Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

Characterization of E-cadherin endocytosis in isolated MCF-7 and Chinese hamster ovary cells - The initial fate of unbound E-cadherin

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Alterations of neocortical pyramidal cell phenotype in the Ts65Dn mouse model of Down syndrome: Effects of environmental enrichment

Mental retardation in individuals with Down syndrome (DS) is thought to result from anomalous development and function of the brain; however, the underlying neuropathological processes have yet to be determined. Early implementation of special care programs result in limited, and temporary, cognitive improvements in DS individuals. In the present study, we investigated the possible neural correlates of these limited improvements. More specifically, we studied cortical pyramidal cells in the frontal cortex of Ts65Dn mice, a partial trisomy of murine chromosome 16 (MMU16) model characterized by cognitive deficits, hyperactivity, behavioral disruption and reduced attention levels similar to those observed in DS, and their control littermates. Animals were raised either in a standard or in an enriched environment. Environmental enrichment had a marked effect on pyramidal cell structure in control animals. Pyramidal cells in environmentally enriched control animals were significantly more branched and more spinous than non-enriched controls. However, environmental enrichment had little effect on pyramidal cell structure in Ts65Dn mice. As each dendritic spine receives at least one excitatory input, differences in the number of spines found in the dendritic arbors of pyramidal cells in the two groups reflect differences in the number of excitatory inputs they receive and, consequently, complexity in cortical circuitry. The present results suggest that behavioral deficits demonstrated in the Ts65Dn model could be attributed to abnormal circuit development.

Parvalbumin-, calbindin-, and calretinin-immunoreactive neurons in the prefrontal cortex of the owl monkey (Aotus trivirgatus): A standardized quantitative comparison with sensory and motor areas

Recent studies have revealed regional variation in the density and distribution of inhibitory neurons in different cortical areas, which are thought to reflect area-specific specializations in cortical circuitry. However, there are as yet few standardized quantitative data regarding how the inhibitory circuitry in prefrontal cortex (PFC), which is thought to be involved in executive functions such as cognition, emotion and decision making, compares to that in other cortical areas. Here we used immunohistochemical techniques to determine the density and distribution of parvalbumin (PV)-, calbindin (CB)-, and calretinin (CR)-immunoreactive (ir) neurons and axon terminals in the dorsolateral and orbital PFC of the owl monkey (Aotus trivirgatus), and compared them directly with data obtained using the same techniques in 11 different visual, somatosensory and motor areas. We found marked differences in the density of PV-ir, CB-ir, and CR-ir interneurons in several cortical areas. One hundred and twenty eight of all 234 possible between-area pairwise comparisons were significantly different. The density of specific subpopulations of these cells also varied among cortical areas, as did the density of axon terminals. Comparison of PFC with other cortical areas revealed that 40 of all 66 possible statistical comparisons of the density of PV-ir, CB-ir, and CR-ir cells were significantly different. We also found evidence for heterogeneity in the pattern of labeling of PV-ir, CB-ir, and CR-ir cells and axon terminals between the dorsolateral and orbital subdivisions of PFC. These data are likely to reflect basic differences in interneuron circuitry, which are likely to influence inhibitory function in the cortex. Copyright (C) 2003 S. Karger AG, Basel.

Cortex, cognition and the cell: New insights into the pyramidal neuron and prefrontal function

Arguably the most complex conical functions are seated in human cognition, the how and why of which have been debated for centuries by theologians, philosophers and scientists alike. In his best-selling book, An Astonishing Hypothesis: A Scientific Search for the Soul, Francis Crick refined the view that these qualities are determined solely by cortical cells and circuitry. Put simply, cognition is nothing more, or less, than a biological function. Accepting this to be the case, it should be possible to identify the mechanisms that subserve cognitive processing. Since the pioneering studies of Lorent de No and Hebb, and the more recent studies of Fuster, Miller and Goldman-Rakic, to mention but a few, much attention has been focused on the role of persistent neural activity in cognitive processes. Application of modern technologies and modelling techniques has led to new hypotheses about the mechanisms of persistent activity. Here I focus on how regional variations in the pyramidal cell phenotype may determine the complexity of cortical circuitry and, in turn, influence neural activity. Data obtained from thousands of individually injected pyramidal cells in sensory, motor, association and executive cortex reveal marked differences in the numbers of putative excitatory inputs received by these cells. Pyramidal cells in prefrontal cortex have, on average, up to 23 times more dendritic spines than those in the primary visual area. I propose that without these specializations in the structure of pyramidal cells, and the circuits they form, human cognitive processing would not have evolved to its present state. I also present data from both New World and Old World monkeys that show varying degrees of complexity in the pyramidal cell phenotype in their prefrontal cortices, suggesting that cortical circuitry and, thus, cognitive styles are evolving independently in different species.

