Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)
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A1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä
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en
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8
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Journal of Visualized Experiments, Volume 2022, issue 180
Abstract
The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.Description
Funding Information: Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain & Mind (A.A.) Funding Information: Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain & Mind (A.A.). Publisher Copyright: © 2022, Journal of Visualized Experiments. All rights reserved.
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Micinski, D, Lahti, L & Abouelezz, A 2022, 'Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)', Journal of Visualized Experiments, vol. 2022, no. 180, e63327. https://doi.org/10.3791/63327