Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.
The Journal of Neuroscience
This work was supported by The Institute of Science and Technology (IST) Austria, the European Union's Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie Grant Agreement No. 793482 (to K.E.) and by the European Research Council (ERC) Grant Agreement No. 694539 (to R.S.). We thank Nicoleta Condruz (IST Austria, Klosterneuburg, Austria) for technical assistance with sample preparation, the Electron Microscopy Facility of IST Austria (Klosterneuburg, Austria) for technical support with EM works, Natalia Baranova (University of Vienna, Vienna, Austria) and Martin Loose (IST Austria, Klosterneuburg, Austria) for advice on liposome preparation, and Yugo Fukazawa (University of Fukui, Fukui, Japan) for comments.
Eguchi K, Le Monnier E, Shigemoto R. Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. The Journal of Neuroscience. 2023;43(23):4197-4216. doi:10.1523/JNEUROSCI.1514-22.2023
Eguchi, K., Le Monnier, E., & Shigemoto, R. (2023). Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. The Journal of Neuroscience. Society for Neuroscience. https://doi.org/10.1523/JNEUROSCI.1514-22.2023
Eguchi, Kohgaku, Elodie Le Monnier, and Ryuichi Shigemoto. “Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.” The Journal of Neuroscience. Society for Neuroscience, 2023. https://doi.org/10.1523/JNEUROSCI.1514-22.2023.
K. Eguchi, E. Le Monnier, and R. Shigemoto, “Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons,” The Journal of Neuroscience, vol. 43, no. 23. Society for Neuroscience, pp. 4197–4216, 2023.
Eguchi K, Le Monnier E, Shigemoto R. 2023. Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons. The Journal of Neuroscience. 43(23), 4197–4216.
Eguchi, Kohgaku, et al. “Nanoscale Phosphoinositide Distribution on Cell Membranes of Mouse Cerebellar Neurons.” The Journal of Neuroscience, vol. 43, no. 23, Society for Neuroscience, 2023, pp. 4197–216, doi:10.1523/JNEUROSCI.1514-22.2023.
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