Pyramidal cell heterogeneity in the visual cortex of the nocturnal new world owl monkey (aotus trivirgatus)

Recent studies have revealed marked variation in pyramidal cell structure in the visual cortex of macaque and marmoset monkeys. In particular, there is a systematic increase in the size of, and number of spines in, the arbours of pyramidal cells with progression through occipitotemporal (OT) visual areas. In the present study we extend the basis for comparison by investigating pyramidal cell structure in visual areas of the nocturnal owl monkey. As in the diurnal macaque and marmoset monkeys, pyramidal cells became progressively larger and more spinous with anterior progression through OT visual areas. These data suggest that: 1. the trend for more complex pyramidal cells with anterior progression through OT visual areas is a fundamental organizational principle in primate cortex; 2. areal specialization of the pyramidal cell phenotype provides an anatomical substrate for the reconstruction of the visual scene in OT areas; 3. evolutionary specialization of different aspects of visual processing may determine the extent of interareal variation in the pyramidal cell phenotype in different species; and 4. pyramidal cell structure is not necessarily related to brain size. Crown Copyright (C) 2003 Published by Elsevier Science Ltd on behalf of IBRO. All rights reserved.

The pyramidal neuron in occipital, temporal and prefrontal cortex of the owl monkey (Aotus trivirgatus): regional specialization in cell structure

Recent studies have revealed marked regional variation in pyramidal cell morphology in primate cortex. In particular, pyramidal cells in human and macaque prefrontal cortex (PFC) are considerably more spinous than those in other cortical regions. PFC pyramidal cells in the New World marmoset monkey, however, are less spinous than those in man and macaques. Taken together, these data suggest that the pyramidal cell has become more branched and more spinous during the evolution of PFC in only some primate lineages. This specialization may be of fundamental importance in determining the cognitive styles of the different species. However, these data are preliminary, with only one New World and two Old World species having been studied. Moreover, the marmoset data were obtained from different cases. In the present study we investigated PFC pyramidal cells in another New World monkey, the owl monkey, to extend the basis for comparison. As in the New World marmoset monkey, prefrontal pyramidal cells in owl monkeys have relatively few spines. These species differences appear to reflect variation in the extent to which PFC circuitry has become specialized during evolution. Highly complex pyramidal cells in PFC appear not to have been a feature of a common prosimian ancestor, but have evolved with the dramatic expansion of PFC in some anthropoid lineages.

Dendritic branching of pyramidal cells in the visual cortex of the nocturnal owl monkey: A fractal analysis

The branching structure of neurones is thought to influence patterns of connectivity and how inputs are integrated within the arbor. Recent studies have revealed a remarkable degree of variation in the branching structure of pyramidal cells in the cerebral cortex of diurnal primates, suggesting regional specialization in neuronal function. Such specialization in pyramidal cell structure may be important for various aspects of visual function, such as object recognition and color processing. To better understand the functional role of regional variation in the pyramidal cell phenotype in visual processing, we determined the complexity of the dendritic branching pattern of pyramidal cells in visual cortex of the nocturnal New World owl monkey. We used the fractal dilation method to quantify the branching structure of pyramidal cells in the primary visual area (V1), the second visual area (V2) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively), which are often associated with color processing. We found that, as in diurnal monkeys, there was a trend for cells of increasing fractal dimension with progression through these cortical areas. The increasing complexity paralleled a trend for increasing symmetry. That we found a similar trend in both diurnal and nocturnal monkeys suggests that it was a feature of a common anthropoid ancestor.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

O combate ?? viol??ncia contra a mulher: a luta entre antigos valores e novos padr??es de pol??ticas p??blicas

