Dataset Information

Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport.

ABSTRACT: Microtubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly oriented minus-end out, while dynamic and tyrosinated microtubules are oriented oppositely. In addition, microtubules with similar orientations and modifications form bundles that bias transport. Importantly, because the plus-end-directed Kinesin-1 selectively interacts with acetylated microtubules, this organization guides this motor out of dendrites and into axons. In contrast, Kinesin-3 prefers tyrosinated microtubules and can enter both axons and dendrites. This separation of distinct microtubule subsets into oppositely oriented bundles constitutes a key architectural principle of the neuronal microtubule cytoskeleton that enables polarized sorting by different motor proteins.

SUBMITTER: Tas RP

PROVIDER: S-EPMC5746200 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport.

Tas Roderick P RP Chazeau Anaël A Cloin Bas M C BMC Lambers Maaike L A MLA Hoogenraad Casper C CC Kapitein Lukas C LC

Neuron 20171130 6

Microtubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly orien ...[more]

PMID: 29198755

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Project description:Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of ?-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged ?-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of ?-actin mRNA granules to the near-side of growth cones within a 4-7 min window. This polarized mRNA trafficking depended on the 3' untranslated region (UTR) since mRNA-?3'UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of ?-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed ?-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated "worm-like" trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of ?-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3'UTR is essential for this cue-induced regulation.

Project description:Differentiated mammalian cells and tissues, such as skeletal muscle fibers, acquire an organization of Golgi complex and microtubules profoundly different from that in proliferating cells and still poorly understood. In adult rodent skeletal muscle, the multinucleated muscle fibers have hundreds of Golgi elements (GE), small stacks of cisternae that serve as microtubule-organizing centers. We are interested in the role of the GE in organizing a peculiar grid of microtubules located in the fiber cortex, against the sarcolemma. Modifications of this grid in the mdx mouse model of Duchenne muscular dystrophy have led to identifying dystrophin, the protein missing in both human disease and mouse model, as a microtubule guide. Compared to wild-type (WT), mdx microtubules are disordered and more dense and they have been linked to the dystrophic pathology. GE themselves are disordered in mdx. Here, to identify the causes of GE and microtubule alterations in the mdx muscle, we follow GFP-tagged microtubule markers in live mdx fibers and investigate the recovery of GE and microtubules after treatment with nocodazole. We find that mdx microtubules grow 10% faster but in 30% shorter bouts and that they begin to form a tangled network, rather than an orthogonal grid, right after nucleation from GE. Strikingly, a large fraction of microtubules in mdx muscle fibers seem to dissociate from GE after nucleation. Moreover, we report that mdx GE are mispositioned and increased in number and size. These results were replicated in WT fibers overexpressing the beta-tubulin tubb6, which is elevated in Duchenne muscular dystrophy, in mdx and in regenerating muscle. Finally, we examine the association of GE with ER exit sites and ER-to-Golgi intermediate compartment, which starts during muscle differentiation, and find it persisting in mdx and tubb6 overexpressing fibers. We conclude that GE are full, small, Golgi complexes anchored, and positioned through ER Exit Sites. We propose a model in which GE mispositioning, together with the absence of microtubule guidance due to the lack of dystrophin, determines the differences in GE and microtubule organization between WT and mdx muscle fibers.

Dataset Information

Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport.

Publications

Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport.

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