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Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles
Published on: November 15, 2019
Regulation of microtubule plus end dynamics by septin 9
Konstantinos Nakos1, Marshall Rosenberg1, Elias T Spiliotis1
1Department of Biology, Drexel University, Philadelphia, Pennsylvania.
Abstract:
Septins are GTP-binding proteins that associate with the microtubule (MT) and actin cytoskeleton. Septins affect MT organization and posttranslational modifications, but their role in MT dynamics is less understood. Here, we reconstituted MT dynamics in the presence of the MT-binding septin (SEPT9) using an in vitro cell-free assay, which images the polymerization of tubulin from guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP)-stabilized MT seeds. We found that submicromolar concentrations of SEPT9 suppress MT catastrophe and enhance the growth of MT plus ends to great lengths, while low micromolar concentrations of SEPT9 stabilize MTs by inhibiting dynamic instability. We show that SEPT9 associates preferentially with the lattice of GMPCPP-stabilized MT seeds and surprisingly recruits soluble tubulin to the MT lattice. Notably, the effects of SEPT9 on MT dynamics are dependent on its G-G dimerization interface, which is formed by the pockets of the GTP-binding domains. A mutation (H530D) that disrupts G-G dimerization abrogates the effects of SEPT9 on MT dynamics and diminishes its ability to recruit tubulin to the MT lattice. Taken together, these results suggest that SEPT9 promotes the formation and maintenance of long stable MTs through a mechanism that may involve recruitment of unpolymerized tubulin to the MT lattice.
Insights
Septin 9 (SEPT9) stabilizes microtubules (MTs) by suppressing catastrophe and promoting growth. This GTP-binding protein recruits soluble tubulin to the MT lattice, enhancing microtubule stability.
Area of Science:
- Cell Biology
- Cytoskeleton Dynamics
- Protein Biochemistry
Background:
- Septins are GTP-binding proteins interacting with actin and microtubule (MT) cytoskeletons.
- While septins influence MT organization, their direct role in MT dynamics remains unclear.
Purpose of the Study:
- To investigate the effect of the MT-binding septin, SEPT9, on microtubule dynamics using an in vitro reconstitution assay.
- To elucidate the mechanism by which SEPT9 influences microtubule polymerization and stability.
Main Methods:
- Utilized an in vitro cell-free assay to image tubulin polymerization from GMPCPP-stabilized MT seeds.
- Quantified the effects of varying SEPT9 concentrations on MT growth, catastrophe, and dynamic instability.
- Investigated SEPT9's interaction with the MT lattice and its dependence on the G-G dimerization interface (H530D mutation).
Main Results:
- Submicromolar SEPT9 suppressed MT catastrophe and enhanced plus-end growth; micromolar concentrations inhibited dynamic instability.
- SEPT9 associated with the MT lattice and recruited soluble tubulin.
- Disruption of SEPT9's G-G dimerization interface abrogated its effects on MT dynamics and tubulin recruitment.
Conclusions:
- SEPT9 promotes the formation and maintenance of long, stable microtubules.
- The mechanism involves SEPT9's G-G dimerization and potential recruitment of soluble tubulin to the MT lattice.
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