Interactions between roughness and topography in hydraulic models

Analysis of river flow using hydraulic modelling and its implications in derived environ-mental applications are inextricably connected with the way in which the river boundary shape is represented. This relationship is scale-dependent upon the modelling resolution which in turn determines the importance of a subscale performance of the model and the way subscale (surface and flow) processes are parameterised. Commonly, the subscale behaviour of the model relies upon a roughness parameterisation whose meaning depends on the dimensionality of the hydraulic model and the resolution of the topographic represen¬tation scale. This latter is, in turn, dependent on the resolution of the computational mesh as well as on the detail of measured topographic data. Flow results are affected by this interactions between scale and subscale parameterisation according to the dimensionality approach. The aim of this dissertation is the evaluation of these interactions upon hy¬draulic modelling results. Current high resolution topographic source availability induce this research which is tackled using a suitable roughness approach according to each di¬mensionality with the purpose of the interaction assessment. A 1D HEC-RAS model, a 2D raster-based diffusion-wave model with a scale-dependent distributed roughness parame-terisation and a 3D finite volume scheme with a porosity algorithm approach to incorporate complex topography have been used. Different topographic sources are assessed using a 1D scheme. LiDAR data are used to isolate the mesh resolution from the topographic content of the DEM effects upon 2D and 3D flow results. A distributed roughness parameterisation, using a roughness height approach dependent upon both mesh resolution and topographic content is developed and evaluated for the 2D scheme. Grain-size data and fractal methods are used for the reconstruction of topography with microscale information, required for some applications but not easily available. Sensitivity of hydraulic parameters to this topographic parameterisation is evaluated in a 3D scheme at different mesh resolu¬tions. Finally, the structural variability of simulated flow is analysed and related to scale interactions. Model simulations demonstrate (i) the importance of the topographic source in a 1D models; (ii) the mesh resolution approach is dominant in 2D and 3D simulations whereas in a 1D model the topographic source and even the roughness parameterisation impacts are more critical; (iii) the increment of the sensitivity to roughness parameterisa-tion in 1D and 2D schemes with detailed topographic sources and finer mesh resolutions; and (iv) the topographic content and microtopography impact throughout the vertical profile of computed 3D velocity in a depth-dependent way, whereas 2D results are not affected by topographic content variations. Finally, the spatial analysis shows that the mesh resolution controls high resolution model scale results, roughness parameterisation control 2D simulation results for a constant mesh resolution; and topographic content and micro-topography variations impacts upon the organisation of flow results depth-dependently in a 3D scheme. Resumen La topografía juega un papel fundamental en la distribución del agua y la energía en los paisajes naturales (Beven and Kirkby 1979; Wood et al. 1997). La simulación hidráulica combinada con métodos de medición del terreno por teledetección constituyen una poderosa herramienta de investigación en la comprensión del comportamiento de los flujos de agua debido a la variabilidad de la superficie sobre la que fluye. La representación e incorporación de la topografía en el esquema hidráulico tiene una importancia crucial en los resultados y determinan el desarrollo de sus aplicaciones al campo medioambiental. Cualquier simulación es una simplificación de un proceso del mundo real, y por tanto el grado de simplificación determinará el significado de los resultados simulados. Este razonamiento es particularmente difícil de trasladar a la simulación hidráulica donde aspectos de la escala tan diferentes como la escala de los procesos de flujo y de representación del contorno son considerados conjuntamente incluso en fases de parametrización (e.g. parametrización de la rugosidad). Por una parte, esto es debido a que las decisiones de escala vienen condicionadas entre ellas (e.g. la dimensionalidad del modelo condiciona la escala de representación del contorno) y por tanto interaccionan en sus resultados estrechamente. Y por otra parte, debido a los altos requerimientos numéricos y computacionales de una representación explícita de alta resolución de los procesos de flujo y discretización de la malla. Además, previo a la modelización hidráulica, la superficie del terreno sobre la que el agua fluye debe ser modelizada y por tanto presenta su propia escala de representación, que a su vez dependerá