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

Abstract

Septins are filamentous GTP-binding proteins, which affect microtubule-dependent functions including membrane trafficking and cell division, but their precise role in microtubule dynamics is poorly understood. Here, in vitro reconstitution of microtubule dynamics with SEPT2/6/7 complexes, the minimal subunits of septin heteromers, shows that SEPT2/6/7 stabilizes and pauses microtubule plus ends in their growth phase, and inhibits tracking of the microtubule plus end protein EB1 in a concentration-dependent manner. We show that SEPT2/6/7 competes with EB1 for binding to GTPγS-stabilized microtubules, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. On dynamic microtubules, however, SEPT2/6/7 rarely binds microtubule plus ends, suggesting that SEPT2/6/7 impacts EB1-binding allosterically through long-range effects on the GDP-bound lattice. Strikingly, plus end pausing and EB1 dissociation also occur during end-on collisions of microtubules with immobilized SEPT2/6/7 filaments, which have a greater impact than actin filaments. In agreement with these in vitro findings, EB1 pausing is enhanced upon contact with septins in primary hippocampal neurons. Taken together, these data demonstrate that SEPT2/6/7 complexes and filaments can directly tune microtubule plus end dynamics and the tracking of plus end proteins, providing a mechanism for capture and guidance of MT plus ends at intracellular regions of septin localization.

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

“…1G). This biphasic effect resembled the increase and diminution of microtubule plus end growth by nanomolar and micromolar Sept2/6/7 concentrations, respectively, which corelates with a transition of Sept2/6/7 from oligomers to higher-order polymers with increasing concentrations (9). Coating microtubules with mCherry-Sept2/6/7 showed that septins are present along the actin-bound microtubule lattice (Fig.…”

“…To test whether septins interact directly and concomitantly with both actin and microtubules, crosslinking them together, we performed in vitro binding assays using stable prepolymerized microtubules, actin filaments and septins. We purified recombinant Sept2/6/7, the minimal septin complex which has been shown to bind separately actin and microtubules (9, 10). Using total internal reflection fluorescence (TIRF) microscopy, we assayed for the association of immobilized glass-bound Sept2/6/7-coated microtubules with actin filaments that were floated into the imaging chamber.…”

Spatial regulation of microtubule-dependent transport by septin GTPases

“…In support of this possibility, septins (SEPT2/6/7, SEPT9) preferentially associate in vitro with microtubule lattices bound to the stable GTP analog GMPCPP (guanylyl-αβ-methylenediphosphonate) which are characterized by a non-compacted longitudinal inter-dimer interface similar to that of taxol-stabilized protofilaments [32][33][34] (Figure 1A). Microtubule bundling may further enhance septin binding, but it is unknown whether the parallel or antiparallel orientation of bundled microtubules plays a role.…”

“…SEPT9 associates with unpolymerized tubulin, and microtubuleassociated SEPT9 recruits tubulin dimers to the microtubule lattice, and thereby may rescue microtubule lattices from depolymerization and also repair sites of damage [33]. In vitro assays show that SEPT9 and SEPT2/6/7 complexes promote microtubule growth and elongation by suppressing catastrophe [32,33] (Figure 1C). In agreement with the in vitro findings, septins associate with microtubule bundles and promote as well as guide microtubule growth in living cells [28].…”

“…Septins associate with microtubules in vitro, and increasing septin concentrations exert biphasic effects on microtubule dynamics, which correlate with a shift from an oligomeric to a higher-order polymeric state [32]. On membrane bilayers, septins form networks of parallel paired filaments whose repeatability and density enable them to function as protein scaffolds [51,52].…”