds SA-RhoGEFspecifically through its unique N-terminal sequence and, together,they localize to actin SFs (Nagata and Inagaki, 2005). SEPT9_i3inhibits SA-RhoGEF activity and, thereby, Rho-induced formationof actin fibers (Nagata and Inagaki, 2005). Although SEPT9_i3binds to rhotekin indirectly, it appears to assemble together withrhotekin onto actin SFs in a Rho-dependent manner (Ito et al.,2005). Functionally, rhotekin plays a key role in switching theoutput of RhoA signaling from actomyosin contractility to actinpolymerization (Chen et al., 2013). Although it is unknown whetherseptins are involved in the modulation of actomyosin contractilityby rhotekin, SEPT2 is assumed to provide a scaffold for thephosphorylation of the non-muscle myosin-II light chain by thecitron Rho-interacting kinase (CIT) and the Rho-associated proteinkinase (ROCK) (Joo et al., 2007). During cytokinesis, thisscaffolding function is required for the contraction of theactomyosin ring that drives cleavage furrow ingression (Joo et al.,2007). Therefore, septins not only modulate actin organizationdirectly, but also integrate signals from pathways that regulate actinassembly and contractility.

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Roles of septins in the spatial organization and guidanceof MTsHistorically, the interactions of mammalian septins with MTs havereceived less attention and are less studied than the roles of septins inactin organization. Like actin-bound septins, MT-associated septinslocalize to specific subsets of MTs, rather than decorating the entireMT network uniformly (Fig. 2A). In non-mitotic epithelial cells,septins associate with perinuclear MT bundles, Golgi-emanatingMTs, peripheral focal adhesion-targeted MTs and/or ciliary MTs(Bowen et al., 2011; Fliegauf et al., 2014; Ghossoub et al., 2013;Nagata et al., 2003; Spiliotis et al., 2008). In dividing cells, septinshave been reported to localize to spindle poles, metaphasekinetochore MTs as well as MTs of the central spindle and themidbody (Menon et al., 2014; Nagata et al., 2003; Qi et al., 2005;Spiliotis et al., 2005; Surka et al., 2002; Zhu et al., 2011, 2008).These MT-specific distributions are not identical for all septinparalogs and complexes, and vary between different cell types.Among the 13 septin paralogs, SEPT9 has been shown to bind to

MTs directly (Bai et al., 2013). In cell-free assays, purifiedrecombinant SEPT9 converted pre-polymerizedMTs into elongatedbundles (Bai et al., 2013), indicating that SEPT9 crosslinks MTslaterally in a staggered fashion (Fig. 2B). However, the orientation(parallel versus antiparallel) and spacing of MTs have not beendetermined. Similar to the charged repeat motifs of bona fidemicrotubule-associated proteins (MAPs) (e.g. MAP1, MAP2), theN-terminal tail of SEPT9 contains a series of tetrapeptide motifswith the sequence Lys/Arg-Arg/x-x-Asp/Glu (Bai et al., 2013;Cravchik et al., 1994; Noble et al., 1989). Detailed biochemicalanalysis suggests that the basic residues (Lys/Arg) of these motifsinteract with the acidic amino acids of the C-terminal tail of βII-tubulin (Bai et al., 2013). Concomitantly, electrostatic interactionsbetween the Lys/Arg residues of the di-basic motifs and the Asp/Gluresidues of the mono-basic motifs of SEPT9 are thought to promotehomophilic interactions between the N-terminal tails of SEPT9,resulting in the crosslinking and bundling of SEPT9-coated MTs(Bai et al., 2013). It is unknown whether other septins associate withMTs through similar repeat-motifs, but the N-terminal tail of SEPT9is unique among all septins. Notably, the N-terminal sequence ofSEPT9 is alternatively spliced, resulting in SEPT9 isoforms thatlack MT-binding motifs (SEPT9_i4, SEPT9_i5) or contain uniqueN-terminal sequences upstream of them (SEPT9_i2, SEPT9_i3).The latter appear to alter the MT-binding properties of SEPT9,pointing to autoregulatory interactions and conformations thatpromote or impede the association of Lys/Arg-Arg/x-x-Asp/Glumotifs with MTs (Verdier-Pinard et al., 2017). Hetero-oligomerization of SEPT9 with septins of the SEPT2, SEPT6 andSEPT7 groups may further modulate MT binding and bundling.Post-translational modifications (PTMs) of the C-terminal tails