de la escala de los datos topográficos medidos con que se elabora el modelo. En última instancia, esta topografía es la que determina el comportamiento espacial del flujo. Por tanto, la escala de la topografía en sus fases de medición y modelización (resolución de los datos y representación topográfica) previas a su incorporación en el modelo hidráulico producirá a su vez un impacto que se acumulará al impacto global resultante debido a la escala computacional del modelo hidráulico y su dimensión. La comprensión de las interacciones entre las complejas geometrías del contorno y la estructura del flujo utilizando la modelización hidráulica depende de las escalas consideradas en la simplificación de los procesos hidráulicos y del terreno (dimensión del modelo, tamaño de escala computacional y escala de los datos topográficos). La naturaleza de la aplicación del modelo hidráulico (e.g. habitat físico, análisis de riesgo de inundaciones, transporte de sedimentos) determina en primer lugar la escala del estudio y por tanto el detalle de los procesos a simular en el modelo (i.e. la dimensionalidad) y, en consecuencia, la escala computacional a la que se realizarán los cálculos (i.e. resolución computacional). Esta última a su vez determina, el detalle geográfico con que deberá representarse el contorno acorde con la resolución de la malla computacional. La parametrización persigue incorporar en el modelo hidráulico la cuantificación de los procesos y condiciones físicas del sistema natural y por tanto debe incluir no solo aquellos procesos que tienen lugar a la escala de modelización, sino también aquellos que tienen lugar a un nivel subescalar y que deben ser definidos mediante relaciones de escalado con las variables modeladas explícitamente. Dicha parametrización se implementa en la práctica mediante la provisión de datos al modelo, por tanto la escala de los datos geográficos utilizados para parametrizar el modelo no sólo influirá en los resultados, sino también determinará la importancia del comportamiento subescalar del modelo y el modo en que estos procesos deban ser parametrizados (e.g. la variabilidad natural del terreno dentro de la celda de discretización o el flujo en las direcciones laterales y verticales en un modelo unidimensional). En esta tesis, se han utilizado el modelo unidimensional HEC-RAS, (HEC 1998b), un modelo ráster bidimensional de propagación de onda, (Yu 2005) y un esquema tridimensional de volúmenes finitos con un algoritmo de porosidad para incorporar la topografía, (Lane et al. 2004; Hardy et al. 2005). La geometría del contorno viene definida por la escala de representación topográfica (resolución de malla y contenido topográfico), la cual a su vez depende de la escala de la fuente cartográfica. Todos estos factores de escala interaccionan en la respuesta del modelo hidráulico a la topografía. En los últimos años, métodos como el análisis fractal y las técnicas geoestadísticas utilizadas para representar y analizar elementos geográficos (e.g. en la caracterización de superficies (Herzfeld and Overbeck 1999; Butler et al. 2001)), están promoviendo nuevos enfoques en la cuantificación de los efectos de escala (Lam et al. 2004; Atkinson and Tate 2000; Lam et al. 2006) por medio del análisis de la estructura espacial de la variable (e.g. Bishop et al. 2006; Ju et al. 2005; Myint et al. 2004; Weng 2002; Bian and Xie 2004; Southworth et al. 2006; Pozd-nyakova et al. 2005; Kyriakidis and Goodchild 2006). Estos métodos cuantifican tanto el rango de valores de la variable presentes a diferentes escalas como la homogeneidad o heterogeneidad de la variable espacialmente distribuida (Lam et al. 2004). En esta tesis, estas técnicas se han utilizado para analizar el impacto de la topografía sobre la estructura de los resultados hidráulicos simulados. Los datos de teledetección de alta resolución y técnicas GIS también están siendo utilizados para la mejor compresión de los efectos de escala en modelos medioambientales (Marceau 1999; Skidmore 2002; Goodchild 2003) y se utilizan en esta tesis. Esta tesis como corpus de investigación aborda las interacciones de esas escalas en la modelización hidráulica desde un punto de vista global e interrelacionado. Sin embargo, la estructura y el foco principal de los experimentos están relacionados con las nociones espaciales de la escala de representación en relación con una visión global de las interacciones entre escalas. En teoría, la representación topográfica debe caracterizar la superficie sobre la que corre el agua a una adecuada (conforme a la finalidad y dimensión del modelo) escala de discretización, de modo que refleje los procesos de interés. La parametrización de la rugosidad debe de reflejar los efectos de la variabilidad de la superficie a escalas de más detalle que aquellas representadas explícitamente en la malla topográfica (i.e. escala de discretización). Claramente, ambos conceptos están físicamente relacionados por un

Analysis of the static and dynamic behaviour of hydraulic fills

En la actualidad existe un gran conocimiento en la caracterización de rellenos hidráulicos, tanto en su caracterización estática, como dinámica. Sin embargo, son escasos en la literatura estudios más generales y globales de estos materiales, muy relacionados con sus usos y principales problemáticas en obras portuarias y mineras. Los procedimientos semi‐empíricos para la evaluación del efecto silo en las celdas de cajones portuarios, así como para el potencial de licuefacción de estos suelos durantes cargas instantáneas y terremotos, se basan en estudios donde la influencia de los parámetros que los rigen no se conocen en gran medida, dando lugar a resultados con considerable dispersión. Este es el caso, por ejemplo, de los daños notificados por el grupo de investigación del Puerto de Barcelona, la rotura de los cajones portuarios en el Puerto de Barcelona en 2007. Por estos motivos y otros, se ha decidido desarrollar un análisis para la evaluación de estos problemas mediante la propuesta de una metodología teórico‐numérica y empírica. El enfoque teórico‐numérico desarrollado en el presente estudio se centra en la determinación del marco teórico y las herramientas numéricas capaces de solventar los retos que presentan estos problemas. La complejidad del problema procede de varios aspectos fundamentales: el comportamiento no lineal de los suelos poco confinados o flojos en procesos de consolidación por preso propio; su alto potencial de licuefacción; la caracterización hidromecánica de los contactos entre estructuras y suelo (camino preferencial para el flujo de agua y consolidación lateral); el punto de partida de los problemas con un estado de tensiones efectivas prácticamente nulo. En cuanto al enfoque experimental, se ha propuesto una metodología de laboratorio muy sencilla para la caracterización hidromecánica del suelo y las interfaces, sin la necesidad de usar complejos aparatos de laboratorio o procedimientos excesivamente complicados. Este trabajo incluye por tanto un breve repaso a los aspectos relacionados con la ejecución de los rellenos hidráulicos, sus usos principales y los fenómenos relacionados, con el fin de establecer un punto de partida para el presente estudio. Este repaso abarca desde la evolución de las ecuaciones de consolidación tradicionales (Terzaghi, 1943), (Gibson, English & Hussey, 1967) y las metodologías de cálculo (Townsend & McVay, 1990) (Fredlund, Donaldson and Gitirana, 2009) hasta las contribuciones en relación al efecto silo (Ranssen, 1985) (Ravenet, 1977) y sobre el fenómeno de la licuefacción (Casagrande, 1936) (Castro, 1969) (Been & Jefferies, 1985) (Pastor & Zienkiewicz, 1986). Con motivo de este estudio se ha desarrollado exclusivamente un código basado en el método de los elementos finitos (MEF) empleando el programa MATLAB. Para ello, se ha esablecido un marco teórico (Biot, 1941) (Zienkiewicz & Shiomi, 1984) (Segura & Caron, 2004) y numérico (Zienkiewicz & Taylor, 1989) (Huerta & Rodríguez, 1992) (Segura & Carol, 2008) para resolver problemas de consolidación multidimensional con condiciones de contorno friccionales, y los correspondientes modelos constitutivos (Pastor & Zienkiewicz, 1986) (Fiu & Liu, 2011). Asimismo, se ha desarrollado una metodología experimental a través de una serie de ensayos de laboratorio para la calibración de los modelos constitutivos y de la caracterización de parámetros índice y de flujo (Castro, 1969) (Bahda 1997) (Been & Jefferies, 2006). Para ello se han empleado arenas de Hostun como material (relleno hidráulico) de referencia. Como principal aportación se incluyen una serie de nuevos ensayos de corte directo para la caracterización hidromecánica de la interfaz suelo – estructura de hormigón, para diferentes tipos de encofrados y rugosidades. Finalmente, se han diseñado una serie de algoritmos específicos para la resolución del set de ecuaciones diferenciales de gobierno que definen este problema. Estos algoritmos son de gran importancia en este problema para tratar el procesamiento transitorio de la consolidación de los rellenos hidráulicos, y de otros efectos relacionados con su implementación en celdas de cajones, como el efecto silo y la licuefacciones autoinducida. Para ello, se ha establecido un modelo 2D axisimétrico, con formulación acoplada u‐p para elementos continuos y elementos interfaz (de espesor cero), que tratan de simular las condiciones de estos rellenos hidráulicos cuando se colocan en las celdas portuarias. Este caso de estudio hace referencia clara a materiales granulares en estado inicial muy suelto y con escasas tensiones efectivas, es decir, con prácticamente todas las sobrepresiones ocasionadas por el proceso de autoconsolidación (por peso propio). Por todo ello se requiere de algoritmos numéricos específicos, así como de modelos constitutivos particulares, para los elementos del continuo y para los elementos interfaz. En el caso de la simulación de diferentes procedimientos de puesta en obra de los rellenos se ha requerido la modificacion de los algoritmos empleados para poder así representar numéricamente la puesta en obra de estos materiales, además de poder realizar una comparativa de los resultados para los distintos procedimientos. La constante actualización de los parámetros del suelo, hace también de este algoritmo una potente herramienta que permite establecer un interesante juego de perfiles de variables, tales como la densidad, el índice de huecos, la fracción de sólidos, el exceso de presiones, y tensiones y