Regulation and function of the cerebral cavernous malformation 2 protein

Cerebral cavernous malformations (CCM) are vascular lesions of the central nervous system characterized as clusters of dilated, thin-walled blood vessels. CCM lesions are fragile and prone to vascular leakiness and rupture, leading to hemorrhages that cause seizure and stroke. Familial CCM has been shown to be genetically linked to three genes: CCM1, CCM2, and CCM3. The proteins encoded by these genes have no apparent catalytic activity, suggesting they are scaffolds to organize and localize functional protein complexes in cells. This scaffolding function has been appreciated for CCM2, which encodes Osmosensing Scaffold for MEKK3 (OSM). CCM2 (OSM) coordinates a signaling complex that consists of Rac1, MEKK3, and MKK3 to activate p38 in response to osmotic stimuli. The studies described here analyze the function of CCM2 in the context of cerebral cavernous malformations. Using proteomic, biochemical, and in vivo models, we characterize CCM2 as a critical regulator of endothelial cell signaling and function. We show that CCM2 binds and localizes the CCM1 protein. The CCM2 phosphotyrosine binding (PTB) domain is necessary for a canonical interaction with NPxY motifs within CCM1. We provide evidence of co-immunoprecipitation and fluorescence resonance energy transfer (FRET) between CCM1 and CCM2, implicating a common genetic and molecular pathway in CCM pathogenesis. We also characterize CCM2 as a Smurf1 binding partner. Through a novel CCM2 PTB domain - Smurf1 HECT domain interaction, CCM2 recruits Smurf1 to specific locations at the plasma membrane where it specifically degrades RhoA. Knockdown of CCM2 in brain endothelial cells leads to increased RhoA protein levels and ROCK signaling. Functionally, this leads to deficiencies in cell migration, tube formation, and maintenance of a permeability barrier. To determine the role of CCM2 in vivo, we used Danio rerio as a model for vertebrate development. Loss of CCM2 expression leads to decreased blood flow due to restrictions and abnormalities of the aortic arch. The findings presented here indicate that CCM2 regulates protein complexes and signaling pathways important in endothelial cell function and provide insight into the molecular mechanisms involved in cerebral cavernous malformation pathogenesis

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Abstract Background The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. Results This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. Conclusion These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope

CCM1 and CCM2 protein interactions in cell signaling: Implications for cerebral cavernous malformations pathogenesis

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and β1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex. © The Author 2005. Published by Oxford University Press. All rights reserved

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

Abstract Background The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. Results This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. Conclusion These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.</p

The coming PIN code epidemic: A survey study of memory of numeric security codes

Most people must remember various numeric passwords, security codes and PIN numbers for banking, credit cards, debit cards, online accounts, mobile phones, door locks, luggage locks, etc. One pilot study (N=13) developed a list of eleven strategies for remembering numeric codes, and another (N=15) optimized the research questionnaire which asked respondents about a) the number of security codes they had, b) the number of self-created codes, c) mnemonic strategies used, d) problems and effort remembering codes, and e) gender, age, and education. Respondents (N=388) had a median of 4 security codes and typically used 2 different memory strategies, the most common of which were based on repetition and on keypad pattern. Difficulties remembering codes were unrelated to gender or education but were positively correlated with age and with number of strategies used. Self-creation of codes slightly reduced difficulties remembering numeric codes

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae-2

<p><b>Copyright information:</b></p><p>Taken from "Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae"</p><p>http://www.biomedcentral.com/1472-6750/7/40</p><p>BMC Biotechnology 2007;7():40-40.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1955438.</p><p></p>, 4 and 5 days post-fertilization from a single zebrafish (dotted line with triangles) and a population average (solid line with squares). (b) Peak velocity, (c) minimum velocity, (d) peak acceleration, (e) peak deceleration, (f) heart rate (no error bars are present for the single fish measurement because beat-to-beat variability is not calculated), (g) stroke volume, and (h) cardiac output. Single-fish data presented are the average obtained by analyzing four to six individual heart beats. Error bars are the SEM and represent intrafish variability. For population average data n = 5 for days 1, 2, and 4; n = 4 for day 5. Error bars are the SEM and represent interfish variability. Laser-Scanning Velocimetry Data Analyzer smoothing parameters used for trace, velocity, and acceleration smoothing were 5, 10, and 20, respectively