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Dive into the research topics where Tobias Corne is active.

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Featured researches published by Tobias Corne.


Scientific Reports | 2016

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

Joke Robijns; F. Molenberghs; Tom Sieprath; Tobias Corne; Marlies Verschuuren; W.H. De Vos

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.


Nucleus | 2015

Sustained accumulation of prelamin A and depletion of lamin A/C both cause oxidative stress and mitochondrial dysfunction but induce different cell fates

Tom Sieprath; Tobias Corne; Marco Nooteboom; Charlotte Grootaert; Andreja Rajkovic; Benjamin Buysschaert; Joke Robijns; Jos L. V. Broers; Frans C. S. Ramaekers; Werner J.H. Koopman; Peter H. G. M. Willems; Winnok H. De Vos

The cell nucleus is structurally and functionally organized by lamins, intermediate filament proteins that form the nuclear lamina. Point mutations in genes that encode a specific subset of lamins, the A-type lamins, cause a spectrum of diseases termed laminopathies. Recent evidence points to a role for A-type lamins in intracellular redox homeostasis. To determine whether lamin A/C depletion and prelamin A accumulation differentially induce oxidative stress, we have performed a quantitative microscopy-based analysis of reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in human fibroblasts subjected to sustained siRNA-mediated knockdown of LMNA and ZMPSTE24, respectively. We measured a highly significant increase in basal ROS levels and an even more prominent rise of induced ROS levels in lamin A/C depleted cells, eventually resulting in Δψm hyperpolarization and apoptosis. Depletion of ZMPSTE24 on the other hand, triggered a senescence pathway that was associated with moderately increased ROS levels and a transient Δψm depolarization. Both knockdowns were accompanied by an upregulation of several ROS detoxifying enzymes. Taken together, our data suggest that both persistent prelamin A accumulation and lamin A/C depletion elevate ROS levels, but to a different extent and with different effects on cell fate. This may contribute to the variety of disease phenotypes witnessed in laminopathies.


Cell Adhesion & Migration | 2017

Deregulation of focal adhesion formation and cytoskeletal tension due to loss of A-type lamins

Tobias Corne; Tom Sieprath; Jonathan Vandenbussche; Danahe Mohammed; Mariska te Lindert; Kris Gevaert; Sylvain Gabriele; Katarina Wolf; Winnok H. De Vos

ABSTRACT The nuclear lamina mechanically integrates the nucleus with the cytoskeleton and extracellular environment and regulates gene expression. These functions are exerted through direct and indirect interactions with the laminas major constituent proteins, the A-type lamins, which are encoded by the LMNA gene. Using quantitative stable isotope labeling-based shotgun proteomics we have analyzed the proteome of human dermal fibroblasts in which we have depleted A-type lamins by means of a sustained siRNA-mediated LMNA knockdown. Gene ontology analysis revealed that the largest fraction of differentially produced proteins was involved in actin cytoskeleton organization, in particular proteins involved in focal adhesion dynamics, such as actin-related protein 2 and 3 (ACTR2/3), subunits of the ARP2/3 complex, and fascin actin-bundling protein 1 (FSCN1). Functional validation using quantitative immunofluorescence showed a significant reduction in the size of focal adhesion points in A-type lamin depleted cells, which correlated with a reduction in early cell adhesion capacity and an increased cell motility. At the same time, loss of A-type lamins led to more pronounced stress fibers and higher traction forces. This phenotype could not be mimicked or reversed by experimental modulation of the STAT3-IL6 pathway, but it was partly recapitulated by chemical inhibition of the ARP2/3 complex. Thus, our data suggest that the loss of A-type lamins perturbs the balance between focal adhesions and cytoskeletal tension. This imbalance may contribute to mechanosensing defects observed in certain laminopathies.


Cell Adhesion & Migration | 2017

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

Marie Versaevel; Maryam Riaz; Tobias Corne; Thomas Grevesse; Joséphine Lantoine; Danahe Mohammed; Céline Bruyère; Laura Alaimo; Winnok H. De Vos; Sylvain Gabriele

ABSTRACT The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.


Advances in Anatomy Embryology and Cell Biology | 2016

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Tom Sieprath; Tobias Corne; Peter H. G. M. Willems; Werner J.H. Koopman; Winnok H. De Vos

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells.


Journal of Visualized Experiments | 2017

Cellular redox profiling using high-content microscopy

Tom Sieprath; Tobias Corne; Joke Robijns; Werner J.H. Koopman; W.H. De Vos

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (ΔΨm) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (ΔΨm and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.


Cell Adhesion and Communication | 2017

Deregulation of focal adhesion and cytoskeletal tension due to loss of A-type lamins

Tobias Corne; Tom Sieprath; Jonathan Vandenbussche; Danahe Mohammed; Mariska te Lindert; Kris Gevaert; Katarina Wolf; Sylvain Gabriele; Winnok De Wos


Nuclear Structure and Dynamics, EMBO conference, Abstracts | 2015

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

Tobias Corne; Tom Sieprath; Jonathan Vandenbussche; Kris Gevaert; Winnok H. De Vos


Systems-Level View of Cytoskeletal Function, EMBO workshop, Abstracts | 2014

Quantitative proteomics reveals differential effects of ZMPSTE24 and LMNA knockdown on human fibroblasts

Tobias Corne; Tom Sieprath; Jonathan Vandenbussche; Kris Gevaert; Winnok H. De Vos


Systems-Level View of Cytoskeletal Function, EMBO workshop, Abstracts | 2014

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

Tom Sieprath; Tobias Corne; Werner J.H. Koopman; Peter H. G. M. Willems; Winnok H. De Vos

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Katarina Wolf

Radboud University Nijmegen

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