cellular-redox-profiling-using-high-content-microscopy

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

University of Antwerp

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.

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

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows.

Joke Robijns

Laboratory of Cell Biology and Histology,
Department of Veterinary Sciences,
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences

University of Antwerp

Cellular Redox Profiling Using High-content Microscopy

Joke Robijns

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Cell Biology and Histology,Department of Veterinary Sciences,Laboratory of Cell Biology and Histology, Department of Veterinary Sciences

University of Antwerp

Cellular Redox Profiling Using High-content Microscopy

Laboratory of Cell Biology and Histology,
Department of Veterinary Sciences,
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences