Venous and arterial endothelial proteomics: mining for markers and mechanisms of endothelial diversity

Endothelial cells (ECs) line the inside of arterial and venous blood vessels in a continuous monolayer and have the important function of responding to environmental cues to regulate vascular tone and new blood vessel formation. They also have well-defined roles in supporting tumorigenesis, and alterations in their function lead to cardiovascular disease. Consequently, ECs have been studied extensively as a cellular model of both normal and abnormal physiology. Despite their importance and the increased utility of proteomic tools in medical research, there are relatively few publications on the topic of vascular endothelial proteomics. A thorough search of the literature mined 52 publications focused exclusively on arterial and/or venous endothelial proteomics. These studies mostly relied upon examination of whole-cell lysates from cultured human umbilical vein ECs to investigate in vitro effects of various molecules, such as VEGF in the context of altering human umbilical vein EC functions related to angiogenesis. Only a few of these publications focused solely on a proteomic characterization of ECs and our analysis further revealed a lack of published studies incorporating proteomic analysis of freshly isolated ECs from tissues or in vitro conditions that mimic in vivo variables, such as oxygen tension and shear stress. It is the purpose of this article to account for the diversity of vascular EC proteomic investigations and comment on the issues that have been and should be addressed in future work.     

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

Summary Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.     

Another role for RNA: a messenger across generations

There has been a recent resurgence of interest in the notion that DNA is not the sole determinant of our inherited phenotype. The strongest evidence for transgenerational epigenetic inheritance has come from studies of paramutation in plants. But few examples have been reported in other species, and the molecular basis for the process has been unclear. A recent paper by Rassoulzadegan and colleagues provides clear evidence of paramutation in mice and strong evidence that RNA is involved.     

Differential roles of Rap1 and Rap2 small GTPases in neurite retraction and synapse elimination in hippocampal spiny neurons

The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.     

Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.     

Working at the interface of phylogenetics and population genetics: a biogeographical analysis of Triaenops spp. (Chiroptera: Hipposideridae)

New applications of genetic data to questions of historical biogeography have revolutionized our understanding of how organisms have come to occupy their present distributions. Phylogenetic methods in combination with divergence time estimation can reveal biogeographical centres of origin, differentiate between hypotheses of vicariance and dispersal, and reveal the directionality of dispersal events. Despite their power, however, phylogenetic methods can sometimes yield patterns that are compatible with multiple, equally well-supported biogeographical hypotheses. In such cases, additional approaches must be integrated to differentiate among conflicting dispersal hypotheses. Here, we use a synthetic approach that draws upon the analytical strengths of coalescent and population genetic methods to augment phylogenetic analyses in order to assess the biogeographical history of Madagascar's Triaenops bats (Chiroptera: Hipposideridae). Phylogenetic analyses of mitochondrial DNA sequence data for Malagasy and east African Triaenops reveal a pattern that equally supports two competing hypotheses. While the phylogeny cannot determine whether Africa or Madagascar was the centre of origin for the species investigated, it serves as the essential backbone for the application of coalescent and population genetic methods. From the application of these methods, we conclude that a hypothesis of two independent but unidirectional dispersal events from Africa to Madagascar is best supported by the data.     

Molecular characterization of the effects of Y-27632

Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.     

Shroom4 (Kiaa1202) is an actin-associated protein implicated in cytoskeletal organization

All animal cells utilize a specialized set of cytoskeletal proteins to determine their overall shape and the organization of their intracellular compartments and organelles. During embryonic development, the dynamic nature of the actin cytoskeleton is critical for virtually all morphogenic events requiring changes in cell shape, migration, adhesion, and division. The behavior of the actin cytoskeleton is modulated by a myriad of accessory proteins. Shroom3 is an actin binding protein that regulates neural tube morphogenesis by eliciting changes in cell shape through a myosin II-dependent pathway. The Shroom-related gene SHROOM4 (formerly called KIAA1202) has also been implicated in neural development, as mutations in this gene are associated with human X-linked mental retardation. To better understand the function of Shrm4 in embryonic development, we have cloned mouse Shroom4 and characterized its protein product in vivo and in vitro. Shroom4 is expressed in a wide range of cell types during mouse development, including vascular endothelium and the polarized epithelium of the neural tube and kidney. In endothelial cells and embryo fibroblasts, endogenous Shroom4 co-distributes with myosin II to a distinct cytoplasmic population of F-actin and ectopic expression of Shroom4 in multiple cell types enhances or induces the formation of this actin-based structure. This localization is mediated, at least in part, by the direct interaction of Shroom4 and F-actin. Our results suggest that Shroom4 is a regulator of cytoskeletal architecture that may play an important role in vertebrate development.     

Enteropathogenic Escherichia coli Tir is an SH2/3 ligand that recruits and activates tyrosine kinases required for pedestal formation

Enteropathogenic Escherichia coli (EPEC) cause intestinal inflammation, severe diarrhoea and mortality, particularly among children in developing nations. Upon attachment to intestinal epithelial cells, EPEC induces actin-filled membrane protrusions called 'pedestals' and disrupts microvilli to form attaching and effacing (A/E) lesions. EPEC also disrupts epithelial barrier function and causes colitis. Here we have investigated how virulence factors which orchestrate formation of actin pedestals interface with host tyrosine kinases. We show that Tec-family tyrosine kinases localize beneath EPEC and, with Abl-family kinases, comprise a set of redundant host kinases utilized by EPEC to form actin pedestals. We also show that Tir, a virulence factor required for pathogenesis, contains a polyproline region (PPR) that interacts with SH3 domains of redundant kinases, and a phosphorylation site (Y474) that interacts with kinase SH2 domains. These interactions are essential for pedestal formation, and mimic activation of kinases by cellular ligands. Our results suggest that a positive feedback loop exists in which initial phosphorylation of Tir on Y474 by tyrosine kinases causes recruitment of additional redundant kinases via PPR-SH3 interactions and PO(3)-Y474-SH2 interactions, which in turn phosphorylate other Tir molecules as well as proteins that catalyse formation of actin pedestals.