Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

Application of Cell Impedance as a Screening Tool to Discover Modulators of Intraocular Pressure.

Purpose: To identify new targets and compounds involved in mediating cellular contractility or relaxation in trabecular meshwork (TM) cells and test their efficacy in an ex vivo model measuring outflow facility. Methods: A low-molecular weight compound library composed of 3,957 compounds was screened for cytoskeletal changes using the Acea xCelligence impedance platform in immortalized human NTM5 TM cells. Hits were confirmed by 8-point concentration response and were subsequently evaluated for impedance changes in 2 primary human TM strains, as well as cross-reactivity in bovine primary cells. A recently described bovine whole eye perfusion system was used to evaluate effects of compounds on aqueous outflow facility. Results: The primary screen conducted was robust, with Z' values >0.5. Fifty-two compounds were identified in the primary screen and confirmed to have concentration-dependent effects on impedance in NTM5 cells. Of these, 9 compounds representing distinct drug classes were confirmed to modulate impedance in both human primary TM cells and bovine cells. One of these compounds, wortmannin, an inhibitor of phosphoinositide 3-kinase, increased outflow facility by 11%. Conclusions: A robust phenotypic assay was developed that enabled identification of contractility modulators in immortalized TM cells. The screening hits were translatable to primary TM cells and modulated outflow facility in an ex vivo perfusion assay.

Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.

Regulation of vascular endothelial junction stability and remodeling through Rap1-Rasip1 signaling.

The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell-cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions.   The vasculature is a dynamic organ that is constantly exposed to a variety of signaling stimuli and mechanical stresses. In embryogenesis, nascent blood vessels form via a process termed vasculogenesis, wherein mesodermally derived endothelial precursor cells aggregate into cords, which subsequently form a lumen that permits trafficking of plasma and erythrocytes. (1)(,) (2) Angiogenesis occurs after establishment of this primitive vascular network, where new vessels sprout from existing vessels, migrate into newly expanded tissues, and anastomose to form a functional and complex circulatory network. (1)(,) (2) In the mouse, this process occurs through the second half of embryogenesis and into postnatal development in some tissues, such as the developing retinal vasculature. (3) Further, angiogenesis occurs in a variety of pathological conditions, such as diabetic retinopathy, age-related macular degeneration, inflammatory diseases such as rheumatoid arthritis, wound healing, and tumor growth. (1)(,) (2)(,) (4) Both vasculogenesis and angiogenesis are driven through signaling by vascular endothelial growth factor (VEGF), and therapeutic agents targeting this pathway have shown efficacy in a number of diseases. (5)(-) (9) Blood vessels must have a sufficient degree of integrity so as to not allow indiscriminate leak of plasma proteins and blood cells into the underlying tissue. However, vessels must be able to sense their environment, respond to local conditions, and mediate the regulated passage of protein, fluid, and cells. For example, endothelial cells are the primary point of attachment for immune cells leaving the blood stream and entering tissue, and leukocytes subsequently migrate either through the endothelial cell body itself (the transcellular route), or through transient disassembly of cell-cell junctions (the paracellular route). (10) Precise regulation of endothelial junctions is critical to the proper maintenance of vascular integrity and related processes, and disruption of vascular cell-cell contacts is an underlying cause or contributor to numerous pathologies such as cerebral cavernous malformations (CCM) and hereditary hemorrhagic telangiectasia (HHT). (11)(-) (13) Understanding the basic mechanisms of endothelial junction formation and maintenance will therefore lead to a greater chance of success of therapeutic intervention in these pathologic conditions, especially in instances where targeting of VEGF signaling is insufficient to resolve vascular abnormalities.

Fused (Stk36) is a ciliary protein required for central pair assembly and motile cilia orientation in the mammalian oviduct.

