Molecular analysis of cortical malformation in mammalian cortex by using proteomics approaches

The layered structure of the cerebral cortex is formed through a complicated sequence of highly controlled stages during corticogenesis. During this process, the perturbation of neuronal migration and cell division can result in a rare disorder called cortical heterotopia. Heterotopia patients can have recurrent epileptic seizures, developmental delays, and mild intellectual disabilities. Heterotopia has been challenging to study because human mutations associated with the disease often fail to form heterotopia in mouse models. Eml1 is a heterotopia-associated gene where the perturbations cause heterotopia formation in humans and mice. During my Ph.D., I focused on the investigation of the functional role of EML1 in heterotopia formation through comprehensive cellular and biochemical analysis, including imaging, BioID proximity labeling, proteomic profiling via label-free proteomics or dimethyl labeling, phosphoproteomics, and microtubule-pelleting assays. In the first part of my thesis, I showed that heterotopia-associated mutant EML1 reduces the protein-microtubule binding affinity. BioID proximity labeling of wild-type and mutant EML1 revealed that EML1 interacts with many microtubule and cytoskeletal organization proteins, most of which are lost in mutant-expressing cells. Thus, the wild-type protein interactome is impacted by a heterotopia-causing mutation. This work identified several novel interactors, along with known interaction partners of EML1. Proteome profiling in cortices and primary neuronal cells in a conditional Eml1 knockout (cKO) mouse versus control showed dysregulations in microtubule organization and protein metabolic processes upon Eml1 depletion. These large-scale proteomic analyses are essential for studying the underlying mechanism in brain development and heterotopia formation. In the second part of my thesis, I present my contributions to the published article, which focuses on the cell cycle-dependent regulation of an adhesion protein, protocadherin-7. In this project, as my side project, I unveiled the spatiotemporal regulation of the protein and its' palmitoylation-dependent regulation in cell division.