deformaciones. En definitiva, el modelo otorga un mejor entendimiento del efecto silo, término comúnmente usado para definir el fenómeno transitorio del gradiente de presiones laterales en las estructuras de contención en forma de silo. Finalmente se incluyen una serie de comparativas entre los resultados del modelo y de diferentes estudios de la literatura técnica, tanto para el fenómeno de las consolidaciones por preso propio (Fredlund, Donaldson & Gitirana, 2009) como para el estudio del efecto silo (Puertos del Estado, 2006, EuroCódigo (2006), Japan Tech, Stands. (2009), etc.). Para concluir, se propone el diseño de un prototipo de columna de decantación con paredes friccionales, como principal propuesta de futura línea de investigación. Wide research is nowadays available on the characterization of hydraulic fills in terms of either static or dynamic behavior. However, reported comprehensive analyses of these soils when meant for port or mining works are scarce. Moreover, the semi‐empirical procedures for assessing the silo effect on cells in floating caissons, and the liquefaction potential of these soils during sudden loads or earthquakes are based on studies where the underlying influence parameters are not well known, yielding results with significant scatter. This is the case, for instance, of hazards reported by the Barcelona Liquefaction working group, with the failure of harbor walls in 2007. By virtue of this, a complex approach has been undertaken to evaluate the problem by a proposal of numerical and laboratory methodology. Within a theoretical and numerical scope, the study is focused on the numerical tools capable to face the different challenges of this problem. The complexity is manifold; the highly non‐linear behavior of consolidating soft soils; their potentially liquefactable nature, the significance of the hydromechanics of the soil‐structure contact, the discontinuities as preferential paths for water flow, setting “negligible” effective stresses as initial conditions. Within an experimental scope, a straightforward laboratory methodology is introduced for the hydromechanical characterization of the soil and the interface without the need of complex laboratory devices or cumbersome procedures. Therefore, this study includes a brief overview of the hydraulic filling execution, main uses (land reclamation, filled cells, tailing dams, etc.) and the underlying phenomena (self‐weight consolidation, silo effect, liquefaction, etc.). It comprises from the evolution of the traditional consolidation equations (Terzaghi, 1943), (Gibson, English, & Hussey, 1967) and solving methodologies (Townsend & McVay, 1990) (Fredlund, Donaldson and Gitirana, 2009) to the contributions in terms of silo effect (Ranssen, 1895) (Ravenet, 1977) and liquefaction phenomena (Casagrande, 1936) (Castro, 1969) (Been & Jefferies, 1985) (Pastor & Zienkiewicz, 1986). The novelty of the study lies on the development of a Finite Element Method (FEM) code, exclusively formulated for this problem. Subsequently, a theoretical (Biot, 1941) (Zienkiewicz and Shiomi, 1984) (Segura and Carol, 2004) and numerical approach (Zienkiewicz and Taylor, 1989) (Huerta, A. & Rodriguez, A., 1992) (Segura, J.M. & Carol, I., 2008) is introduced for multidimensional consolidation problems with frictional contacts and the corresponding constitutive models (Pastor & Zienkiewicz, 1986) (Fu & Liu, 2011). An experimental methodology is presented for the laboratory test and material characterization (Castro 1969) (Bahda 1997) (Been & Jefferies 2006) using Hostun sands as reference hydraulic fill. A series of singular interaction shear tests for the interface calibration is included. Finally, a specific model algorithm for the solution of the set of differential equations governing the problem is presented. The process of consolidation and settlements involves a comprehensive simulation of the transient process of decantation and the build‐up of the silo effect in cells and certain phenomena related to self‐compaction and liquefaction. For this, an implementation of a 2D axi‐syimmetric coupled model with continuum and interface elements, aimed at simulating conditions and self‐weight consolidation of hydraulic fills once placed into floating caisson cells or close to retaining structures. This basically concerns a loose granular soil with a negligible initial effective stress level at the onset of the process. The implementation requires a specific numerical algorithm as well as specific constitutive models for both the continuum and the interface elements. The simulation of implementation procedures for the fills has required the modification of the algorithm so that a numerical representation of these procedures is carried out. A comparison of the results for the different procedures is interesting for the global analysis. Furthermore, the continuous updating of the model provides an insightful logging of variable profiles such as density, void ratio and solid fraction profiles, total and excess pore pressure, stresses and strains. This will lead to a better understanding of complex phenomena such as the transient gradient in lateral pressures due to silo effect in saturated soils. Interesting model and literature comparisons for the self‐weight consolidation (Fredlund, Donaldson, & Gitirana, 2009) and the silo effect results (Puertos del Estado (2006), EuroCode (2006), Japan Tech, Stands. (2009)). This study closes with the design of a decantation column prototype with frictional walls as the main future line of research.

