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Final Report Summary - NEUROSMLM (The molecular mechanism behind the axonal initial segment diffusion barrier)

Cell polarization is a decisive step in neuronal development and once axonal and somatodendritic domains are generated, they generally persist over the lifetime of an organism. The axonal initial segment (AIS), a stretch of 50 – 100 μm length close to the cell body, bears a specialized cytoskeleton and maintains the polarized distribution of neuronal molecules by blocking the exchange of molecules between axon and soma. The project helped resolving THE MOLECULAR MECHANISM BEHIND THE AXONAL INITIAL SEGMENT DIFFUSION BARRIER which was hitherto poorly understood.
The first project goal of performing superresolution imaging of the major components of the AIS in mature hippocampal neurons was addressed by the establishment of a powerful dual color dSTORM imaging modality (Winterflood et al. ACS Chemical Biology 2015). The dual color imaging mode addressed many difficulties involved in multicolour dSTORM including good and compatible fluorophores, no requirement for color registration, very low chromatic aberration, low crosstalk, simplicity of experimental system and imaging and analysis procedure. The dual color imaging mode was extended to 3D capability and used to determine the relative spatial organization of the key AIS cytoskeletal components Ankyrin G and Beta IV spectrin (Winterflood et al. Biophys. J. 2015). A second task was to develop a dual color single-particle tracking assay to study the diffusion barrier at the AIS with respect to the nature of the membrane probe during its establishment (transmembrane protein vs GPI-anchored in outer leaflet). The task was successfully completed and the novel tracking methodology itself was published (Winterflood et al. Methods and Applications in Fluorescene 2015). The method showed that in U2OS cells that lipid anchored or transmembrane proteins do not experience friction by the sub-membrane cytoskeleton differently. Together with later important findings this provided experimental support for the anchored transmembrane “picket fence” model. During the project a single-particle tracking assay with increased spatio-temporal resolution was developed. Single-particle tracking was now performed with brighter and more photostable quantum dots of GPI-GFP and revealed a strikingly periodic diffusional pattern. A highly periodic arrangement was recently shown for the cytoskeleton in axons (Xu et al. Science 2012). During the project, the compartmentalized single-molecule diffusion was successfully correlated with the periodical cytoskeleton of the axonal initial segment using a sophisticated assay which involved live-cell tracking and post-hoc super-resolution imaging of the cytoskeleton. Such correlation between the submembrane cytoskeleton and membrane diffusion is unparalleled in its clarity and preciseness and has so far not been shown. It showed that the corralling of the membrane motion happens between two actin rings which further supports the “picket fence” model which suggests that molecules anchored to the actin cytoskeleton act as obstacles to membrane motion. The projects findings have laid the foundation to our understanding of the molecular mechanism of the diffusion barrier. It has paved the way for further research into the AIS and its role in cell polarisation which currently gains substantial attention, not least because of its association with diseases such as bipolar disorder and depression.

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United Kingdom
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