PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum.

Tspan18 is a novel regulator of the Ca channel Orai1 and von Willebrand factor release in endothelial cells.

Ca entry Orai1 store-operated Ca channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.

Sunitinib Treatment Enhances Metastasis of Innately Drug-Resistant Breast Tumors.

Antiangiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date, there has not been an inquiry into the roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the antiangiogenic tyrosine kinase inhibitor sunitinib.

Tube-Forming Assays.

Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis.

Glutamate dependent NMDA receptor 2D is a novel angiogenic tumour endothelial marker in colorectal cancer.

Current vascular-targeted therapies in colorectal cancer (CRC) have shown limited benefit. The lack of novel, specific treatment in CRC has been hampered by a dearth of specific endothelial markers. Microarray comparison of endothelial gene expression in patient-matched CRC and normal colon identified a panel of putative colorectal tumour endothelial markers.

MCAM and LAMA4 Are Highly Enriched in Tumor Blood Vessels of Renal Cell Carcinoma and Predict Patient Outcome.

The structure and molecular signature of tumor-associated vasculature are distinct from those of the host tissue, offering an opportunity to selectively target the tumor blood vessels. To identify tumor-specific endothelial markers, we performed a microarray on tumor-associated and nonmalignant endothelium collected from patients with renal cell carcinoma (RCC), colorectal carcinoma, or colorectal liver metastasis. We identified a panel of genes consistently upregulated by tumor blood vessels, of which melanoma cell adhesion molecule (MCAM) and its extracellular matrix interaction partner laminin alpha 4 (LAMA4) emerged as the most consistently expressed genes.

RhoJ interacts with the GIT-PIX complex and regulates focal adhesion disassembly.

RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it.

The role of RhoJ in endothelial cell biology and angiogenesis.

RhoJ is an endothelially expressed member of the Cdc42 (cell division cycle 42) subfamily of small Rho GTPases. It is expressed in both the developing mammalian vasculature and the vascular beds of a number of adult tissues, with its expression regulated by the endothelial transcription factor ERG (ETS-related gene). RhoJ has been shown to regulate endothelial motility, tubulogenesis and lumen formation in vitro, and modulates the vascularization of Matrigel plugs in vivo.

Shear stress, tip cells and regulators of endothelial migration.

We have in recent years described several endothelial-specific genes that mediate cell migration. These include Robo4 (roundabout 4), CLEC14A (C-type lectin 14A) and ECSCR (endothelial cell-specific chemotaxis regulator) [formerly known as ECSM2 (endothelial cell-specific molecule 2)]. Loss of laminar shear stress induces Robo4 and CLEC14A expression and an endothelial 'tip cell' phenotype.

RhoJ/TCL regulates endothelial motility and tube formation and modulates actomyosin contractility and focal adhesion numbers.

Objective: RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation.

Methods And Results: Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence.

Functionally defining the endothelial transcriptome, from Robo4 to ECSCR.

We have applied search algorithms to expression databases to identify genes whose expression is restricted to the endothelial cell. Such genes frequently play a critical role in endothelial biology and angiogenesis. Two such genes are the roundabout receptor Robo4 and the ECSCR (endothelial-cell-specific chemotaxis regulator).

Anticancer strategies involving the vasculature.

The growth and metastasis of solid tumors critically depends on their ability to develop their own blood supply, a process known as tumor angiogenesis. Over the past decade much work has been performed to understand this process, and modifying this process provides a key point of therapeutic intervention in the fight against cancer. This Review explores the development of anti-VEGF-based antiangiogenic therapies, of which there are currently three licensed for clinical use worldwide.

Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors.

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays.

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains.

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets.

ECSM2, an endothelial specific filamin a binding protein that mediates chemotaxis.

Objective: We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2).

Methods And Results: A full-length cDNA was isolated and predicted ECSM2 to be a putative 205-amino acid transmembrane protein that bears no homology to any known protein. Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown.

A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes.

Background: In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers.

A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein.

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis.

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease.

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT.

Differentiation of murine committed megakaryocytic progenitors isolated by a novel strategy reveals the complexity of GATA and Ets factor involvement in megakaryocytopoiesis and an unexpected potential role for GATA-6.

Objective: The differentiation of megakaryocytes is characterized by polyploidization and cytoplasmic maturation leading to platelet production. Studying these processes is hindered by the paucity of bone marrow megakaryocytes and their precursors. We describe a method for the expansion and purification of committed megakaryocyte progenitors and demonstrate their usefulness by studying changes in the expression of Ets and GATA family transcription factors throughout megakaryocytopoiesis.

Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions.

Polyphosphoinositides (PPIn) are low-abundance membrane phospholipids that each bind to a distinctive set of effector proteins and, thereby, regulate a characteristic suite of cellular processes. Major functions of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)] are in membrane and protein trafficking, and in pH control in the endosome-lysosome axis. Recently identified PtdIns(3,5)P(2) effectors include a family of novel beta-propeller proteins, for which we propose the name PROPPINs [for beta-propeller(s) that binds PPIn], and possibly proteins of the epsin and CHMP (charged multi-vesicular body proteins) families.

SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase.

The Tec family of protein-tyrosine kinases (PTKs), that includes Tec, Itk, Btk, Bmx, and Txk, plays an essential role in phospholipase Cgamma (PLCgamma) activation following antigen receptor stimulation. This function requires activation of phosphatidylinositol 3-kinase (PI 3-kinase), which promotes Tec membrane localization through phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) generation. The mechanism of negative regulation of Tec family PTKs is poorly understood.

Hph1p and Hph2p, novel components of calcineurin-mediated stress responses in Saccharomyces cerevisiae.

Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase that plays a key role in animal and yeast physiology. In the yeast Saccharomyces cerevisiae, calcineurin is required for survival during several environmental stresses, including high concentrations of Na+, Li+, and Mn2+ ions and alkaline pH. One role of calcineurin under these conditions is to activate gene expression through its regulation of the Crz1p transcription factor.