Prototype emitter for use in subsurface drip irrigation: Manufacturing, hydraulic evaluation and experimental analyses

The current research aims to analyse theoretically and evaluate a self-manufactured simple design for subsurface drip irrigation (SDI) emitter to avoid root and soil intrusion. It was composed of three concentric cylindrical elements: an elastic silicone membrane; a polyethylene tube with two holes drilled on its wall for water discharge; and a vinyl polychloride protector system to wrap the other elements. The discharge of the emitter depends on the change in the membrane diameter when it is deformed by the water pressure. The study of the operation of this emitter is a new approach that considers mechanical and hydraulic principles. Thus, the estimation on the membrane deformation was based on classical mechanical stress theories in composite cylinders. The hydraulic principles considered the solid deformation due to force based on water pressure and the general Darcy–Weisbach head-loss equation. Twenty emitter units, with the selected design, were handcrafted in a lathe and were used in this study. The measured pressure/discharge relationship for the emitters showed good agreement with that calculated by the theoretical approach. The variation coefficient of the handcrafted emitters was high compared to commercial emitters. Results from field evaluations showed variable values for the relative flow variation, water emission uniformity and relative flow rate coefficients, but no emitter was obstructed. Therefore, the current emitter design could be suitable for SDI following further studies to develop a final prototype.

Development of a PWR-W and an AP1000® containment building 3D model with a CFD code for Best-Estimate Thermal-Hydraulic Analysis

The simulation of design basis accidents in a containment building is usually conducted with a lumped parameter model. The codes normally used by Westinghouse Electric Company (WEC) for that license analysis are WGOTHIC or COCO, which are suitable to provide an adequate estimation of the overall peak temperature and pressure of the containment. However, for the detailed study of the thermal-hydraulic behavior in every room and compartment of the containment building, it could be more convenient to model the containment with a more detailed 3D representation of the geometry of the whole building. The main objective of this project is to obtain a standard PWR Westinghouse as well as an AP1000® containment model for a CFD code to analyze the thermal-hydraulic detailed behavior during a design basis accident. In this paper the development and testing of both containment models is presented.

Two-dimensional direct rainfall hydraulic models applied to direct calculation of hydrologic events in dams to prevent overtopping

One of the main concerns when conducting a dam test is the acute determination of the hydrograph for a specific flood event. The use of 2D direct rainfall hydraulic mathematical models on a finite elements mesh, combined with the efficiency of vector calculus that provides CUDA (Compute Unified Device Architecture) technology, enables nowadays the simulation of complex hydrological models without the need for terrain subbasin and transit splitting (as in HEC-HMS). Both the Spanish PNOA (National Plan of Aereal Orthophotography) Digital Terrain Model GRID with a 5 x 5 m accuracy and the CORINE GIS Land Cover (Coordination of INformation of the Environment) that allows assessment of the ground roughness, provide enough data to easily build these kind of models

