Early host responses toward pathogens are essential for defense against infection. In Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets, which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In addition, activating p38/MAPK signaling in the absence of pathogens, is sufficient to drive SKN-1–dependent loss of somatic fat. These data define a SKN-1– and p38-dependent axis for coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic consequences of aberrant transcriptional activity.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Fluid shear stress provided by blood flow instigates a transition from active blood vessel network expansion during development, to vascular homeostasis and quiescence that is important for mature blood vessel function. Here we show that SMAD6 is required for endothelial cell flow-mediated responses leading to maintenance of vascular homeostasis. Concomitant manipulation of the mechanosensor Notch1 pathway and SMAD6 expression levels revealed that SMAD6 functions downstream of ligand-induced Notch signaling and transcription regulation. Mechanistically, full-length SMAD6 protein was needed to rescue Notch loss-induced flow misalignment. Endothelial cells depleted for SMAD6 had defective barrier function accompanied by upregulation of proliferation-associated genes and down regulation of junction-associated genes. The vascular protocadherin PCDH12 was upregulated by SMAD6 and required for proper flow-mediated endothelial cell alignment, placing it downstream of SMAD6. Thus, SMAD6 is a required transducer of flow-mediated signaling inputs downstream of Notch1 and upstream of PCDH12, as vessels transition from an angiogenic phenotype to maintenance of a homeostatic phenotype.

Exposure to environmental stress is clinically established to influence male reproductive health, but the impact of normal cellular metabolism on sperm quality is less well-defined. Here we show that impaired mitochondrial proline catabolism, reduces energy-storing flavin adenine dinucleotide (FAD) levels, alters mitochondrial dynamics toward fusion, and leads to age-related loss of sperm quality (size and activity), which diminishes competitive fitness of the animal. Loss of the 1-pyrroline-5-carboxylate dehydrogenase enzyme alh-6 that catalyzes the second step in mitochondrial proline catabolism leads to premature male reproductive senescence. Reducing the expression of the proline catabolism enzyme alh-6 or FAD biosynthesis pathway genes in the germline is sufficient to recapitulate the sperm-related phenotypes observed in alh-6 loss-of-function mutants. These sperm-specific defects are suppressed by feeding diets that restore FAD levels. Our results define a cell autonomous role for mitochondrial proline catabolism and FAD homeostasis on sperm function and specify strategies to pharmacologically reverse these defects.

Objective: Endothelial cells (ECs) that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to EC quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level ECs regulate overall expression heterogeneity during this transition is poorly understood. Here, we profiled EC transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow. Approach and Results: Single-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human ECs under homeostatic laminar flow compared to nonflow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous, although small subsets of cells did not follow this pattern. Analysis of a subset of genes for relative protein expression revealed little congruence between RNA and protein heterogeneity changes under flow. In contrast, the magnitude of expression level changes in RNA and protein was more coordinated among ECs in flow versus nonflow conditions. Conclusions: ECs exposed to homeostatic laminar flow showed overall increased heterogeneity in RNA expression levels, while expression heterogeneity of selected cognate proteins did not follow RNA heterogeneity changes closely. These findings suggest that EC homeostasis is imposed post-transcriptionally in response to laminar flow.

20Exposure to environmental stress is clinically established to influence male reproductive health, but the 21 impact of normal cellular metabolism on sperm quality is less well-defined. Here we show that impaired 22 mitochondrial proline catabolism, reduces energy-storing flavin adenine dinucleotide (FAD) levels, 23 alters mitochondrial dynamics toward fusion, and leads to age-related loss of sperm quality (size and 24 activity), which reduces competitive fitness. Loss of the 1-pyrroline-5-carboxylate dehydrogenase 25 enzyme alh-6 that catalyzes the second step in mitochondrial proline catabolism, leads to premature 26 male reproductive senescence. Reducing the expression of the proline catabolism enzyme alh-6 or 27 FAD biosynthesis pathway genes in the germline is sufficient to recapitulate the sperm-related 28 phenotypes observed in alh-6 loss-of-function mutants. These sperm-specific defects are suppressed 29 by feeding diets that restore FAD levels. Our results define a cell autonomous role for mitochondrial 30 proline catabolism and FAD homeostasis on sperm function and specify strategies to pharmacologically 31 reverse these defects.

ObjectiveEndothelial cells that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to endothelial cell quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level endothelial cells regulate overall expression heterogeneity during this transition is poorly understood. Here we profiled endothelial cell transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow.Approach and ResultsSingle-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human endothelial cells under homeostatic laminar flow compared to non-flow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous. Analysis of a subset of genes for relative protein expression revealed that most protein profiles showed decreased heterogeneity under flow. In contrast, the magnitude of expression level changes in RNA and protein was coordinated among endothelial cells in flow vs. non-flow conditions.ConclusionsEndothelial cells exposed to homeostatic laminar flow showed increased cohort heterogeneity in RNA expression levels, while cohort expression heterogeneity of selected cognate proteins decreased under laminar flow. These findings suggest that EC homeostasis is imposed at the level of protein translation and/or stability rather than transcriptionally.

Tumor blood vessels support tumor growth and progression. Centrosomes are microtubule organization centers in cells, and often up to 30% of tumor endothelial cells (ECs) acquire excess (>2) centrosomes. Although excess centrosomes can lead to aneuploidy and chromosome instability in tumor cells, how untransformed ECs respond to excess centrosomes is poorly understood. We found that the frequency of primary human ECs with excess centrosomes was quickly reduced in a p53-dependent manner. Excess centrosomes in ECs were associated with p53 phosphorylation at Ser33, increased p21 levels, and decreased cell proliferation and expression of senescence markers, but independent of DNA damage and apoptosis. Aspects of the senescence-associated phenotype were also observed in mouse ECs that were isolated from tumors with excess centrosomes. Primary ECs with excess centrosomes in vascular sprouts also had elevated Ser33 p53 phosphorylation and expressed senescence markers. Our work demonstrates that nontransformed ECs respond differently to excess centrosomes than do most tumor cells-they undergo senescence in vascular sprouts and vessels, which suggests that pathologic outcomes of centrosome overduplication depend on the transformation status of cells.-Yu, Z., Ruter, D. L., Kushner, E. J., Bautch, V. L. Excess centrosomes induce p53-dependent senescence without DNA damage in endothelial cells.