39 research outputs found

    Intermolecular complexes of MT1-MMP, Cdc42 and 2 1 EC lumen formation in 3D collagen matrices [abstract]

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    A major focus of our laboratory is to elucidate molecular mechanisms controlling the ability of endothelial cells (ECs) to form luminal structures during vascular morphogenesis in 3D collagen matrices. EC luminal and tube formation is particularly dependent on a signaling axis involving the collagen-binding integrin 2 1, the Rho GTPases, Cdc42 and Rac1 and the membrane- type 1 metalloproteinase, MT1-MMP. This information was obtained using blocking antibody experiments directed to 2 1, dominant negative mutants for Cdc42 and Rac1, siRNA suppression for all four molecules and proteinase inhibitors for MT1- MMP. In all four cases, blockade of function of these molecules inhibits EC lumen formation. How these molecules signal together to control vascular morphogenesis in collagen matrices is not well understood and is the subject of considerable investigation in our laboratory. To begin to address this question, we hypothesized that these proteins may exist in intermolecular complexes that might be regulated during EC lumen and tube formation. Using either epitope-tagged MT1-MMP or Cdc42 we were able to specifically capture each of the above endogenous proteins from ECs, suggesting that they work in conjuction to promote lumen formation. Furthermore, we see a stronger association of these intermolecular complexes during EC tube formation in 3D collagen matrices (compared to 2D matrices) which is in part controlled by EC interactions with collagen and other matrix components. These data suggest that intermolecular complexes of MT1-MMP, Cdc42 and integrins control EC lumen formation during vascular morphogenesis

    Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes

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    Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature

    Molecular control of endothelial tube formation from single and aggregated cells by lumen signalling complexes that contain MT1-MMP and CDC42 in 3D collagen matrices

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    The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from PDF of title page (University of Missouri--Columbia, viewed on August 3, 2010).Vita.Thesis advisor: George E. Davis."May 2010"Ph. D. University of Missouri-Columbia 2010.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] The first blood vessels to form in the embryo are generated by vasculogenesis. New insight into vasculogenesis in mammals is emerging from studies of various transgenic mice or the use of in vitro systems. The Davis lab for many years now has established the use of unique in vitro models that have the ability of elucidating the molecular controls underlying this vascular event. (Davis, 1996, Davis, 2002, Davis, 2003, Kamei et al, 2006) Prior work has revealed a critical role for extracellular matrices and matrix metalloproteinases as well as RhoGTPases in the molecular control of vascular morphogenesis in three-dimensional (3D) tissue environments. Formation of intracellular vacuoles has also been implicated to be a major mechanism regulating EC lumen development both in vivo and in vitro (Davis et al, 2002, Folkman, 1980, Montesamo, 1988, Nicosia et al, 1982) Even though there is considerable work done in identifying key regulators of EC vasculogenesis there are still many unanswered questions in regards to lumen formation from single cells or from aggregated cells. The experiments described in this dissertation were designed to identify new molecular requirements and further our understanding of EC single and aggregated cell lumen formation in 3D collagen type I matrices.Includes bibliographical references

    Formation of endothelial lumens requires a coordinated PKCε-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation

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    In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCε in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCε during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCε- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCε, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices

    MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices

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    Complex signaling events control tumor invasion in three-dimensional (3D) extracellular matrices. Recent evidence suggests that cells utilize both matrix metalloproteinase (MMP)-dependent and MMP-independent means to traverse 3D matrices. Herein, we demonstrate that lysophosphatidic-acid-induced HT1080 cell invasion requires membrane-type-1 (MT1)-MMP-mediated collagenolysis to generate matrix conduits the width of a cellular nucleus. We define these spaces as single-cell invasion tunnels (SCITs). Once established, cells can migrate within SCITs in an MMP-independent manner. Endothelial cells, smooth muscle cells and fibroblasts also generate SCITs during invasive events, suggesting that SCIT formation represents a fundamental mechanism of cellular motility within 3D matrices. Coordinated cellular signaling events are required during SCIT formation. MT1-MMP, Cdc42 and its associated downstream effectors such as MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) and Pak4 (p21 protein-activated kinase 4), protein kinase Cα and the Rho-associated coiled-coil-containing protein kinases (ROCK-1 and ROCK-2) coordinate signaling necessary for SCIT formation. Finally, we show that MT1-MMP and Cdc42 are fundamental components of a co-associated invasion-signaling complex that controls directed single-cell invasion of 3D collagen matrices

    RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG

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    ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knockdown on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knockdown of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver. (Blood. 2011; 118(4):1145-1153)</p
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