EML1'in mikrotübül organizasyonu ve hücre bölünmesindeki düzenleyici rolü

Microtubules and microtubule-associated proteins (MAPs) are crucial for regulating diverse cellular processes, including intracellular transport, spindle formation, and cell division, all of which are critical for ensuring proper brain development and cortical organization. Among these, EML1 is a MAP that has been associated with subcortical heterotopia, yet its molecular role in microtubule organization and the consequences of disease-associated mutations remain incompletely understood. To investigate the regulatory role of EML1, CRISPR-Cas9-mediated knockout in HeLa cells and Eml1 conditional knockout neural progenitors obtained from E14.5 mouse forebrain were used. Exogenous expression of GFP-tagged wild-type or disease-causing T243A mutant EML1 was employed to assess the impact of the mutation on microtubule binding and cellular phenotypes. A combination of microtubule pelleting assays, confocal immunofluorescence imaging, label-free quantitative proteomics, and live-cell imaging was applied to evaluate microtubule stability, MAP recruitment, spindle organization, and mitotic progression. Loss of EML1 resulted in a reduction of taxol-stabilized microtubules, an increase in soluble tubulin, reduced α-tubulin acetylation, and enhanced vulnerability to nocodazole-induced microtubule depolymerization. These changes were accompanied by diminished recruitment of other MAPs such as EML4 and SEPTIN2 to microtubules, despite unchanged total protein levels. EML1 was found to be required for EML4's association with the microtubule network; its absence led to a significant reduction in EML4 binding, potentially contributing to impaired microtubule organization and mitotic fidelity. Notably, mitotic abnormalities such as spindle disorganization and prolonged division time were rescued by wild-type EML1 but not by the T243A mutant, which exhibited impaired microtubule binding and aggregation. Cell cycle-specific proteomic analysis revealed that EML1 associates with partially distinct sets of proteins during interphase and mitosis, indicating dynamic regulation of its interactome throughout the cell cycle. In addition, NEK9, a mitotic kinase not previously reported as an EML1 interactor, was reproducibly identified in Eml1-GFP pull-down samples, suggesting a potential new association. Importantly, EML1 regulates the dynamic localization of EML4 during cell division. In EML1 knockout cells, EML4 shows aberrant accumulation on spindle and midzone microtubules, whereas Eml1-GFP rescue restores its normal distribution. Overall, EML1 is established as a central scaffold-like regulator of microtubule stability, MAP interactions, and mitotic organization. Loss or mutation of EML1 compromises cytoskeletal integrity and mitotic fidelity, providing mechanistic insight into the pathogenesis of EML1-related heterotopia and suggesting potential targets for therapeutic intervention.