Image-based identification and isolation of micronucleated cells to dissect cellular consequences

Abstract

Micronuclei are aberrant nuclear compartments that trap a portion of a cell's chromatin in a distinct organelle separate from the nucleus and are drivers of inflammation, DNA damage, chromosome instability, and chromothripsis. Many of the consequences of micronucleus formation stem from micronucleus rupture: the sudden loss of micronucleus compartmentalization, resulting in mislocalization of nuclear factors and the exposure of chromatin to the cytosol for the remainder of interphase. Micronuclei form primarily from segregation errors during mitosis, errors that also give rise to other, non-exclusive phenotypes, including aneuploidy and chromatin bridges. The stochastic formation of micronuclei and phenotypic overlap confounds the use of population-level assays or hypothesis discovery, requiring labor-intensive techniques to visually identify and follow micronucleated cells individually. In this study, we present a novel technique for automatically identifying and isolating micronucleated cells generally and cells with ruptured micronuclei specifically using ade novoneural net combined with Visual Cell Sorting. As a proof of concept, we compare the early transcriptomic responses to micronucleation and micronucleus rupture with previously published responses to aneuploidy, revealing micronucleus rupture to be a potential driver of the aneuploidy response.

Pathogenic Morphological Signatures of Perturbations in Mitochondrial-Related Genes Revealed by Pooled Imaging Assay

“…Similarly, a recent advent has been the introduction of screening techniques designed to investigate the morphologies of specific organelles and their links to human disease 12,25 . Deep cellular phenotyping techniques have also emerged exploring molecular characterization, cellular morphology, functional single cell analysis, and even cellular interactions [26][27][28][29][30][31] . Such techniques signify a critical shift beyond cell survival and toward a deeper exploration of organelle structure and function.…”

A high-content screen reveals new regulators of nuclear membrane stability

“…However, they can cause DNA damage, gene expression changes, and activation of cell invasion and inflammation pathways, especially under conditions of mechanical stress 2,4-9 . In addition, persistent rupture of micronuclear membranes can lead to catastrophic changes in chromosome structure, ongoing chromatin defects, and altered gene expression patterns that are proposed to be initiating events in some tumors and drivers of metastasis [10][11][12][13][14][15] . Nuclear membrane rupture occurs in a range of mechanically challenging conditions in vivo, including migration through dense extracellular matrices, at the leading edge of tumors, and during nuclear migration in fission yeast, C. elegans, and mouse models of laminopathies 4,5,8,[16][17][18][19][20][21][22] .…”