Quantitative analysis of 3D heterochromatin structures within and between neuronal cell types
Date
2025
Authors
Smith, Rory
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Abstract
Heterochromatin organization within neuronal nuclei plays a critical role in maintaining genomic stability and regulating gene expression, yet its 3D morphological variability between neuron types remains poorly understood. Using confocal microscopy, we quantitatively analyzed heterochromatin 3D structures within neuronal nuclei. Existing methods in the literature were re-created to measure the distribution of nuclear intensity into predefined classes, as well as basic morphological features of the foci (volume, eccentricity, etc.). However, these morphological descriptors impose rigid, preconceived geometric assumptions that do not consider 3D spatial positioning. We attempt to address these limitations with a novel computational method that directly assays both spatial and morphological "similarity" of heterochromatin formations in nuclei. This approach was applied to a range of neuron types to assess variation within and between groups. Our results demonstrate significant differences between spatial heterochromatin foci positioning between neural types. If coupled with immunohistochemistry and increased replicates, we conclude with a demonstration of how this analysis could be applied to effectively study single-genotype differences in heterochromatin formation. This methodology offers a generalizable framework for studying morphological changes in other genotypes and nuclear protein disorders, which is of interest to various neurodevelopmental disorders.