O caso relata uma situa????o de viol??ncia dom??stica vivenciada por uma mulher negra e os obst??culos por ela enfrentados para denunciar o agressor e fazer valer seus direitos. Mostra a contradi????o entre normas e sua efetiva aplica????o, quando o comportamento de agentes p??blicos ainda conserva padr??es e valores de um Estado autorit??rio, patriarcal e escravocrata. O caso ?? fict??cio, mas espelha situa????es reais coletadas em documentos e relat??rios da Ouvidoria da Secretaria de Pol??ticas para as Mulheres- SPM/PR. O estudo suscita discuss??es sobre a dimens??o ??tica da atividade de agentes p??blicos de um Estado democr??tico, englobando quest??es referentes aos direitos humanos, direitos e deveres do Estado e da sociedade, o papel da transpar??ncia, do controle social e da responsabiliza????o por resultados (accountability) etc. Pode ser aplicado em cursos sobre ??tica e servi??o p??blico, pol??ticas p??blicas de g??nero e ra??a e atendimento ao cidad??o

Data Mart para apoio ?? capacita????o em gest??o de pol??ticas p??blicas voltado para uma Escola de Administra????o P??blica

Este trabalho consiste num estudo de caso que se destina ao desenvolvimento de um Data Mart que possibilite a Escola Nacional de Administra????o P??blica ??? ENAP conhecer o perfil e o panorama geral da situa????o funcional dos servidores p??blicos federais que se capacitaram na Escola nos ??ltimos 7 anos. O aplicativo foi desenvolvido cruzando o banco de dados do sistema gerenciador dos cursos ministrados pela ENAP, onde est??o armazenadas informa????es sobre os alunos capacitados, os cursos realizados, os resultados alcan??ados, o perfil dos docentes e demais informa????es relativas ??s atividades da Escola, com os dados gerados pelo Sistema Integrado de Administra????o de Recursos Humanos ??? SIAPE, cuja extra????o de dados foi direcionada para os registros sobre a situa????o funcional, cargos, carreiras, fun????es, ??rg??os e alguns dados pessoais dos alunos, servidores p??blicos federais que se encontram registrados no SIAPE

Plano de implementa????o do projeto de desenvolvimento de capacidade de governan??a

