Nucleus is the largest cellular organelle in animal cells. Due to its bulky nature and the stiffness of nuclear lamina the nucleus constitutes the substantial problem for migrating cells where nucleus has to move. The actomyosin generated forces and LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, that is composed of SUN and nesprin proteins, play key role in nuclear movement. LINC complex mechanically couples nuclear lamina to the cytoskeleton and allows the forces exerted by the cytoskeleton to move the nucleus. Perinuclear actin fibers, also termed actin cap, mechanically link focal adhesions with nucleus and they may generate forces that position the nucleus in a way that is optimal for cellular movement. However, molecular mechanism of how perinuclear actin fibers and LINC complex orchestrate the nuclear movement and functional significance of this process remain poorly understood. The specific aim was to determine the mechanisms by which perinuclear actin fibers are formed and how are these mechanisms employed to facilitate cell migration. The role of LPA-RhoA signaling axis and LINC complex in the formation of perinuclear actin fibers was also examined. It was confirmed that LPA is essencial stimulus during actin cap formation. On the other hand, FAK kinase was found necessary for...

Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T05:53:22Z (GMT). No. of bitstreams: 1 Silveira_AngelicaAparecidaAntoniellis_M.pdf: 1268408 bytes, checksum: 22c750391706cfa07cb1fd66d9e8a9c1 (MD5) Previous issue date: 2012 / Resumo: Durante a resposta inflamatória, os neutrófilos e outros leucócitos aderem ao endotélio, deixando os vasos sanguíneos e movimentando-se ativamente em direção ao foco inflamatório. A migração dos neutrófilos para sítios inflamatórios depende de uma série de eventos adesivos e quimiotáticos, resultantes da ativação de moléculas de adesão como as selectinas e integrinas e receptores de quimiocinas. Devido às suas propriedades, os neutrófilos podem ser ativados por proteínas de sinalização intracelular, as Rho GTPases, que auxiliam os neutrófilos a desempenhar esta função por interferirem em mudanças no citoesqueleto. Estas proteínas também estão envolvidas na adesão e proliferação celular. Os neutrófilos são capazes de sintetizar óxido nítrico (NO), sendo que esta produção de NO é um importante componente da resposta imune inata durante a inflamação. Estudos demonstraram que os neutrófilos têm papel indutor na geração de inflamação e esforços visando compreender o mecanismo adesivo destas células nos processos inflamatórios podem ser um ponto chave para intervenções farmacológicas em doenças que são caracterizadas por inflamação vascular com consequente obstrução de fluxo sanguíneo. Diante disso, este estudo objetivou avaliar o papel da via do NO e das Rho GTPases no mecanismo pelo qual os estímulos inflamatórios aumentam a adesão de neutrófilos. Também foi avaliado os efeitos da sinvastatina na modulação das propriedades adesivas de neutrófilos, como ferramenta para auxiliar no estudo do envolvimento das Rho GTPases, Rac1 e RhoA, no processo adesivo destas células. Este mecanismo foi estudado a partir de neutrófilos isolados do sangue periférico por ensaios de adesão estática e em fluxo e citometria de fluxo. Além disso, foi analisada a expressão gênica das Rho GTPases, Rac1 e RhoA, através de PCR em tempo real. Sob potente estímulo de TNF-?, as propriedades adesivas dos neutrófilos aumentam significativamente. Inibidores de NO sintase e doadores de NO não alteraram as propriedades adesivas de neutrófilos quando estimulados com TNF-?. Não observamos grande diferença quanto à adesão e expressão das moléculas de adesão na superfície dos neutrófilos usando inibidor de Rac1, porém o composto Y-27632, inibidor de ROCK (Rho-associated coiled coil forming protein serine/threonine kinase), proteína efetora de RhoA, mostrou aumentar a adesão dos neutrófilos sob condições basais. O uso da sinvastatina modulou as propriedades adesivas e a expressão de Mac-1 nos neutrófilos na presença de um estímulo inflamatório, apoiando evidências de seu uso como anti-inflamatório. Em destaque foi observado que o Y-27632 reverteu o efeito da sinvastatina sob estímulo de TNF-? e que o mevalonato e os