The presence of tRNA genes in bacteriophages has been explained on the basis of codon usage (tRNA genes are retained in the phage genome if they correspond to codons more common in the phage than in its host) or amino acid usage (independent of codon, the amino acid corresponding to the retained tRNA gene is more common in the phage genome than in the bacterial host). The existence of a large database of sequenced mycobacteriophages, isolated on the common host Mycobacterium smegmatis, allows us to test the above hypotheses as well as explore other hypotheses for the presence of tRNA genes. Our analyses suggest that amino acid rather than codon usage better explains the presence of tRNA genes in mycobacteriophages. However, closely related phages that differ in the presence of tRNA genes in their genomes are capable of lysing the common bacterial host and do not differ in codon or amino acid usage. This suggests that the benefits of having tRNA genes may be associated with either growth in the host or the ability to infect more hosts (i.e., host range) rather than simply infecting a particular host.

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.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Endothelial cells line all blood vessels and coordinate blood vessel formation and the blood-tissue barrier via endothelial cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but the mechanisms are poorly understood. Here we show that nuclear-localized SUN1, a LINC complex component that connects the nucleus to the cytoskeleton, regulates endothelial cell-cell junction communication and blood vessel formation. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions. At the cellular level, SUN1 stabilized endothelial cell-cell junctions and promoted barrier function. Abnormal SUN1-depleted junctions resembled those seen with loss of microtubules, and they were accompanied by impaired microtubule dynamics and actomyosin hypercontractility. Angiogenic sprouts formed but retracted in SUN1-depleted endothelial cells, and vessels of zebrafish lacking SUN1 had abnormal extension and were defective in forming connections. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus, likely via microtubule-based interactions, and this long-range regulation is important for blood vessel formation and barrier function.

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Vascular endothelial cells regulate their cell cycle as blood vessels remodel and mature, and as they transition from active angiogenesis to quiescence. Mechanical cues provided by fluid shear stress orchestrate this transition, and laminar blood flow instigates a quiescent (G0) state and homeostasis. However, how flow-mediated quiescence is set up and maintained is poorly understood. We found that flow-mediated endothelial cell quiescence has unique properties and temporal regulation of quiescence depth. Flow-exposed endothelial cells had a distinct transcriptome, and quiescent endothelial cells re-entered the cell cycle more rapidly after extended flow exposure compared to contact inhibition, indicating a shallow quiescence depth. The cell cycle inhibitor CDKN1B (p27) was required for endothelial cell flow-mediated quiescence but was not significantly expressed after extended flow exposure. Rather, flow-exposed endothelial cells first established a deep quiescence that subsequently became shallow, and p27 levels positively correlated with these distinct quiescent states. HES1 and ID3, transcriptional repressors of p27 downstream of flow-regulated Notch and BMP signaling, were required for flow-mediated quiescence depth changes and the reduced p27 levels associated with shallow quiescence. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of transcriptional regulation of p27.