Quantitative morphological analysis of Deinococcus radiodurans elucidates complex dose-dependent nucleoid condensation during recovery from ionizing radiation.

The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

Single-cell morphology encodes functional subtypes of senescence in aging human dermal fibroblasts.

Cellular senescence is an established driver of aging, exhibiting context-dependent phenotypes across multiple biological length-scales. Despite its mechanistic importance, profiling senescence within cell populations is challenging. This is in part due to the limitations of current biomarkers to robustly identify senescent cells across biological settings, and the heterogeneous, non-binary phenotypes exhibited by senescent cells. Using a panel of primary dermal fibroblasts, we combined live single-cell imaging, machine learning, multiple senescence induction conditions, and multiple protein-based senescence biomarkers to show the emergence of functional subtypes of senescence. Leveraging single-cell morphologies, we defined eleven distinct morphology clusters, with the abundance of cells in each cluster being dependent on the mode of senescence induction, the time post-induction, and the age of the donor. Of these eleven clusters, we identified three bona-fide senescence subtypes (C7, C10, C11), with C10 showing the strongest age-dependence across a cohort of fifty aging individuals. To determine the functional significance of these senescence subtypes, we profiled their responses to senotherapies, specifically focusing on Dasatinib + Quercetin (D+Q). Results indicated subtype-dependent responses, with senescent cells in C7 being most responsive to D+Q. Altogether, we provide a robust single-cell framework to identify and classify functional senescence subtypes with applications for next-generation senotherapy screens, and the potential to explain heterogeneous senescence phenotypes across biological settings based on the presence and abundance of distinct senescence subtypes.

E-cadherin interacts with EGFR resulting in hyper-activation of ERK in multiple models of breast cancer.

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success.

Substrate stiffness modulates the emergence and magnitude of senescence phenotypes in dermal fibroblasts.

Cellular senescence is a major driver of aging and disease. Here we show that substrate stiffness modulates the emergence and magnitude of senescence phenotypes after exposure to senescence inducers. Using a primary dermal fibroblast model, we show that decreased substrate stiffness accelerates senescence-associated cell-cycle arrest and regulates the expression of conventional protein-based biomarkers of senescence. We found that the expression of these senescence biomarkers, namely p21WAF1/CIP1 and p16INK4a are mechanosensitive and are in-part regulated by myosin contractility through focal adhesion kinase (FAK)-ROCK signaling. Interestingly, at the protein level senescence-induced dermal fibroblasts on soft substrates (0.5 kPa) do not express p21WAF1/CIP1 and p16INK4a at comparable levels to induced cells on stiff substrates (4GPa). However, cells express CDKN1a, CDKN2a, and IL6 at the RNA level across both stiff and soft substrates. Moreover, when cells are transferred from soft to stiff substrates, senescent cells recover an elevated expression of p21WAF1/CIP1 and p16INK4a at levels comparable to senescence cells on stiff substrates, pointing to a mechanosensitive regulation of the senescence phenotype. Together, our results indicate that the emergent senescence phenotype depends critically on the local mechanical environments of cells and that senescent cells actively respond to changing mechanical cues.

Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells.

Particulate matter (PM) is a ubiquitous component of air pollution that is epidemiologically linked to human pulmonary diseases. PM chemical composition varies widely, and the development of high-throughput experimental techniques enables direct profiling of cellular effects using compositionally unique PM mixtures. Here, we show that in a human bronchial epithelial cell model, exposure to three chemically distinct PM mixtures drive unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability, DNA damage responses, and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Finally, we observed that PM mixtures with higher cadmium content induced increased DNA damage and drove redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of individual cellular morphologies provides a robust, high-throughput approach to gauge the effects of environmental stressors on biological systems and score cellular susceptibilities to pollution.

Particulate matter composition drives differential molecular and morphological responses in lung epithelial cells.

Particulate matter (PM) is a ubiquitous component of indoor and outdoor air pollution that is epidemiologically linked to many human pulmonary diseases. PM has many emission sources, making it challenging to understand the biological effects of exposure due to the high variance in chemical composition. However, the effects of compositionally unique particulate matter mixtures on cells have not been analyzed using both biophysical and biomolecular approaches. Here, we show that in a human bronchial epithelial cell model (BEAS-2B), exposure to three chemically distinct PM mixtures drives unique cell viability patterns, transcriptional remodeling, and the emergence of distinct morphological subtypes. Specifically, PM mixtures modulate cell viability and DNA damage responses and induce the remodeling of gene expression associated with cell morphology, extracellular matrix organization and structure, and cellular motility. Profiling cellular responses showed that cell morphologies change in a PM composition-dependent manner. Lastly, we observed that particulate matter mixtures with high contents of heavy metals, such as cadmium and lead, induced larger drops in viability, increased DNA damage, and drove a redistribution among morphological subtypes. Our results demonstrate that quantitative measurement of cellular morphology provides a robust approach to gauge the effects of environmental stressors on biological systems and determine cellular susceptibilities to pollution.

Senescent stroma induces nuclear deformations in cancer cells via the inhibition of RhoA/ROCK/myosin II-based cytoskeletal tension.

The presence of senescent cells within tissues has been functionally linked to malignant transformations. Here, using tension-gauge tethers technology, particle-tracking microrheology, and quantitative microscopy, we demonstrate that senescent-associated secretory phenotype (SASP) derived from senescent fibroblasts impose nuclear lobulations and volume shrinkage on malignant cells, which stems from the loss of RhoA/ROCK/myosin II-based cortical tension. This loss in cytoskeletal tension induces decreased cellular contractility, adhesion, and increased mechanical compliance. These SASP-induced morphological changes are, in part, mediated by Lamin A/C. These findings suggest that SASP induces defective outside-in mechanotransduction from actomyosin fibers in the cytoplasm to the nuclear lamina, thereby triggering a cascade of biophysical and biomolecular changes in cells that associate with malignant transformations.