isoprenóides intermediários da via do colesterol, GGPP e FPP, não foram capazes de reverter o efeito da sinvastatina. Dados indicam que a via de sinalização dependente em NO-GMPc aparentemente não modula as propriedades adesivas dos neutrófilos sob condições inflamatórias. Por outro lado, resultados indicam que as Rho GTPases parecem estar envolvidas na regulação das propriedades adesivas dos neutrófilos sob condições inflamatórias. O envolvimento de ROCK na adesão celular ainda não está completamente compreendido, mas de acordo com nossos resultados podemos sugerir a hipótese de que esta enzima efetora tenha um papel na inducão de adesão dos neutrófilos na presença de um estímulo inflamatório. A sinvastatina foi capaz de inibir as propriedades adesivas de neutrófilos quando ativados indicando mais uma utilidade desta classe de drogas no tratamento de doenças inflamatórias. O papel das Rho GTPases nas propriedades adesivas dos neutrófilos sob condições inflamatórios ainda precisa ser melhor elucidado / Abstract: During the inflammatory response, neutrophils and other leukocytes adhere to the endothelium leaving the blood vessels and actively moving towards the inflammatory focus. The migration of neutrophils to inflammatory sites depends on a variety of chemotactic and adhesive events resulting from the activation of adhesion molecules such as selectins and integrins and chemokine receptors. Due to its properties, neutrophils may be activated by small intracellular signaling proteins, the Rho GTPases, which help neutrophils to fulfill this function by interfering in cytoskeletal changes. These proteins are also involved in cell adhesion and proliferation. The neutrophils are able to synthesize nitric oxide (NO), and this production of NO is an important component of innate immunity during inflammation. Studies have shown that neutrophils play a role in inducing inflammation and generation of efforts to understand the adhesive mechanism exerted by neutrophils in inflammatory processes may be a key point for pharmacological interventions for diseases that are characterized by vascular inflammation with consequent obstruction of blood flow. Thus, this study aimed to evaluate the role of the NO pathway and the Rho GTPases in the mechanism by which inflammatory stimuli increases neutrophil adhesion. We also assessed the effects of simvastatin on neutrophil adhesive properties as a tool to aid in studying the involvement of Rho GTPases, RhoA and Rac1 in these mechansims. Neutrophils were isolated from peripheral blood and aspects of adhesion studied by static and flow adhesion assays as well as flow cytometry. In addition, we analyzed the gene expression of Rho GTPases, Rac1 and RhoA by real time - PCR. Following a strong stimulation with TNF-?, the adhesive properties of neutrophils increase significantly. NO synthase inhibitors and NO donors did not modify the adhesive properties of neutrophils when stimulated with TNF-?. We did not observe any significant differences in the adhesion of neutrophils and the expression of adhesion molecules on their surface in the presence of a Rac1 inhibitor. However, an inhibitor of ROCK (Rho-associated coiled coil forming protein serine/threonine kinase, an efector protin for the RhoA), Y-27632, was shown to increase the adhesion of neutrophils under basal conditions. The use of simvastatin decreased adhesive properties and modulated the expression of Mac-1 of neutrophils in the presence of an inflammatory stimulus, supporting the use of this class of drugs as anti-inflammatory agents. Importantly, the attenuating effects of simvastatin on TNF-? stimulated neutrophil adhesion were reversed by Y-27632, whereas the cholesterol pathway intermediates, mevalonate, and the isoprenoids, GGPP FPP, were unable to reverse the effects of this drug. Data indicate that the NO-cGMP signaling pathway does not appear to modulate the adhesive properties of neutrophils under inflammatory conditions. Moreover, results suggest that Rho GTPases may be involved in the regulat ion of the adhesive properties of neutrophils. The involvement of ROCK in cellular adhesion is not yet fully understood but, according to our findings, it may be hypothesized that this protein effector has a role in the induction of neutrophil adhesion. Simvastatin was able to inhibit the adhesive properties of neutrophils when activated, indicating another use of this class of drugs for the treatment of inflammatory diseases. The role of Rho GTPases in the adhesive properties of neutrophils under inflammatory conditions should be further elucidated / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

