Septin 9 Exhibits Polymorphic Binding to F-Actin and Inhibits Myosin and Cofilin Activity

Abstract

Septins are a highly conserved family of proteins in eukaryotes that is recognized as a novel component of the cytoskeleton. Septin 9 (SEPT9) interacts directly with actin filaments and functions as an actin stress fiber cross-linking protein that promotes the maturation of nascent focal adhesions and cell migration. However, the molecular details of how SEPT9 interacts with F-actin remain unknown. Here, we use electron microscopy and image analysis to show that SEPT9 binds to F-actin in a highly polymorphic fashion. We demonstrate that the basic domain (B-domain) of the N-terminal tail of SEPT9 is responsible for actin cross-linking, while the GTP-binding domain (G-domain) does not bundle F-actin. We show that the B-domain of SEPT9 binds to three sites on F-actin, and the two of these sites overlap with the binding regions of myosin and cofilin. SEPT9 inhibits actin-dependent ATPase activity of myosin and competes with the weakly-bound state of myosin for binding to F-actin. At the same time, SEPT9 significantly reduces the extent of F-actin depolymerization by cofilin. Taken together, these data suggest that SEPT9 protects actin filaments from depolymerization by cofilin and myosin, and indicate a mechanism by which SEPT9 could maintain the integrity of growing and contracting actin filaments.

Septin Mutations in Human Cancers

“…As reviewed above, SEPT9 is linked to the molecular mechanisms of proliferation, angiogenesis, cell invasion and resistance to anti-cancer drugs. SEPT9 contains a unique N-terminal extension with a basic domain, which binds microtubules, actin and the angiogenic HIF1α, as well as an acidic proline-rich domain, which may interact with proteins that contain Src homology 3 (SH3) domains (Golan and Mabjeesh, 2013; Diesenberg et al, 2015; Smith et al, 2015; Bai et al, 2016). In addition, the N-terminal domain of SEPT9 is phosphorylated by the cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis (Estey et al, 2013).…”

“…Septins vary mainly in the N- and C-terminal sequences that flank the GTP-binding domain. SEPT7 and septins of the SEPT6 group contain α-helical coiled-coil domains in their C-terminal tails, while SEPT9 contains an elongated N-terminal tail, which is enriched with prolines and interacts with microtubules and actin microfilaments (Bai et al, 2013; Smith et al, 2015). With the exception of the SEPT6 group, all septins contain a polybasic domain, which has been shown to interact with membrane phosphoinositides (Zhang et al, 1999; Casamayor and Snyder, 2003).…”

Cancer-Related Functions and Subcellular Localizations of Septins

“…Septin filaments bind to and stabilize the transverse arc and radial stress fibers in the lamellipodia of migrating cells (Dolat et al, 2014). Such stabilization could involve SEPT9-mediated prevention of actin depolymerisation by myosin and cofilin (Smith et al, 2015). Septins also contribute to the stabilization of nascent focal adhesions (Dolat et al, 2014), which is necessary for their turnover and thus for effective migration.…”

“…Septins have been found to bind actin directly (Mavrakis et al, 2014; Smith et al, 2015). The N-terminal tail of SEPT9 was recently evidenced to cross-link actin filaments by binding to three different sites on F-actin (Smith et al, 2015).…”

“…The N-terminal tail of SEPT9 was recently evidenced to cross-link actin filaments by binding to three different sites on F-actin (Smith et al, 2015). While anillin has been evidenced to bridge septin filaments with actin during mitosis, BORG2 was recently shown to play a similar role in non-dividing cells (Calvo et al, 2015).…”