(CTTs) of α- and β-tubulin bestow a biochemical and functionaldiversity within the MT network, and modulate MT interactionswith MAPs and motor proteins (Janke, 2014; Roll-Mecak, 2015).As SEPT9-MT binding involves the C-terminal tails of β-tubulin,septins could associate with MT subsets that exhibit unique PTMs.In non-polarized MDCK epithelia, juxtanuclear and/or Golgi-proximal septins colocalize with polyglutamylated MTs (Spiliotiset al., 2008). Intriguingly, MT polyglutamylation is reduced uponseptin depletion or overexpression of MAP2, which competes withseptins for MT binding (Spiliotis et al., 2008). In breast cancer cellsthat have developed resistance to theMT-stabilizing drug Paclitaxel,septins associate with polyglutamylated MTs (Froidevaux-Klipfelet al., 2015) (Fig. 2C). In particular, septins interact preferentiallywith polyglutamylated chains of three or more glutamate residues

and provide a scaffold for tubulin tyrosine ligase-like 1 and 11proteins (TTLL1 and TTLL11, respectively), which elongatethe polyglutamylated chains (Froidevaux-Klipfel et al., 2015).Concomitantly, septins scaffold the cytosolic carboxypeptidase 1(AGTPBP1; hereafter referred to as CCP1), which trimspolyglutamylated chains (Froidevaux-Klipfel et al., 2015). Noticethat the C-terminal Tyr residue of α-tubulin is critical for theseseptin roles as detyrosination reduces the binding of septins to MTs(Froidevaux-Klipfel et al., 2015). Although this positive feedbackloop between MT tyrosination, septins and MT polyglutamylation/deglutamylation was found in cells with acquired resistance toPaclitaxel, it is possible that septins have similar roles in normalcells and tissues.

Acetylation of Lys40 of α-tubulin which is positioned in thelumen of MT polymers, is another PTM that is affected by septins(Ageta-Ishihara et al., 2013) (Fig. 2D). Septin knockdown increasesMT acetylation in HeLa cells and primary neurons, indicating a rolein MT deacetylation (Ageta-Ishihara et al., 2013; Kremer et al.,2005). Indeed, cytoplasmic SEPT7 interacts directly with the MTdeacetylase HDAC6 (Ageta-Ishihara et al., 2013). SEPT7 does notaffect the expression or enzymatic activity of HDAC6, but promotesits interaction with soluble non-polymerized α-tubulin (Ageta-Ishihara et al., 2013). It is unknown whether MT-associated septinscan scaffold the interaction of HDAC6 with soluble α-tubulin, butseptins have been reported to colocalize weakly with acetylatedMTs and decorate segments that are less acetylated (Verdier-Pinardet al., 2017). Hence, it might be possible that MT-associated septinsprovide a scaffold for HDAC6 that increases the concentration ofcytoplasmic deacetylated α-tubulin around septin-coated MTs.

As MT-binding proteins, septins have the capacity to regulate MTdynamics. In MDCK epithelia, SEPT2 depletion increases thecatastrophic shrinkage of perinuclear and peripheral MTs, butdecreases the growth rates of only peripheral MTs (Bowen et al.,2011). In primary neurons, MT plus-end growth rates were similarlyreduced by SEPT7 knockdown (Ageta-Ishihara et al., 2013),suggesting that septins suppress MT catastrophe events and promoteMT growth. However, this notion is difficult to reconcile with thefindings that SEPT7-depleted cells exhibit increased MT ‘stability’ asinferred by MT acetylation (Ageta-Ishihara et al., 2013; Kremer et al.,2005), MT resistance to the depolymerizing drug nocodazole (Kremeret al., 2005) and rescue of SEPT7 knockout phenotypes withstathmins, inhibitors of MT polymerization (Menon et al., 2014).Given that septins modulate the interaction of MTs with MAP4 and,possibly, other MAPs, it is plausible that these MAPs alter MTdynamics in the absence of septins (Kremer et al., 2005; Spiliotis et al.,2008). Thus, further studies that utilize in vitro cell-free assays arenecessary to clarify the direct effects of septins on MT dynamics.

