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Spatiotemporal organization of branched microtubule networks.

ABSTRACT: To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.

SUBMITTER: Thawani A

PROVIDER: S-EPMC6519983 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Spatiotemporal organization of branched microtubule networks.

Thawani Akanksha A Stone Howard A HA Shaevitz Joshua W JW Petry Sabine S

eLife 20190508

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use <i>Xenopus</i> egg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, f ...[more]

PMID: 31066674

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Project description:Rhodamine-phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ?40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ?13 kBT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an "order parameter," peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.

Project description:A class of self-assembling branched amphiphilic peptide capsules (BAPCs) was recently developed that could serve as a new drug delivery vehicle. BAPCs can encapsulate solutes up to ∼12 kDa during assembly, are unusually stable, and are readily taken up by cells with low cytotoxicity. Coarse-grained simulations have supported that BAPCs are defined by bilayers that resemble those formed by diacyl phospholipids. Here, atomistic simulations were performed to characterize the structure and organization of bilayers formed by three branched amphiphilic peptides (BAPs): bis(Ac-FLIVIGSII)-K-K4-CO-NH2, bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2, and bis(Ac-FLIVI)-K-K4-CO-NH2. The results show BAPs form a network of intra- and intermolecular backbone hydrogen bonds within the same leaflet in addition to hydrophobic side-chain interactions. The terminal residues of two leaflets form an interdigitation region locking two leaflets together. The phenyl groups in bis(Ac-FLIVIGSII)-K-K4-CO-NH2 and bis(Ac-FLIVI)-K-K4-CO-NH2 are tightly packed near the bilayer center but do not formed ordered structures with specific π-π stacking. Replacing phenyl groups with the cyclohexane side chain only slightly increases the level of disorder in bilayer structures and thus should not significantly affect the stability, consistent with experimental results on bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2 BAPCs. Self-assembly simulations further suggest that leaflet interdigitation likely occurs at early stages of BAPC formation. Atomistic simulations also reveal that the BAPC bilayers are highly permeable to water. This prediction was validated using fluorescence measurements of encapsulated self-quenching dye upon transferring BAPCs to buffers with different salt concentrations. Improved understanding of the organization and structure of BAPC bilayers at the atomic level will provide a basis for future rational modifications of BAP sequence to improve BAPC properties as a new class of delivery vehicle.

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Spatiotemporal organization of branched microtubule networks.

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Spatiotemporal organization of branched microtubule networks.

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