Evaluation/Optimization of reservoir operation rules for flood management using an integrated hydrologic-hydraulic framework

Se ha presentado la evaluación y optimización de las reglas de operación de un embalse para gestión de avenidas usando un entorno integrado hidrológico- hidráulico de tipo Monte Carlo. Some reservoirs play a major role in flood protection, managing the floods and reducing or delaying the peak discharges in the river downstream. However, the changing environment (natural and anthropological changes) requires the development of more elaborated strategies for reservoir operation. Three factors are relevant: 1) the natural variability of inflow hydrographs, 2) the competition for reservoir storage capacity between flood control and other uses, and 3) the existence of built-up areas on downstream river reaches. A framework for evaluation/optimization of reservoir operation rules for flood management in a changing environment is presented in this study. The study was carried out using an integrated hydrologic – hydraulic model in a Monte Carlo framework.

Advanced Control Strategies for a 6 DoF Hydraulic Parallel Robot Based on the Dynamic Model

Nowadays robots have made their way into real applications that were prohibitive and unthinkable thirty years ago. This is mainly due to the increase in power computations and the evolution in the theoretical field of robotics and control. Even though there is plenty of information in the current literature on this topics, it is not easy to find clear concepts of how to proceed in order to design and implement a controller for a robot. In general, the design of a controller requires of a complete understanding and knowledge of the system to be controlled. Therefore, for advanced control techniques the systems must be first identified. Once again this particular objective is cumbersome and is never straight forward requiring of great expertise and some criteria must be adopted. On the other hand, the particular problem of designing a controller is even more complex when dealing with Parallel Manipulators (PM), since their closed-loop structures give rise to a highly nonlinear system. Under this basis the current work is developed, which intends to resume and gather all the concepts and experiences involve for the control of an Hydraulic Parallel Manipulator. The main objective of this thesis is to provide a guide remarking all the steps involve in the designing of advanced control technique for PMs. The analysis of the PM under study is minced up to the core of the mechanism: the hydraulic actuators. The actuators are modeled and experimental identified. Additionally, some consideration regarding traditional PID controllers are presented and an adaptive controller is finally implemented. From a macro perspective the kinematic and dynamic model of the PM are presented. Based on the model of the system and extending the adaptive controller of the actuator, a control strategy for the PM is developed and its performance is analyzed with simulation.

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 ± 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 ± 0.2 μM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 μM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Redistribution of phosphatidylethanolamine at the cleavage furrow of dividing cells during cytokinesis

Ro09-0198 is a tetracyclic polypeptide of 19 amino acids that recognizes strictly the structure of phosphatidylethanolamine (PE) and forms a tight equimolar complex with PE on biological membranes. Using the cyclic peptide coupled with fluorescence-labeled streptavidin, we have analyzed the cell surface localization of PE in dividing Chinese hamster ovary cells. We found that PE was exposed on the cell surface specifically at the cleavage furrow during the late telophase of cytokinesis. PE was exposed on the cell surface only during the late telophase and no alteration in the distribution of the plasma membrane-bound cyclic peptide was observed during the cytokinesis, suggesting that the surface exposure of PE reflects the enhanced scrambling of PE at the cleavage furrow. Furthermore, cell surface immobilization of PE induced by adding the cyclic peptide coupled with streptavidin to prometaphase cells effectively blocked the cytokinesis at late telophase. The peptide-streptavidin complex treatment had no effect on furrowing, rearrangement of microtubules, and nuclear reconstitution, but specifically inhibited both actin filament disassembly at the cleavage furrow and subsequent membrane fusion. These results suggest that the redistribution of the plasma membrane phospholipids is a crucial step for cytokinesis and the cell surface PE may play a pivotal role in mediating a coordinate movement between the contractile ring and plasma membrane to achieve successful cell division.

WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

Electric Field–directed Cell Motility Involves Up-regulated Expression and Asymmetric Redistribution of the Epidermal Growth Factor Receptors and Is Enhanced by Fibronectin and Laminin

Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell–substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell–substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor β and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor β and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.

The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the β2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the β2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Gαq C terminus antibody; and (iv) was associated with a net increase in Gαq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Gα subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Gαq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.