Septins also appear to steer MT growth by guiding MT movementto specific intracellular regions and compartments. Two independentstudies have reported that MT plus-ends move along septin-coatedMT bundles (Bowen et al., 2011; Nölke et al., 2016). Strikingly, MTplus-ends dock and turn onto septin-coated MTs, which provide apath for reaching the cell edge (Bowen et al., 2011). Septinknockdown decreases these turning events and significantly altersthe trajectories of MT plus-ends, which become rather entangledinstead of moving vectorially to the cell periphery (Bowen et al.,2011). Consequently, epithelial MTs fail to position correctly in thesubapical cytoplasm or to target FAs (Bowen et al., 2011). Therefore,by localizing to distinct MT subsets and promoting MT growth,septins direct spatial MT growth and MT-MT collisions. Becauseseptins are more stable thanMTs (Hu et al., 2008), theymay provide aspatial ‘memory’ for the organization of the MT cytoskeleton,

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similarly to the function of vimentin intermediate filaments, whichhave been shown to template, i.e. guide, MT growth and orientationin directionally migrating cells (Gan et al., 2016).Spatial guidance of MTs requires interactions with MT plus-end

tracking proteins (+TIPs). Although no septin has been reported totrack or localize specifically to MT plus-ends, it has recently beenshown that the +TIP EB1 (UniProt Q15691) interacts with SEPT2,SEPT6 and SEPT7, and that the EB1-binding kinesin-like proteinKIF17 binds to SEPT9 (Bai et al., 2016; Jaulin and Kreitzer, 2010;Nölke et al., 2016). These interactions provide a starting point tounderstand how MT ends can be captured and guided by septinsassociated with MTs, membranes or actin. Given that septinslocalize to the membrane curvature that is present at the neck andbase of various protrusive structures (e.g. filipodia, dendritic spines;see below), septin interactions with +TIPs could guide MTs to cellprotrusions in different cellular processes (Hu et al., 2012; Tadaet al., 2007; Xie et al., 2007). Indeed, SEPT7 is required for MTentry into the axonal filipodia that give rise to the axon collateralbranches of dorsal root ganglia neurons (Hu et al., 2012). Moreover,septins guide the formation of MT-driven protrusions that arecaused by the pathogen Clostridium difficile in order to enhanceadhesion to host cells (Nölke et al., 2016). Future work may revealsimilar roles of septins in MT targeting to dendritic spines − whichis crucial for synaptic plasticity − and/or to sites of cortical polarity(Dent et al., 2011; Siegrist and Doe, 2007).

The cytoskeletal topology of septinsmight be determined bytheir membrane specificitySeptins preferentially associate with lipid bilayers of positivecurvature of micron-scale and with selective phospholipids that areenriched in specific membrane compartments and domains (Akil

et al., 2016; Bridges et al., 2016; Dolat and Spiliotis, 2016; Steelset al., 2007; Zhang et al., 1999). New evidence suggests that themembrane specificities of septins determine the subsets of actinstructures and MTs that they bind (Fig. 3). In sub-confluent epithelialcells, which lack apicobasal polarity, septin localization to the actinSFs of peripheral lamellae resembles the distribution ofphosphatidylinositide 3,5-bisphosphate [PI(3,5)P2], a phospholipidthat is enriched in late endosomes and lysosomes (Dolat and Spiliotis,2016; Jin et al., 2016). Interestingly, septins colocalizewith both actinfilaments and the membranes of macropinosomes and/or endosomesat a zone between transverse arc and dorsal (radial) SFs (Dolat andSpiliotis, 2016). This septin population is significantly diminishedafter pharmacological inhibition of the phosphoinositide kinasePIKfyve that converts phosphatidylinositide 3-phosphate to PI(3,5)P2 (Dolat and Spiliotis, 2016). In hepatocytes infected with thehepatitis C virus, inhibition of PIKfyve diminishes SEPT9colocalization with MTs and disrupts the accumulation ofperinuclear lipid droplets (Akil et al., 2016). Interestingly,introduction of phosphatidylinositide 5-phosphate into hepatocytesenhances the colocalization of SEPT9 with MTs, which –surprisingly − is dependent on the phosphatidylinositide-bindingdomain of SEPT9 (Akil et al., 2016).

