Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

CXCR3-CXCL11 signaling restricts angiogenesis and promotes pericyte recruitment.

Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces, yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs-the chemokine receptor, CXCR3, and one of its ligands, CXCL11-that delimits EC angiogenic potential and suppresses pericyte recruitment during development through regulation of pdgfb expression in ECs. In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with and expansion of the vasculature in zebrafish treated with the Cxcr3 inhibitor AMG487. We also demonstrate using flow modeling platforms that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared to their control counterparts. Together these data suggest that CXCR3 signaling in ECs drives vascular stabilization events during development.

Characterization of Bud3 domains sufficient for bud neck targeting in S. cerevisiae.

The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds in vivo that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the 'bud neck' is critical for completion of cytokinesis and execution of numerous other cellular events. One such pathway includes bud site selection and the recruitment of proteins such as Bud4 and Bud3 that are responsible for promoting an axial budding pattern in haploid yeast. While Bud4 appears to be recruited to the septins independently of the presence of Bud3, it is likely that Bud3 can localize to the bud neck using both Bud4-dependent and Bud4-independent mechanisms. Furthermore, it remains unclear which precise domain or domains within Bud3 is/are both necessary and sufficient for optimal association at the septin structure. In this study, we examined the localization of GFP-Bud3 constructs in otherwise wild-type (WT) haploid yeast cells expressing Cdc10-mCherry using fluorescence microscopy; we tested a collection of N- and C-terminal truncations and fusions of separate Bud3 protein elements to identify the smallest domain(s) responsible for bud neck localization. We found that the coordinate action of the central amphipathic helix (residues 847-865) and a partially conserved C-terminal motif (residues 1172-1273) was sufficient to promote bud neck recruitment in the presence of endogenous Bud3. This domain is considerably smaller than the previously characterized C-terminal portion required to physically interact with Bud4 (1221-1636) and utilizes a similar mechanism of pairing membrane association, with a separate localization domain, similar to other non-septin proteins targeted to the division site during cell division.

Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae.

BACKGROUND: The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. Additional research into molecular mechanisms that would allow for control, titration, and inhibition of drive systems is needed. RESULTS: In this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells. CONCLUSIONS: These findings have implications for control of traditional nuclease-dependent editing and use of gene drive systems within other organisms. For instance, initiation of a nuclear export mechanism to Cas9 could serve as a molecular safeguard within an active gene drive to reduce or eliminate editing.

Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of 'anti-CRISPR' proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the 'gene drive'. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.