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      Microtubule-Associated Protein EB3 Regulates IP 3 Receptor Clustering and Ca 2+ Signaling in Endothelial Cells

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          Summary

          The mechanisms by which the microtubule cytoskeleton regulates the permeability of endothelial barrier are not well understood. Here, we demonstrate that microtubule-associated end-binding protein 3 (EB3), a core component of the microtubule plus-end protein complex, binds to inositol 1,4,5-trisphosphate receptors (IP 3Rs) through an S/TxIP EB-binding motif. In endothelial cells, α-thrombin, a pro-inflammatory mediator that stimulates phospholipase Cβ, increases the cytosolic Ca 2+ concentration and elicits clustering of IP 3R3s. These responses, and the resulting Ca 2+-dependent phosphorylation of myosin light chain, are prevented by depletion of either EB3 or mutation of the TxIP motif of IP 3R3 responsible for mediating its binding to EB3. We also show that selective EB3 gene deletion in endothelial cells of mice abrogates α-thrombin-induced increase in endothelial permeability. We conclude that the EB3-mediated interaction of IP 3Rs with microtubules controls the assembly of IP 3Rs into effective Ca 2+ signaling clusters, which thereby regulate microtubule-dependent endothelial permeability.

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          Highlights

          • IP 3 receptors (IP 3Rs) bind to microtubule end-binding protein EB3

          • EB3, in turn, promotes IP 3R clustering and Ca 2+ signals in endothelial cells

          • IP 3R-EB3 interaction thereby contributes to endothelial barrier disruption

          • In vivo EB3 deletion in endothelial cells protects from vascular hyperpermeability

          Abstract

          End binding proteins (EBs) mediate interactions between growing microtubules and intracellular structures. Geyer et al. demonstrate that interactions between EB3 and IP 3 receptors control clustering of IP 3 receptors in endothelial cells and Ca 2+ signaling, and thus permeability of endothelial barrier in inflammatory diseases.

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          Most cited references 47

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          Inositol trisphosphate receptor Ca2+ release channels.

          The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.
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            Endothelial cell-cell junctions: happy together.

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              An EB1-binding motif acts as a microtubule tip localization signal.

              Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
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                Author and article information

                Contributors
                Journal
                Cell Rep
                Cell Rep
                Cell Reports
                Cell Press
                2211-1247
                25 June 2015
                07 July 2015
                25 June 2015
                : 12
                : 1
                : 79-89
                Affiliations
                [1 ]Department of Pharmacology and The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA
                [2 ]Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, UK
                Author notes
                []Corresponding author ykomarov@ 123456uic.edu
                Article
                S2211-1247(15)00584-7
                10.1016/j.celrep.2015.06.001
                4487770
                26119739
                © 2015 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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                Cell biology

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