BACKGROUND: Motile cilia on the inner lining of the oviductal epithelium play a central role in ovum transport toward the uterus and subsequent fertilization by sperm. While the basic ultrastructure of 9+2 motile cilia (nine peripheral microtubule doublets surrounding a central pair) has been characterized, many important steps of ciliogenesis remain poorly understood. RESULTS: Our previous studies on mammalian Fused (Fu) (Stk36), a putative serine-threonine kinase, reveal a critical function of Fu in central pair construction and cilia orientation of motile cilia that line the tracheal and ependymal epithelia. These findings identify a novel regulatory component for these processes. In this study, we show that Fu is expressed in the multi-ciliated oviductal epithelium in several vertebrates, suggesting a conserved function of Fu in the oviduct. In support of this, analysis of Fu-deficient mouse oviducts uncovers a similar role of Fu in central pair construction and cilia orientation. We also demonstrate that Fu localizes to motile cilia and physically associates with kinesin Kif27 located at the cilium base and known central pair components Spag16 and Pcdp1. CONCLUSIONS: Our results delineate a novel pathway for central pair apparatus assembly and add important insight to the biogenesis and function of oviductal motile cilia.

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling.

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.

Impact of alternative fuels on emissions characteristics of a gas turbine engine - part 1: gaseous and particulate matter emissions.

Growing concern over emissions from increased airport operations has resulted in a need to assess the impact of aviation related activities on local air quality in and around airports, and to develop strategies to mitigate these effects. One such strategy being investigated is the use of alternative fuels in aircraft engines and auxiliary power units (APUs) as a means to diversify fuel supplies and reduce emissions. This paper summarizes the results of a study to characterize the emissions of an APU, a small gas turbine engine, burning conventional Jet A-1, a fully synthetic jet fuel, and other alternative fuels with varying compositions. Gas phase emissions were measured at the engine exit plane while PM emissions were recorded at the exit plane as well as 10 m downstream of the engine. Five percent reduction in NO(x) emissions and 5-10% reduction in CO emissions were observed for the alternative fuels. Significant reductions in PM emissions at the engine exit plane were achieved with the alternative fuels. However, as the exhaust plume expanded and cooled, organic species were found to condense on the PM. This increase in organic PM elevated the PM mass but had little impact on PM number.

A cell-based screen for inhibitors of flagella-driven motility in Chlamydomonas reveals a novel modulator of ciliary length and retrograde actin flow.

Cilia are motile and sensory organelles with critical roles in physiology. Ciliary defects can cause numerous human disease symptoms including polycystic kidneys, hydrocephalus, and retinal degeneration. Despite the importance of these organelles, their assembly and function is not fully understood. The unicellular green alga Chlamydomonas reinhardtii has many advantages as a model system for studies of ciliary assembly and function. Here we describe our initial efforts to build a chemical-biology toolkit to augment the genetic tools available for studying cilia in this organism, with the goal of being able to reversibly perturb ciliary function on a rapid time-scale compared to that available with traditional genetic methods. We screened a set of 5520 compounds from which we identified four candidate compounds with reproducible effects on flagella at nontoxic doses. Three of these compounds resulted in flagellar paralysis and one induced flagellar shortening in a reversible and dose-dependent fashion, accompanied by a reduction in the speed of intraflagellar transport. This latter compound also reduced the length of cilia in mammalian cells, hence we named the compound "ciliabrevin" due to its ability to shorten cilia. This compound also robustly and reversibly inhibited microtubule movement and retrograde actin flow in Drosophila S2 cells. Ciliabrevin may prove especially useful for the study of retrograde actin flow at the leading edge of cells, as it slows the retrograde flow in a tunable dose-dependent fashion until flow completely stops at high concentrations, and these effects are quickly reversed upon washout of the drug.

Vertebrate Hedgehog signaling: cilia rule.

The Hedgehog (Hh) signaling pathway differentially utilizes the primary cilium in mammals and fruit flies. Recent work, including a study in BMC Biology, demonstrates that Hh signals through the cilium in zebrafish, clarifying the evolution of Hh signal transduction. See research article: http://www.biomedcentral.com/1741-7007/8/65.

