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Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

ABSTRACT: Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.

SUBMITTER: Marx A

PROVIDER: S-EPMC3655815 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

Marx Astrid A Godinez William J WJ Tsimashchuk Vasil V Bankhead Peter P Rohr Karl K Engel Ulrike U

Molecular biology of the cell 20130320 10

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protei ...[more]

PMID: 23515224

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Project description:Sprouty2 (Spry2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) are both well-established regulators of receptor tyrosine kinase (RTK) signaling, and knockdown of Spry2 or PTEN enhances axon regeneration of dorsal root ganglia (DRG) neurons. The major role of Spry2 is the inhibition of the rat sarcoma RAS/extracellular signal-regulated kinase (ERK) pathway, whereas PTEN acts mainly as an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In non-neuronal cells, Spry2 increases the expression and activity of PTEN, and PTEN enhances the amount of Spry2 by the inhibition of the microRNA-21 (miR-21) that downregulates Spry2. Applying dissociated DRG neuron cultures from wild-type (WT) or Spry2 deficient mice, we demonstrate that PTEN protein was reduced after 72 h during rapid axonal outgrowth on the laminin substrate. Furthermore, PTEN protein was decreased in DRG cultures obtained from homozygous Spry2-/- knockout mice. Vice versa, Spry2 protein was reduced by PTEN siRNA in WT and heterozygous Spry2+/- neurons. Knockdown of PTEN in DRG cultures obtained from homozygous Spry2-/- knockout mice promoted axon elongation without increasing axonal branching. Activation of Akt, but not ERK, was stronger in response to PTEN knockdown in homozygous Spry2-/- DRG neurons than in WT neurons. Together, our study confirms the important role of the signaling modulators Spry2 and PTEN in axon growth of adult DRG neurons. Both function as endogenous inhibitors of neuronal growth factor signaling and their simultaneous knockdown promotes axon elongation more efficiently than the single knockdown of each inhibitor. Furthermore, Spry2 and PTEN are reciprocally downregulated in adult DRG neuron cultures. Axon growth is influenced by multiple factors and our results demonstrate that the endogenous inhibitors of axon growth, Spry2 and PTEN, are co-regulated in adult DRG neuron cultures. Together, our data demonstrate that combined approaches may be more useful to improve nerve regeneration than targeting one single inhibitor of axon growth.

Dataset Information

Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

Publications

Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.

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