The nuclear lamina is a critical regulator of nuclear structure and function. Nuclei from laminopathy patient cells experience repetitive disruptions of the nuclear envelope, causing transient intermingling of nuclear and cytoplasmic components. The exact causes and consequences of these events are not fully understood, but their stochastic occurrence complicates in-depth analyses. To resolve this, we have established a method that enables quantitative investigation of spontaneous nuclear ruptures, based on co-expression of a firmly bound nuclear reference marker and a fluorescent protein that shuttles between the nucleus and cytoplasm during ruptures. Minimally invasive imaging of both reporters, combined with automated tracking and in silico synchronization of individual rupture events, allowed extracting information on rupture frequency and recovery kinetics. Using this approach, we found that rupture frequency correlates inversely with lamin A/C levels, and can be reduced in genome-edited LMNA knockout cells by blocking actomyosin contractility or inhibiting the acetyl-transferase protein NAT10. Nuclear signal recovery followed a kinetic that is co-determined by the severity of the rupture event, and could be prolonged by knockdown of the ESCRT-III complex component CHMP4B. In conclusion, our approach reveals regulators of nuclear rupture induction and repair, which may have critical roles in disease development.

Human papillomaviruses (HPV) are small, non‐enveloped DNA viruses, which upon chronic infection can provoke cervical and head‐and‐neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer‐causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high‐content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time‐resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image‐based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

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Human papillomavirus (HPV) infection is the prime elicitor of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope dismantles. Since lamins safeguard nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. We challenged human cervical cancer cells knocked out for the major lamin genes with a HPV16 pseudovirus (PsV) encoding an EGFP reporter and found that loss of lamin B1 amplified infection rate. A prolonged mitotic window and extensive nuclear rupture propensity during interphase led to a higher nuclear PsV load in LMNB1 knockout cells, but unchanged EGFP transcript levels pointed to an additional defect in protein turnover. We found a strong decrease in autophagic capacity in LMNB1 knockout cells, which we connect to the persistent activation of cGAS. Thus, loss of lamin B1 increases nuclear perviousness and blunts the autophagic capacity, which primes cells for unrestrained buildup of HPV capsids.