In silico synchronization reveals regulators of nuclear ruptures in lamin A/C deficient model cells

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 rmly bound nuclear reference marker and a uorescent 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

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations

Quantitative proteomics reveals altered expression of actin binding proteins after LMNA knockdown in human dermal fibroblasts

The nuclear lamina physically supports the cell nucleus and has a central role in gene regulation. Mutations in the LMNA gene, which encodes A-type lamins, cause a wide spectrum of tissue-specific and systemic diseases collectively called laminopathies. To elucidate the molecular mechanisms underlying this phenotypic diversity, we set out to identify changes in the proteome upon specific lamin perturbations. More specifically, mature lamin A was reduced by sustained knockdown of LMNA in human dermal fibroblasts. To quantitatively compare protein composition, we made use of SILAC-based shotgun proteomics. The expression of 48 proteins was altered after LMNA knockdown. Gene Ontology analysis of the most significant hits revealed that the largest fraction of the differentially produced proteins in lamin A/C depleted cells were cytoskeletal proteins, more specifically those involved in actin cytoskeleton organization, such as ACTR2 and ACTR3 which are components of the ARP2/3 complex and FSCN1 which plays a critical role in cell migration, motility, adhesion and cellular interactions. Indeed, impaired wound healing and focal adhesion was observed in lamin A/C depleted fibroblasts. Furthermore, decreased expression of FSCN1 was correlated with a downregulation of IL6 and STAT3, which might be the instigators of the altered FSCN1 expression

Chemically and genetically induced accumulation of farnesylated prelamin A differentially affect oxidative stress and mitochondrial potential

The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that jointly make up the nuclear lamina. Point mutations that interfere with proper maturation of a specific subset of lamins, the A-type lamins, are the prime cause for a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox management. To decipher whether different lamin perturbations differentially affect intracellular oxidative stress, we have analyzed basal levels of reactive oxygen species (ROS), sensitivity towards oxidative stress, mitochondrial potential and expression of ROS defusing enzymes in human fibroblasts in which we experimentally induced accumulation of different prelamin A variants. Using a quantitative single cell imaging workflow, we measured a significant increase in basal ROS levels upon chemically induced, but not upon genetically induced accumulation of prelamin A. Since both chemical and genetic induction of prelamin A lead to mitochondrial hyperpolarization, chemical treatments may have lamin-independent effects that aggravate ROS production or impair ROS defusing capacity. In contrast, reduction of mature lamin A via siRNA-mediated knockdown caused a highly significant rise in basal ROS levels and an even more prominent increase in sensitivity towards ROS, but had no effect on mitochondrial potential, consistent with a direct ROS-buffering capacity of mature lamins. Hence, different lamin intermediates exert distinct roles in cellular redox management

High-content analysis of cellular oxidative stress

Reactive oxygen species (ROS) are small, short lived molecules that mediate various cellular responses including cell proliferation, differentiation, gene expression and migration. Excessive accumulation of ROS however, can lead to DNA damage and the build-up of irreversibly oxidized proteins. To counter the potential damaging effects of ROS, cells have evolved several antioxidant systems, including ROS defusing enzymes and vitamins. An imbalance between production of oxidative radicals and antioxidant mechanisms induces a state of oxidative stress, a phenomenon associated with various systemic diseases and aging. One of the major sources of ROS is the mitochondrion, which harbours the electron transport chain, handling highly active electrons in close proximity to oxygen. To study the relationship between ROS levels and mitochondrial (dys-) function in various contexts, we have developed an integrated workflow for measurement of intracellular ROS levels, mitochondrial potential and mitochondrial morphology. To this end, human dermal fibroblasts, cultured in glass-bottom 96-well plates, were stained with a fluorescent ROS-sensitive dye (pan-cellular (CM-H2DCFDA) or mitochondrially targeted (MitoSOX)), or a mitochondrial membrane potential-sensitive dye (TMRM), after which they were acquired in a fully automated manner and analysed using home-written image analysis protocols (RedoxMetrics and MitoMetrics). In fluxo chemical perturbation allowed determining the dynamic range and sensitivity towards the applied stressors. In case of mitochondrial analysis, mitochondria were specifically enhanced using a multiscale Laplacian operator and an exhaustive morphological and textural feature extraction was performed. Using this approach, we showed that specific chemical perturbations selectively affect cellular ROS levels or mitochondrial morphology