La voie RhoA/Rho-kinases (ROCK) joue un rôle important dans la physiopathologie de l’hypertension pulmonaire (HTP) par son implication dans le dysfonctionnement endothélial, la constriction et le remodelage des vaisseaux pulmonaires. Selon les classifications internationales, la bronchopneumopathie chronique obstructive (BPCO) et la pneumopathie infiltrante diffuse (PID) sont deux causes fréquentes d’HTP ayant en commun plusieurs mécanismes physiopathologiques dont le dysfonctionnement endothélial, le remodelage vasculaire et la fibrose parenchymateuse. Les objectifs de ce travail étaient d’étudier le rôle de la voie RhoA/ROCK dans la physiopathologie de la BPCO et de la PID avec ou sans HTP et de préciser les anomalies moléculaires liées à la perturbation de la signalisation de cette voie dans chacune de ces situations.Le dysfonctionnement endothélial est un événement essentiel dans l’initiation et la progression de la BPCO. L’activation de la voie RhoA/ROCK dans le dysfonctionnement endothélial systémique et pulmonaire a été mise en évidence chez les tabagiques avec ou sans BPCO. Les résultats de notre première étude montrent l’existence d’une activation de la voie RhoA/ROCK au niveau des artères pulmonaires chez les patients BPCO ayant un dysfonctionnement endothélial, et une corrélation entre l’activité de la RhoA et l’expression génique et l’activité de la NO synthase endothéliale (NOS-3).L’HTP est une complication grave des PID. Nous avons montré dans notre deuxième étude l’implication de la voie RhoA/ROCK dans la réponse inflammatoire et la fibrose pulmonaire (FP) dans un modèle murin de PID induite par injection intratrachéale de bléomycine (BLM). Nous avons ensuite testé l’effet préventif du fasudil, un inhibiteur des ROCK, sur l’apparition de la FP et l’HTP expérimentales induites par la BLM. Les résultats de cette deuxième étude montrent que la FP et l’HTP sont associées à une activation de la voie RhoA/ROCK dans ce modèle murin et que le fasudil inhibe la réponse inflammatoire, la FP et l’HTP, via l’inhibition de la phosphorylation de Smad2/3 de la voie de signalisation par le TGF-β1.La FP et l’HTP représentent deux causes principales de mortalité liée à la sclérodermie systémique (ScS). Nous avons étudié le rôle de la voie RhoA/ROCK dans la physiopathologie de la fibrose cutanée et l’atteinte pulmonaire dans un modèle murin de ScS induite par injection intradermique d’acide hypochloreux (HOCl). Les résultats de cette troisième étude montrent l’association entre la fibrose cutanée induite par l’HOCl et l’activation de la voie RhoA/ROCK au niveau de la peau, et l’effet préventif du fasudil sur la fibrose cutanée et pulmonaire, en partie via l’inhibition de la phosphorylation de Smad2/3 et de l’activation des protéines ERK1/2. Ces résultats suggèrent l’implication de la voie RhoA/ROCK dans la physiopathologie de la BPCO et de la PID avec ou sans HTP. La voie RhoA/ROCK pourrait de ce fait représenter une nouvelle cible thérapeutique dans la BPCO et la PID avec ou sans HTP.Mots-clés : RhoA, Rho-kinases, fasudil, BPCO, fibrose pulmonaire, hypertension pulmonaire. / The RhoA/Rho-kinases (ROCK) pathway plays a pivotal role in the pathophysiology of pulmonary hypertension (PH) as its abnormal activation leads to endothelial dysfunction, sustained vasoconstriction and pulmonary vascular remodeling. According to the international classification of PH, chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) represent two main causes of PH associated with chronic respiratory diseases. These two causes have in common major pathophysiological mechanisms such as endothelial dysfunction, vascular remodeling and interstitial fibrosis. The aims of the present study were to investigate the role of the RhoA/ROCK pathway in the pathophysiology of lung vascular and interstitial injuries in COPD and ILD with or without development of