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

Abstract

Septins (SEPTs) are filamentous guanosine-5′-triphosphate (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 modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5′- O-[γ-thio]triphosphate (GTPγS)-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. [Media: see text]

Cellular functions of actin- and microtubule-associated septins

“…It is unclear whether septins exist as oligomers or filamentous polymers on the surface of intracellular actin filaments, but the oligomeric and polymeric states of septins correlate with the in vitro formation of curved and linear bundles, respectively 52 . Hence, the actin-binding, -bending and -crosslinking properties of septins may depend on their oligomeric and polymeric states, which also have differential effects on microtubule dynamics 54,55 .…”

“…In addition, septin-microtubule binding involves the carboxy-terminal tyrosine of a-tubulin and polyglutamylation of the carboxy-terminal tail 109,115 . SEPT9_i1 and SEPT2-SEPT6-SEPT7 complexes associate preferentially with microtubule lattices bound to the GTP analog GMPCPP 54,55 . This is consistent with increased septin-microtubule binding in cells treated with taxol 107,114,116 , which stabilizes microtubules by inducing a GMPCPP-like conformational state 117 .…”

Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

“…Our in vitro reconstitution experiments results are the first one to recapitulate the specific binding of Sept9_i1 octamers to microtubules observed in cells here and by others 40,52 . Of note, although recombinant Sept2-Sept6-Sept7-Sept7-Sept6-Sept2 hexamers were found to interact with microtubules in vitro 58 , this is not consistent with the absence of co-localization of septin hexamers with microtubules, when Sept9 was knocked down in cells 40 (Fig 8a and Supplementary Fig. 1d).…”

Novel Functions of the Septin Cytoskeleton

“…36 SEPT9_i1 splice isoform directly binds to b-tubulin and induces microtubule bundling, 42 whereas the SEPT2/6/7 complex preferentially associates with microtubule plus ends and modulates their dynamics. 43 The functional relevance of these septin-microtubule interactions was highlighted by the findings that either SEPT2, or SEPT7 depletion disrupts different microtubule populations in polarized and migrating epithelial cells. 44,45 It should be noted that the ability of septins to form multiprotein complexes is not limited to their oligomerization and interactions with actin filaments and microtubules.…”