Abstract
ATR responds to mechanical stress at the nuclear envelope and mediates envelope-associated repair of aberrant topological DNA states. By combining microscopy, electron microscopic analysis, biophysical and in vivo models, we report that ATR-defective cells exhibit altered nuclear plasticity and YAP delocalization. When subjected to mechanical stress or undergoing interstitial migration, ATR-defective nuclei collapse accumulating nuclear envelope ruptures and perinuclear cGAS, which indicate loss of nuclear envelope integrity, and aberrant perinuclear chromatin status. ATR-defective cells also are defective in neuronal migration during development and in metastatic dissemination from circulating tumor cells. Our findings indicate that ATR ensures mechanical coupling of the cytoskeleton to the nuclear envelope and accompanying regulation of envelope-chromosome association. Thus the repertoire of ATR-regulated biological processes extends well beyond its canonical role in triggering biochemical implementation of the DNA damage response.
Highlights
ATR responds to mechanical stress at the nuclear envelope and mediates envelopeassociated repair of aberrant topological DNA states
ATR (18.8%) was bound to actin filaments, in the proximity of the nuclear envelope (NE) and more than 20% was bound to cellular membranes (Fig. 1a)
Membrane fractionation analysis confirmed that 17% of ATR co-fractionated with membranes, when nucleic acids were degraded by Benzonase treatment (Supplementary Fig. 1b)
Summary
ATR is enriched at membranes and actin filaments around the nucleus. We visualized ATR distribution in exponentially growing HeLa cells by electron microscope (EM) and found ATR in the nucleus, cytosol, and other organelles, including endoplasmic reticulum (ER), Golgi, and mitochondria (Supplementary Fig. 1a). ATR-defective cells exhibited a 20% increase in K9 trimethylated histone H3 compared to control (Supplementary Fig. 2d), implying that ATR depletion promotes increased heterochromatization This conclusion is confirmed by the fluorescence energy transfer (FRET)-based fluorescent lifetime imaging microscopy (FLIM) assay utilizing GFP- and mCherry-tagged histone H2B to measure chromatin compaction[23]. Cells lacking functional ATR, either through acute inhibition or following longterm depletion, exhibited an increased FRET signal at the NE (Fig. 2e) This result indicates that the actin-binding and the membrane-binding domains of the linker molecule are in close proximity, presumably due to an altered NE-cytoskeleton connection. This finding is consistent with Nesprin 2 influencing the NE tension, suggesting that ATR defects may cause a reduced NE tension.
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