Regulation of microtubule plus end dynamics by septin 9

Nakos, Konstantinos; Rosenberg, Marshall; Spiliotis, Elias T.

doi:10.1002/cm.21488

Cited by 38 publications

(42 citation statements)

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“…This is therefore likely to be a 'promiscuous' interaction as seen in many other septin crystal structures. Nevertheless, recent evidence suggests that a homotypic G interface formed by SEPT9 may be physiologically important in the control of microtubule dynamics (Nakos et al, 2019).…”

Section: The G and Nc Interfaces Of The Sept3 Subgroupmentioning

confidence: 99%

A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface

Castro

Silva

Pereira

et al. 2020

IUCrJ

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Human septins 3, 9 and 12 are the only members of a specific subgroup of septins that display several unusual features, including the absence of a C-terminal coiled coil. This particular subgroup (the SEPT3 septins) are present in rod-like octameric protofilaments but are lacking in similar hexameric assemblies, which only contain representatives of the three remaining subgroups. Both hexamers and octamers can self-assemble into mixed filaments by end-to-end association, implying that the SEPT3 septins may facilitate polymerization but not necessarily function. These filaments frequently associate into higher order complexes which associate with biological membranes, triggering a wide range of cellular events. In the present work, a complete compendium of crystal structures for the GTP-binding domains of all of the SEPT3 subgroup members when bound to either GDP or to a GTP analogue is provided. The structures reveal a unique degree of plasticity at one of the filamentous interfaces (dubbed NC). Specifically, structures of the GDP and GTPγS complexes of SEPT9 reveal a squeezing mechanism at the NC interface which would expel a polybasic region from its binding site and render it free to interact with negatively charged membranes. On the other hand, a polyacidic region associated with helix α5′, the orientation of which is particular to this subgroup, provides a safe haven for the polybasic region when retracted within the interface. Together, these results suggest a mechanism which couples GTP binding and hydrolysis to membrane association and implies a unique role for the SEPT3 subgroup in this process. These observations can be accounted for by constellations of specific amino-acid residues that are found only in this subgroup and by the absence of the C-terminal coiled coil. Such conclusions can only be reached owing to the completeness of the structural studies presented here.

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Section: The G and Nc Interfaces Of The Sept3 Subgroupmentioning

confidence: 99%

A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface

Castro

Silva

Pereira

et al. 2020

IUCrJ

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“…Given that MT growth and dynamics are inhibited by higher concentrations of SEPT2/6/7 (>800 nM), we asked whether higher-order SEPT2/6/7 complexes associate with free unpolymerized tubulin, lowering the effective concentration of tubulin that is available to polymerize. This is plausible as we recently found that SEPT9 binds and recruits free tubulin to the MT lattice (Nakos et al, 2019). To test this possibility, SEPT2/6/7 and SEPT9 complexes were immobilized on functionalized glass chambers and fluorescent tubulin was added in the buffer conditions of the MT dynamics assay ( Figure S2a).…”

mentioning

confidence: 86%

“…(Nakos et al, 2019; Reid et al, 2016;Tanenbaum et al, 2013). TIRF imaging was performed as described before(Nakos et al, 2019).…”

mentioning

confidence: 99%

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

Nakos

Radler

Spiliotis

2019

Preprint

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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.

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“…Understanding of these mechanistic differences is important as the assembly and association of these higher order components with cell membranes, actin filaments, and microtubules are fundamentally tied to septin cellular function. 24,[49][50][51][52][53][54][55] Septin hetero-hexamers and hetero-octamers both form in mammalian cells with the heterooctamers typically being the dominant species. 41,42 While the cell biology of mammalian septins is advancing rapidly (see above) and the biophysical basis of yeast septin assembly is progressing, [56][57][58][59][60][61] biochemical and biophysical studies of mammalian septins have lagged due to the lack of a mammalian hetero-octamer purification.…”

Section: Introductionmentioning

confidence: 99%

Production and analysis of a mammalian septin hetero-octamer complex

DeRose

Kelley

Ravi

et al. 2020

Preprint

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supported by NIH/NIGMS grants 5RO1 GM097664-9 and 1R35 GM136337. We thank Robert Fairman and Ronen Marmorstein for access to analytical ultracentrifugation instrumentation. We thank Dr. Patrick Loll for critical reading of the manuscript and the p97 vector. We thank Meagan Tomasso for a critical reading of the manuscript and the SEPT2/6/7 complex. We thank the College of Medicine and the Department of Pharmacology and Physiology access to the Olympus FV3000 laser scanning confocal microscope. AbstractThe 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 heterooctamers 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.

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Regulation of microtubule plus end dynamics by septin 9

Cited by 38 publications

References 38 publications

A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface

A complete compendium of crystal structures for the human SEPT3 subgroup reveals functional plasticity at a specific septin interface

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

Production and analysis of a mammalian septin hetero-octamer complex

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