The investigation into protein Willin/FRMD6 in neuronal polarization and associated molecular mechanisms

Abstract

Neuronal polarization is defined as the biological process where neurons obtain their molecular, morphological, and functional asymmetries between dendritic and axonal compartments. Establishing neuronal polarity, especially the growth of the axon, is of fundamental importance. By utilising a range of optical imaging methods, this thesis investigates how Willin, a cell architecture regulatory Hippo signalling component, might affect the polarization process of neuronal cells.

By creating different levels of Willin expression in neuronal cells using molecular gene-editing, specifically in the HT-22 cell line, it was observed that the modification of Willin expression levels changed the number and length of axons as observed under conventional fluorescent microscopes. As the cell cytoskeleton dynamics greatly shape how neuronal cells adapt to a new morphology, the organization of the F-actin filaments was examined at the sub-diffraction level (<200 nm) by applying a super-resolution 3D-Structured Illumination Microscope. Additionally, modifications of Willin expression led to the cytoskeleton becoming more mobile, a loss of the actin cortex, and the formation of irregular actin protrusions at the cell membrane of HT-22 cells, as well as changes in cell proliferation, migration, and adhesion of SHSY-5Y cells. Lastly, by applying an all-optical calcium imaging technique, OptoCaMP imaging, it was shown that changing Willin expression levels can affect the activity of rat cortical neurons. Surprisingly, the reduction of Willin expression in mouse cortical neurons caused axon disintegration and impaired neuronal health, supporting the notion that Willin might regulate the growth and integrity of the axonal compartment. What is more, the biochemical analysis suggested that the above changes might be independent of the PI3K/aPKC signalling pathway but relies on the Hippo signalling pathway; however, in a cell type specific manner.

Collectively, the evidence presented in this thesis supports the involvement of Willin in various neuronal behaviours, specifically in neuronal polarization, and therefore opens a new and promising area for future studies involved in understanding the roles of Willin in the central nervous system.