Abstract
Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / metabolism*
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Animals
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Cell Membrane / metabolism
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Cells, Cultured
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Endocytosis*
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Endosomes / metabolism
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Growth Cones / metabolism*
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Growth Cones / radiation effects
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HEK293 Cells
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Hippocampus / cytology
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Hippocampus / embryology
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Humans
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Light
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Mice
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Microscopy, Confocal
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Neurites / physiology
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Neurites / radiation effects
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Neurons / metabolism
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Neurons / radiation effects
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Optogenetics / methods
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphatidylinositol Phosphates / metabolism*
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Proto-Oncogene Proteins c-akt / genetics
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Proto-Oncogene Proteins c-akt / metabolism
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Pseudopodia / metabolism
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Time-Lapse Imaging
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rab5 GTP-Binding Proteins / genetics
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rab5 GTP-Binding Proteins / metabolism
Substances
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Actins
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Luminescent Proteins
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Phosphatidylinositol Phosphates
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phosphatidylinositol 3,4,5-triphosphate
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Phosphatidylinositol 3-Kinases
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Proto-Oncogene Proteins c-akt
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rab5 GTP-Binding Proteins
Grants and funding
T.K. is supported by a Japan Society for the Promotion of Science Fellowship for Young Scientists. This work was supported by a grant from the Research Foundation for Opto-Science and Technology to T.N., a grant from the Yamada Science Foundation to T.N., a grant from The Uehara Memorial Foundation to T.N., a grant from the Brain Science Foundation to T.N., and the Ministry of Education, Culture, Sports, Science and Technology, Japan under a Grant in Aid for Scientific Research B (No. 25293042) to T.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.