Abstract
Axon development requires membrane addition from the intracellular supply, which has been shown to be mediated by Rab10-positive plasmalemmal precursor vesicles (PPVs). However, the molecular mechanisms underlying the membrane trafficking processes of PPVs remain unclear. Here, we show that myristoylated alanine-rich C-kinase substrate (MARCKS) mediates membrane targeting of Rab10-positive PPVs, and this regulation is critical for axon development. We found that the GTP-locked active form of Rab10 binds to membrane-associated MARCKS, whose affinity depends on the phosphorylation status of the MARCKS effector domain. Either genetic silencing of MARCKS or disruption of its interaction with Rab10 inhibited axon growth of cortical neurons, impaired docking and fusion of Rab10 vesicles with the plasma membrane, and consequently caused a loss of membrane insertion of axonal receptors responsive to extracellular axon growth factors. Thus, this study has identified a novel function of MARCKS in mediating membrane targeting of PPVs during axon development.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (31330032, 31321091, and 61327902 to ZG L and 81171190 to XH X) and the National Key Basic Research Program of China (2014CB910203). We thank Dr Q Hu of ION Imaging Facility with microscope analysis and Dr Y Kong with EM analysis. XH X was supported by SA-SIBS Scholarship Program, Shanghai Postdoctoral Scientific Program, China Postdoctoral Science Foundation funded Project, and KC Wong Education Foundation.
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Supplementary information
Supplementary information, Figure S1
Localization of MARCKS and Rab10 in the brain and cultured cortical neurons. (PDF 136 kb)
Supplementary information, Figure S2
Effects of siMARCKS or MARCKS-D3 on cell health. (PDF 219 kb)
Supplementary information, Figure S3
Effects of MARCKS wild type or mutants on axon development. (PDF 254 kb)
Supplementary information, Figure S4
MARCKS mediates docking and fusion of Rab10 vesicles. (PDF 127 kb)
Supplementary information, Figure S5
Membrane localization of MARCKS-D3. (PDF 81 kb)
Supplementary information, Figure S6
Analysis of the TD-Rab10 binding with MARCKS-eGFP in fibroblasts with FRET. (PDF 137 kb)
Supplementary information, Figure S7
The effect of MARCKS-D3 on the endocytosis of transferrin ligand. (PDF 229 kb)
Supplementary information, Figure S8
The role of MARCKS in anterograde transport of Rab10 vesicles. (PDF 102 kb)
Supplementary information, Figure S9
Colocalization between Rab10 and various membrane proteins at axonal growth cones of cultured cortical neurons. (PDF 361 kb)
Supplementary information, Movie S1
TIRFM imaging for the behavior of TD-Rab10 vesicles in fibroblasts transfected with vehicle plasmid. (AVI 4830 kb)
Supplementary information, Movie S2
TIRFM imaging for the behavior of TD-Rab10 vesicles in fibroblasts transfected with siMARCKS plasmid. (AVI 4652 kb)
Supplementary information, Movie S3
Behavior of TD-Rab10 TIRF signals in fibroblasts transfected with siMARCKS. Note the dynamic trajectory of Rab10 signals on the membrane plane. (AVI 1217 kb)
Supplementary information, Movie S4
Live imaging of TD-Rab10 TIRF signals in fibroblasts co-transfected with MARCKS-D3 plasmids. (AVI 4801 kb)
Supplementary information, Movie S5
TIRFM imaging for TD-Rab10 vesicle fusion in the axonal growth cone of a cortical neuron at DIV2. (AVI 1648 kb)
Supplementary information, Movie S6
Live imaging of TD-Rab10 vesicles in the axons or longest neurites of a DIV3 cortical neuron transfected with siScramble or siMARCKS. Scale bar, 10 μm. (AVI 3538 kb)
Supplementary information, Movie S7
Live imaging of TD-Rab10 vesicles in the axonal growth cones of a DIV3 cortical neuron transfected with siScramble or siMARCKS. Scale bar, 10 μm. (AVI 1035 kb)
Supplementary information, Movie S8
Live imaging of TD-Rab10 vesicles in the axonal growth cone of a DIV3 cortical neuron transfected with MARCKS-D3 (S4N)-YFP. Scale bar, 10 μm. (AVI 7022 kb)
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Xu, XH., Deng, CY., Liu, Y. et al. MARCKS regulates membrane targeting of Rab10 vesicles to promote axon development. Cell Res 24, 576–594 (2014). https://doi.org/10.1038/cr.2014.33
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DOI: https://doi.org/10.1038/cr.2014.33
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