Collectively, these data suggest that the cytoskeletal topology ofseptins is controlled by their membrane-binding properties andspecificities. Phosphoinositide-rich membranes provide a favorableenvironment for the assembly of cytoplasmic proteins includingseptins (Bertin et al., 2010; Yogurtcu and Johnson, 2017 preprint).Thus, a dynamic association with membrane organelles or domainswith a specific lipid composition could result in binding of septins toMTs and actin filaments in the proximity. Alternatively, membrane-bound septins may traffic to distant cytoskeletal regions. In neurons,

Y -E-E-EE-E- Y -E-E-EE-E-

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Fig. 2. Septins crosslink MTs into bundles, and regulate MT polyglutamylation and acetylation. (A) Confocal image shows SEPT9 (red) localization to asubset of MTs (grayscale) in MDCK cells. Scale bar: 10 µm. (B) Illustration of the crosslinking of MTs into elongated bundles by SEPT9-containing complexes (red).The N-terminal MT-binding domain (MTBD) of SEPT9 consists of repeat motifs that interact electrostatically with the C-terminal tails of β-tubulin. (C) Model for theregulation of MT polyglutamylation through MT-associated septins based on recent findings in taxane-resistant cancer cells. (Left) Binding of septins to MTs ispromoted by the C-terminal Tyr residue (Y) of α-tubulin (α) and side-chains of 1−3 Glu residues (E). β, β-tubulin. (Middle) Upon association with MTs, septins arethought to scaffold the recruitment of the enzymes TTLL1 and TTLL11, which elongate the glutamate chains of α-tubulin. (Right) Concomitantly, septins interactwith CCP1, which trims the length of the polyglutamate chains. (D) Deacetylation of cytosolic α-tubulin requires the interaction of HDAC6 with SEPT7. Cytosolicseptin complexes that contain SEPT7 are thought to provide a scaffold for the deacetylation of non-polymerized tubulin.

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for example,MAP6 is palmitoylated and, thereby, integrates into themembranes of secretory vesicles that traffic from the Golgi complexof the cell body to axons, where it associates with MTs upondepalmitoylation (Tortosa et al., 2017); other MAPs, such as tau andMAP1B are also known to associate with membranes (Georgievaet al., 2014; Surridge and Burns, 1994).The preferential association of septins with membranes of

positive curvature and unique phospholipid content is expected toimpact the organization of membrane-bound actin andMTs. Septinslocalize to actin filaments of the plasma membrane skeleton and,thus, could modulate actin-membrane binding and actinpolymerization into linear or branched networks (Hagiwara et al.,2011). Biasing actin organization towards linear bundles with aparallel orientation to the membrane plane might be crucial tosuppress protrusive activity and enhance rigidity at the neck andbase of protrusive structures (e.g. filopodia, dendritic spines), whichare characterized by micrometer-scale positive curvature. It is

unkown whether membrane-bound septins can simultaneouslyinteract with actin or MTs, but several ER- and Golgi-residentmembrane proteins interact with MTs directly or through MT end-binding proteins, raising the possibility that septins are similarlyinvolved in anchoring or organizing MTs at subcellular membranecompartments (Gurel et al., 2014).