Mechanism and evolution of cytosolic Hedgehog signal transduction.

Hedgehog (Hh) signaling is required for embryonic patterning and postnatal physiology in invertebrates and vertebrates. With the revelation that the primary cilium is crucial for mammalian Hh signaling, the prevailing view that Hh signal transduction mechanisms are conserved across species has been challenged. However, more recent progress on elucidating the function of core Hh pathway cytosolic regulators in Drosophila, zebrafish and mice has confirmed that the essential logic of Hh transduction is similar between species. Here, we review Hh signaling events at the membrane and in the cytosol, and focus on parallel and divergent functions of cytosolic Hh regulators in Drosophila and mammals.

Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved.

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.

Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium.

Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

Fused has evolved divergent roles in vertebrate Hedgehog signalling and motile ciliogenesis.

Hedgehog (Hh) signalling is essential for several aspects of embryogenesis. In Drosophila, Hh transduction is mediated by a cytoplasmic signalling complex that includes the putative serine-threonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2, also known as Cos), yet Fu does not have a conserved role in Hh signalling in mammals. Mouse Fu (also known as Stk36) mutants are viable and seem to respond normally to Hh signalling. Here we show that mouse Fu is essential for construction of the central pair apparatus of motile, 9+2 cilia and offers a new model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 orthologue, and linked Fu to known structural components of the central pair apparatus, providing evidence for the first regulatory component involved in central pair construction. We also demonstrated that zebrafish Fu is required both for Hh signalling and cilia biogenesis in Kupffer's vesicle. Mouse Fu rescued both Hh-dependent and -independent defects in zebrafish. Our results delineate a new pathway for central pair apparatus assembly, identify common regulators of Hh signalling and motile ciliogenesis, and provide insights into the evolution of the Hh cascade.

New "hogs" in Hedgehog transport and signal reception.

A recent paper in Cell (Yao et al., 2006) and two papers in Developmental Cell (Tenzen et al., 2006; Zhang et al., 2006) identify a new receptor component for Hedgehog, a key morphogen in embryonic development. Many other proteins that bind to Hedgehog in the extracellular matrix or on the cell surface have been identified. In light of these recent discoveries, we discuss how these factors control the stability, transport, reception, and availability of Hedgehog in modulating Hedgehog-mediated responses.

GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis.

Truncating mutations in Gli3, an intracellular effector in the SHH-SMO-GLI signaling pathway, cause renal aplasia/dysplasia in humans and mice. Yet, the pathogenic mechanisms are undefined. Here, we report the effect of decreased SHH-SMO signaling on renal morphogenesis, the expression of SHH target genes and GLI binding to Shh target genes. Shh deficiency or cyclopamine-mediated SMO inhibition disrupted renal organogenesis, decreased expression of GLI1 and GLI2 proteins, but increased expression of GLI3 repressor relative to GLI3 activator. Shh deficiency decreased expression of kidney patterning genes (Pax2 and Sall1) and cell cycle regulators (cyclin D1 and MYCN). Elimination of Gli3 in Shh(-/-) mice rescued kidney malformation and restored expression of Pax2, Sall1, cyclin D1, MYCN, Gli1 and Gli2. To define mechanisms by which SHH-SMO signaling controls gene expression, we determined the binding of GLI proteins to 5' flanking regions containing GLI consensus binding sequences in Shh target genes using chromatin immunoprecipitation. In normal embryonic kidney tissue, GLI1 and/or GLI2 were bound to each target gene. By contrast, treatment of embryonic kidney explants with cyclopamine decreased GLI1 and/or GLI2 binding, and induced binding of GLI3. However, cyclopamine failed to decrease Gli1 and Gli2 expression and branching morphogenesis in Gli3-deficient embryonic kidney tissue. Together, these results demonstrate that SHH-SMO signaling controls renal morphogenesis via transcriptional control of Gli, renal patterning and cell cycle regulator genes in a manner that is opposed by GLI3.