High-volume, label-free imaging for quantifying single-cell dynamics in induced pluripotent stem cell colonies.

To facilitate the characterization of unlabeled induced pluripotent stem cells (iPSCs) during culture and expansion, we developed an AI pipeline for nuclear segmentation and mitosis detection from phase contrast images of individual cells within iPSC colonies. The analysis uses a 2D convolutional neural network (U-Net) plus a 3D U-Net applied on time lapse images to detect and segment nuclei, mitotic events, and daughter nuclei to enable tracking of large numbers of individual cells over long times in culture. The analysis uses fluorescence data to train models for segmenting nuclei in phase contrast images. The use of classical image processing routines to segment fluorescent nuclei precludes the need for manual annotation. We optimize and evaluate the accuracy of automated annotation to assure the reliability of the training. The model is generalizable in that it performs well on different datasets with an average F1 score of 0.94, on cells at different densities, and on cells from different pluripotent cell lines. The method allows us to assess, in a non-invasive manner, rates of mitosis and cell division which serve as indicators of cell state and cell health. We assess these parameters in up to hundreds of thousands of cells in culture for more than 36 hours, at different locations in the colonies, and as a function of excitation light exposure.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development.

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-ßPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation.

Reversible modification of proteins with linkage-specific ubiquitin chains is critical for intracellular signaling. Information on physiological roles and underlying mechanisms of particular ubiquitin linkages during human development are limited. Here, relying on genomic constraint scores, we identify 10 patients with multiple congenital anomalies caused by hemizygous variants in OTUD5, encoding a K48/K63 linkage-specific deubiquitylase. By studying these mutations, we find that OTUD5 controls neuroectodermal differentiation through cleaving K48-linked ubiquitin chains to counteract degradation of select chromatin regulators (e.g., ARID1A/B, histone deacetylase 2, and HCF1), mutations of which underlie diseases that exhibit phenotypic overlap with OTUD5 patients. Loss of OTUD5 during differentiation leads to less accessible chromatin at neuroectodermal enhancers and aberrant gene expression. Our study describes a previously unidentified disorder we name LINKED (LINKage-specific deubiquitylation deficiency-induced Embryonic Defects) syndrome and reveals linkage-specific ubiquitin cleavage from chromatin remodelers as an essential signaling mode that coordinates chromatin remodeling during embryogenesis.

Somatic Mutations in UBA1 and Severe Adult-Onset Autoinflammatory Disease.

BACKGROUND: Adult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODS: We analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9-edited zebrafish were used as an in vivo model to assess gene function. RESULTS: We identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene UBA1 lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONS: Using a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. (Funded by the NIH Intramural Research Programs and the EU Horizon 2020 Research and Innovation Program.).

Control of craniofacial and brain development by Cullin3-RING ubiquitin ligases: Lessons from human disease genetics.

Metazoan development relies on intricate cell differentiation, communication, and migration pathways, which ensure proper formation of specialized cell types, tissues, and organs. These pathways are crucially controlled by ubiquitylation, a reversible post-translational modification that regulates the stability, activity, localization, or interaction landscape of substrate proteins. Specificity of ubiquitylation is ensured by E3 ligases, which bind substrates and co-operate with E1 and E2 enzymes to mediate ubiquitin transfer. Cullin3-RING ligases (CRL3s) are a large class of multi-subunit E3s that have emerged as important regulators of cell differentiation and development. In particular, recent evidence from human disease genetics, animal models, and mechanistic studies have established their involvement in the control of craniofacial and brain development. Here, we summarize regulatory principles of CRL3 assembly, substrate recruitment, and ubiquitylation that allow this class of E3s to fulfill their manifold functions in development. We further review our current mechanistic understanding of how specific CRL3 complexes orchestrate neuroectodermal differentiation and highlight diseases associated with their dysregulation. Based on evidence from human disease genetics, we propose that other unknown CRL3 complexes must help coordinate craniofacial and brain development and discuss how combining emerging strategies from the field of disease gene discovery with biochemical and human pluripotent stem cell approaches will likely facilitate their identification.

Transient ALT activation protects human primary cells from chromosome instability induced by low chronic oxidative stress.

Cells are often subjected to the effect of reactive oxygen species (ROS) as a result of both intracellular metabolism and exposure to exogenous factors. ROS-dependent oxidative stress can induce 8-oxodG within the GGG triplet found in the G-rich human telomeric sequence (TTAGGG), making telomeres highly susceptible to ROS-induced oxidative damage. Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes and their dysfunction is believed to affect a wide range of cellular and/or organismal processes. Acute oxidative stress was shown to affect telomere integrity, but how prolonged low level oxidative stress, which may be more physiologically relevant, affects telomeres is still poorly investigated. Here, we explored this issue by chronically exposing human primary fibroblasts to a low dose of hydrogen peroxide. We observed fluctuating changes in telomere length and fluctuations in the rates of chromosome instability phenotypes, such that when telomeres shortened, chromosome instability increased and when telomeres lengthened, chromosome instability decreased. We found that telomere length fluctuation is associated with transient activation of an alternative lengthening of telomere (ALT) pathway, but found no evidence of cell death, impaired proliferation, or cell cycle arrest, suggesting that ALT activation may prevent oxidative damage from reaching levels that threaten cell survival.

Differential dielectric responses of chondrocyte and Jurkat cells in electromanipulation buffers.

Electromanipulation of cells as a label-free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCBs) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance, and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component.