Abstract
Neurite initiation is critical for neuronal morphogenesis and early neural circuit development. Recent studies showed that local actin aggregation underneath the cell membrane determined the site of neurite initiation. An immediately arising question is what signaling mechanism initiated actin aggregation. Here we demonstrate that local clustering of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), a phospholipid with relatively few known signaling functions, is necessary and sufficient for aggregating actin and promoting neuritogenesis. In contrast, the related and more extensively studied phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol (3,4,5)-trisphosphate (PIP3) molecules did not have such functions. Specifically, we showed that beads coated with PI(3,4)P2 promoted actin aggregation and neurite initiation, while pharmacological interference with PI(3,4)P2 synthesis inhibited both processes. PI(3,4)P2 clustering occurred even when actin aggregation was pharmacologically blocked, demonstrating that PI(3,4)P2 functioned as the upstream signaling molecule. Two enzymes critical for PI(3,4)P2 generation, namely, SH2 domain-containing inositol 5-phosphatase and class II phosphoinositide 3-kinase α, were complementarily and non-redundantly required for actin aggregation and neuritogenesis, as well as for subsequent dendritogenesis. Finally, we demonstrate that neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex functioned downstream of PI(3,4)P2 to mediate neuritogenesis and dendritogenesis. Together, our results identify PI(3,4)P2 as an important signaling molecule during early development and demonstrate its critical role in regulating actin aggregation and neuritogenesis.
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Acknowledgements
We thank Professors Volker Haucke, Marc Kirschner, Alexander Sorisky and Jane Y Wu for constructs. We thank Yuan Lu and Zong-Fang Wan for excellent technical assistance. We thank Dr Qian Hu and ION Optical Imaging Core Facility for confocal microscopy and data analysis support, and Dr Min Jiang and IOBS-Nikon Biological Imaging Center for use of the N-SIM microscope. We are grateful to Drs Xueliang Zhu and Xiumin Yan, colleagues at ION, and members of the Yu laboratory for suggestions and comments. This work was supported by the National Natural Science Foundation of China (31321091 and 31530030), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB02010000) and the Science and Technology Commission of Shanghai Municipality (16XD1404800).
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Supplementary information, Figure S1
The PIP antibodies used in this study are specific. (PDF 402 kb)
Supplementary information, Figure S2
High concentration of Wortmannin blocked actin aggregation and neurite initiation, and reduced PI(3,4)P2 level. (PDF 370 kb)
Supplementary information, Figure S3
SHIP2 RNAi and PI3K C2α RNAi specifically reduced the level of correspondent proteins. (PDF 268 kb)
Supplementary information, Figure S4
Lamellipodin contributes to dendrite morphogenesis, but is not necessary for primary dendrite initiation. (PDF 273 kb)
Supplementary information, Figure S5
PI(3,4)P2 co-localizes with actin aggregates, while PI(4,5)P2 is mostly nuclear. (PDF 320 kb)
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Zhang, SX., Duan, LH., He, SJ. et al. Phosphatidylinositol 3,4-bisphosphate regulates neurite initiation and dendrite morphogenesis via actin aggregation. Cell Res 27, 253–273 (2017). https://doi.org/10.1038/cr.2017.13
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DOI: https://doi.org/10.1038/cr.2017.13
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