Conclusions and future directionsAlthough it has been nearly 20 years since observing for the firsttime that septins are associated with the mammalian cytoskeleton,we are still in the early days of understanding how septins regulatethe organization and functions of actin and MTs. Nevertheless, fourkey points have emerged that lay the framework for future work.First, septins localize to distinct subsets and/or segments of actinfilaments and MTs. In part, this might be due to preferentialassociation with specific membrane compartments and/or domains.Additionally, septins may possess stronger affinity for polymerswith distinct post-translational modifications, orientation (i.e.parallel, anti-parallel) or isotypes of actin or tubulin, and theymight be sterically excluded from others by certain ABPs or MAPs.Second, septins crosslink actin filaments and MTs into bundles.This property appears to be driven by specific septin paralogs andisoforms with actin- and MT-binding domains, and may be furtherinfluenced by the subunit identity of septin complexes. Third,septins are more stable than actin andMTs and, thereby, septins mayprovide a spatial memory for the re-growth of dynamic actinfilaments and MTs at specific intracellular regions. In this regard,septins could serve as landmarks for a regio-specific organizationand guidance of actin and MTs. Finally, actin- and MT-associatedseptins exert scaffolding roles in both positive- and negative-feedback loops that modulate actomyosin polymerization andcontractility, and affect the post-translational modifications of MTs.

Undoubtedly, much remains unknown. At a fundamental level,we need a better understanding of how septins interact with actinand MTs. To that end, the actin- and MT-binding properties ofseptin paralogs and isoforms need to be examined both individuallyand in their heteromeric complexes. In vitro reconstitutionexperiments, super-resolution imaging and 3D EM will providemore clarity on how septins affect the dynamics of actin and MTs,and on whether they are organized as complexes or filaments on thesurface of actin and MTs. Moreover, it is important to determinewhether septins bind to actin and MTs in a mutually exclusivemanner or can mediate crosstalk, which is crucial for many cellularprocesses. Given that septins are present on a subset of actinfilaments and MTs, an obvious but completely unexplored questionis whether septins regulate membrane traffic on these tracks. Couldseptins restrict or promote the transport of certain motors and theircargo? Septins have been reported to interact directly with somekinesin motors (Bai et al., 2016; Zhu et al., 2008), but it is unknownwhether cytoskeleton-bound septins affect the velocity, processivityand directionality of myosins, kinesins and dynein. Growingevidence suggests that kinesin- and dynein-driven transport isspatially controlled by MAPs (Atherton et al., 2013). Thus, septinscould be part of the cellular machinery that directs intracellulartraffic.

In conclusion, septins comprise an intricate network that may beof central importance in the spatial regulation and coordination ofthe actin and MT networks. Akin to the monomeric Rab GTPases,which provide a code for the spatial organization of theendomembrane system, septins may function similarly ingenerating and maintaining actin and MT networks of distinctorganization and composition at specific intracellular areas. With 13

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Fig. 3. The cytoskeletal topology of septins might be determined by theirmembrane-binding properties. (A) Septin accumulation to peripherallamellar regions with transverse arc SFs are in part dependent on PI(3,5)P2-containing endolysosomes. LE, late endosome. (B) Septin association withER-derived phosphatidylinositol 5-phosphate (PI5P)-containing lipid dropletsmay result in septin binding to MT subsets that are spatially proximal to thesemembranes. (C) Septins associate preferentially with plasma membranedomains that have a positive curvature at the micron-scale, e.g. domains thatoutline the neck and base of protrusive structures, such as filopodia,lammelipodia and dendritic spines. At these locations, septins might crosslinkactin filaments and, possibly, capture or guide MTs.

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paralogs and several isoforms, which can heteromerize into amultitude of complexes, a potential septin code will be difficult tocrack. However, on-going and future studies are poised to providemore clarity on how the actin and microtubule cytoskeletons areregulated by the septin GTPases.

AcknowledgementsOmission of any references related to the topic of this Review was unintentionalor due to space limitations. I thank members of my laboratory for comments onthe manuscript, and especially Dr Lee Dolat for his feedback and theimmunofluorescence images, which were taken at Drexel University’s Cell ImagingCenter.

Competing interestsThe author declares no competing or financial interests.

FundingThis work was supported by the National Institute of Health (NIH)/National Instituteof General Medical Sciences (NIGMS) [grant no. GM097664]. Deposited in PMC forrelease after 12 months.

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