Abstract
Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through β1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.
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Acknowledgements
We thank Dr MM Poo (Institute of Neuroscience, SIBS, CAS) for suggestions on this work and critical reading of the manuscript. This work was supported by the National Basic Research Program (2011CBA00403 to ZGL), and the National Natural Science Foundation of China (31021063 and 30825013 to ZGL, 21025520 to XYJ). We also thank Dr JF Chen (Institute of Biochemistry and Cell Biology, SIBS, CAS) for providing reagents and Dr Q Hu of ION Imaging Facility with microscope analysis.
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Supplementary information
Supplementary information, Figure S1
Polarized activation of Itgb1 in cultured hippocampal neurons. (PDF 76 kb)
Supplementary information, Figure S2
Effects of Itgb1 function-blocking antibodies on axon development in cultured hippocampal neurons. (PDF 83 kb)
Supplementary information, Figure S3
Itgb1 is dispensable for the growth of minor neurites. (PDF 40 kb)
Supplementary information, Figure S4
Definition and angular distribution of axon-initiation sites. (PDF 79 kb)
Supplementary information, Figure S5
Effects of Itgb1 function-blocking antibodies on axon initiation on laminin-stripes. (PDF 103 kb)
Supplementary information, Figure S6
Effects of Itgb1 function-blocking antibodies on axon initiation and turning on laminin gradients. (PDF 105 kb)
Supplementary information, Figure S7
Homogenates of cortex from littermate P0 mice with indicated genotypes were subject to IB with indicated antibodies. (PDF 31 kb)
Supplementary information, Figure S8
Taxol stabilization of microtubules partially prevents the loss of neuronal polarity caused by Itgb1 function-blocking antibodies. (PDF 130 kb)
Supplementary information, Figure S9
Cortical sections from Itgb1f/f or Itgb1f/f; Emx1-Cre mice at P20 were stained with antibody against Tbr1 (A) to mark layers II-IV and VI, or Cux1 (B) to mark layers II-III. Scale bar, 1 mm. (PDF 92 kb)
Supplementary information, Figure S10
Cortical sections of control (Itgb1f/f) or Itgb1 mutant (Itgb1f/f; Emx1-Cre) mice at E14.5 were stained with antibody against TAG1 or MAP2. (PDF 143 kb)
Supplementary information, Figure S11
Itgb1f/f or Itgb1f/+ mice at E15.5 were subject to in utero electroporation with pCAG-IRES-GFP plasmid together with pTurbo-Cre. (PDF 97 kb)
Supplementary information, Movie S1
Hippocampal neurons were transfected with EGFP-EB3, together with plasmids encoding scrambled siRNA, and then cultured on substrates coated with laminin stripes. At DIV 2, movement of individual EGFP-EB3 puncta was recorded using live imaging fluorescence microscope. (AVI 6036 kb)
Supplementary information, Movie S2
Hippocampal neurons were transfected with EGFP-EB3, together with plasmids encoding Itgb1 siRNA, and cultured on substrates coated with laminin stripes. At DIV2, movement of individual EGFP-EB3 puncta was recorded using live imaging fluorescence microscope. (AVI 6488 kb)
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Lei, WL., Xing, SG., Deng, CY. et al. Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization. Cell Res 22, 954–972 (2012). https://doi.org/10.1038/cr.2012.40
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DOI: https://doi.org/10.1038/cr.2012.40
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