Desde a d??cada de 1990, o Governo Federal brasileiro vem implementando uma agenda ambiciosa de reformas do Estado, centradas na redu????o da pobreza e na melhoria da efici??ncia dos servi??os p??blicos. As principais prioridades, conforme previstas no Plano Plurianual (PPA) para o per??odo 2003-2007, s??o as seguintes: inclus??o social e redu????o da desigualdade; crescimento econ??mico com gera????o de emprego; distribui????o de renda e respeito ao meio ambiente; promo????o e amplia????o dos direitos de cidadania; e fortalecimento da democracia. No in??cio de 2006, o Governo criou a Pol??tica Nacional de Desenvolvimento de Pessoal (Decreto 5.707), com o objetivo de melhorar e aumentar a efici??ncia e a efic??cia na presta????o de servi??os p??blicos. No marco dessa pol??tica recente, as escolas de administra????o p??blica desempenham um papel fundamental na identifica????o das compet??ncias que precisam ser desenvolvidas nas institui????es do governo, bem como na implementa????o de pol??ticas de capacita????o para os servidores p??blicos, diretamente e/ou em parceria com escolas de governo nos n??veis federal, estadual ou local. O Canad?? tamb??m est?? criando uma estrutura para levantar as compet??ncias necess??rias para os servidores p??blicos e desenvolv??-las como um componente da Renova????o do Servi??o P??blico em todo o governo. Como institui????es l??deres no desenvolvimento de compet??ncias de servidores p??blicos, a Canada School of Public Service (CSPS) e a Escola Nacional de Administra????o P??blica (ENAP) firmaram uma parceria para implementar o Projeto de Desenvolvimento de Capacidade de Governan??a no Brasil. A finalidade do Projeto ?? melhorar a capacidade de servidores p??blicos federais, estaduais e municipais do Brasil para desenvolver e implementar programas de capacita????o e gerenciar pol??ticas p??blicas descentralizadas. Espera-se que essa parceria e o resultante compartilhamento de experi??ncias em capacita????o para governan??a efetiva contribuam para a redu????o da pobreza e das desigualdades no Brasil, por meio do desenvolvimento de compet??ncias de servidores na presta????o de servi??os p??blicos eficazes e eficientes, voltados para o cidad??o. O Projeto re??ne, al??m das duas principais Escolas de Governo no Canad?? e no Brasil, seis Escolas Brasileiras de Administra????o P??blica regionais e duas renomadas Institui????es Acad??micas Canadenses ??? a Queen???s University e a Western Ontario University. O Minist??rio do Desenvolvimento Social e Combate ?? Fome (MDS) e tr??s Secretarias Especiais do Governo Federal ??? Ra??a (SEPPIR), Direitos Humanos (SEDH) e Pol??ticas para as Mulheres (SPM) ??? tamb??m se envolver??o nas atividades de compartilhamento de conhecimentos com o Human Resources and Skills Development Canada (HRSDC) e a Canada Public Service Agency (CPSA). A CIDA fornecer?? CND$1.700.000 por meio do Programa Brasil-Canad?? de Interc??mbio de Conhecimentos para a Promo????o da Equidade (PIPE). A contribui????o da ENAP ser?? de CND$1.069.707 em esp??cie. A CSPS contribuir?? com cursos, al??m de conhecimentos e suporte t??cnicos, avaliados em CND$1.000.000. Aproveitando a parceria entre a CSPS e a ENAP, que resultou na transfer??ncia e na adapta????o bem sucedidas de cursos e metodologias canadenses, o novo projeto extrapola o n??cleo do servi??o p??blico em Bras??lia, alcan??ando escolas de governo em regi??es brasileiras em situa????o de desvantagem. ?? semelhan??a do papel da CSPS no primeiro projeto, a ENAP fortalecer?? a capacidade das escolas parceiras regionais para capacitar servidores p??blicos envolvidos na presta????o de servi??os aos brasileiros. O interc??mbio estruturado entre Minist??rios dos Governos canadense e brasileiro tamb??m aplicar?? a aprendizagem mais diretamente a quest??es de pol??ticas e programas sociais do Brasil. O desafio assumido neste Projeto ?? a adapta????o de conhecimentos e aprendizagem, com vistas a melhorar a implementa????o de pol??ticas e programas sociais. Para tanto, a CSPS e a ENAP introduzir??o novos cursos nos curr??culos das escolas parceiras e incorporar??o novos m??todos e tecnologias de aprendizagem como, por exemplo, comunidades de pr??tica virtuais e um componente de tutoria (mentoring) envolvendo o Human Resources and Skills Development Canada e o Minist??rio do Desenvolvimento Social e Combate ?? Fome do Brasil. Seis institui????es da Rede Nacional de Escolas de Governo do Brasil e do Programa de Parceria da ENAP foram selecionadas e convidadas a se unir ?? CSPS e ?? ENAP nesse novo Projeto: a Universidade Federal do Par?? (UFPA), de Bel??m (estado do Par?? ??? regi??o Norte); a Funda????o Joaquim Nabuco (FUNDAJ), de Recife (Pernambuco ??? Nordeste); a Universidade Corporativa do Servi??o P??blico / Secretaria de Administra????o do Estado da Bahia (UCS/SAEB), Salvador (Bahia ??? Nordeste); a Escola de Governo do Mato Grosso do Sul (ESCOLAGOV), Campo Grande (estado do Mato Grosso do Sul ??? Centro-Oeste); a Escola Nacional de Ci??ncias Estat??sticas / Instituto Brasileiro de Geografia e Estat??stica (ENCE/IBGE), Rio de Janeiro (estado do Rio de Janeiro ??? Sudeste); e o Instituto Municipal de Administra????o P??blica (IMAP) de Curitiba (Paran?? ??? Sul). Essas escolas de refer??ncia foram escolhidas segundo sua capacidade de trabalhar como p??los de pr??ticas inovadoras em pol??ticas p??blicas e disseminar os benef??cios do Projeto para outras escolas em suas regi??es, por meio da Rede Nacional coordenada pela ENAP. O objetivo dessa parceria ?? fortalecer as escolas de governo locais, para que estas desenvolvam, por meio de eventos de aprendizagem, compet??ncias em servidores p??blicos, a fim de aumentar a capacidade do governo na implementa????o e gest??o de pol??ticas p??blicas. O Plano de Implementa????o do Projeto (PIP) descreve o trabalho a ser realizado por essas institui????es nos pr??ximos 30 meses, ao tempo em que serve de guia para os Parceiros do Projeto no que se refere ??s a????es e aos recursos necess??rios para a obten????o dos resultados acordados. Na medida em que o Projeto estiver em andamento e os parceiros iniciarem um interc??mbio produtivo de conhecimentos, o Plano de Trabalho Anual ser?? atualizado e revisto por meio de reuni??es anuais de avalia????o e encontros do Comit?? Diretor do Projeto, com vistas a assegurar que os resultados descritos no PIP sejam alcan??ados com sucesso