PH, and to study the molecular mechanisms associated with regulation of the RhoA/ROCK pathway in each of these situations.The pulmonary endothelial dysfunction is an essential event in the initiation and progression of COPD. Although the role of the RhoA/Rho-kinase pathway in pulmonary endothelial dysfunction has been demonstrated in smokers with normal lung function, little is known about its role in patients with COPD. The results of our first study demonstrated an increase in RhoA and ROCK activity in pulmonary arteries of patients with COPD, simultaneously with an altered pulmonary endothelial-dependent vasodilation. The increased RhoA activity in patients with COPD was correlated with an impairment of the gene expression and activity of endothelial NO synthase (eNOS).PH associated with pulmonary fibrosis (PF) considerably worsens prognosis of ILD. The results of our second study showed an activation of the RhoA/ROCK pathway in lung tissues of mice intoxicated by intratracheal instillation of bleomycin (BLM). BLM induced severe PF and PH in mice, associated with an increased RhoA and ROCK activity in the lung. We further demonstrated that long-term treatment with fasudil, a selective ROCK inhibitor, reduced BLM-induced lung inflammation, lung fibrosis and PH in mice, at least in part, via inhibition of Smad2/3 phosphorylation in TGF-β1 signaling.PF and PH represent two leading causes of death in patients with systemic sclerosis (SSc). In our third study, we investigated the role of the RhoA/ROCK pathway in the pathophysiology of skin fibrosis and lung injuries in a murine model of SSc induced by intradermal injection of hypochlorous acid (HOCl). We demonstrated that HOCl-induced skin fibrosis was associated with an activation of the RhoA/ROCK pathway in the fibrotic skin, and that long-term treatment with fasudil reduced both skin and lung fibrosis through inhibition of the phosphorylation of Smad2/3 and ERK1/2 in the fibrotic skin.These results suggest the implications of the RhoA/ROCK pathway in the pathophysiology of lung vascular and interstitial injuries in COPD and ILD with and without development of PH. The RhoA/ROCK pathway might be a promising therapeutic target for patients with COPD or ILD with and without PH.

Candida albicans est un pathogène fongique opportuniste de l’Homme, qui peut causer des infections superficielles mais aussi systémiques chez les patients immunodéprimés. Sa virulence est associée à sa capacité de changer d’une forme bourgeonnante à une forme hyphale. La petite GTPase de type Rho, Cdc42, est critique pour la croissance filamenteuse et, sous forme activée, sa localisation est restreinte à l’extrémité des hyphes. J’ai utilisé un système photoactivable, constitué des domaines d’Arabidopsis thaliana Cry2PHR-CibN, pour contrôler le recrutement de Cdc42 constitutivement actif à la membrane plasmique. J'ai déterminé comment le photo-recrutement de Cdc42 constitutivement actif perturbe la croissance filamenteuse et où, quand et comment une nouvelle croissance filamenteuse est ré-initiée. Mes résultats démontrent que, lors du photo-recrutement de Cdc42 constitutivement actif, l'extension du filament cesse puis un nouveau site de croissance s’établit dans la cellule. La localisation de ce nouveau site de croissance est corrélée à la longueur du filament. J'ai étudié les mécanismes moléculaires qui sous-tendent le désassemblage du site de croissance initial et l'emplacement spécifique du nouveau site de croissance filamenteuse. Dans les hyphes en croissance, un «cluster» de vésicules, appelé Spitzenkörper, est localisé à l'extrémité du filament. Lors du photo-recrutement de Cdc42 constitutivement actif, un nouveau «cluster» de vésicules, de composition similaire à celui du Spitzenkörper initial, apparaît dans la cellule mère. J'ai suivi la dynamique du Spitzenkörper et la localisation de Cdc42 sous forme activée, des sites d'endocytose, des vésicules de sécrétion et des câbles d’actine suite à la perturbation du site de croissance initial dans le filament. Dans l’ensemble, mes résultats indiquent qu'il existe une compétition pour la croissance entre le Spitzenkörper et le «cluster» de vésicules qui se forme immédiatement après le photo-recrutement de Cdc42 constitutivement actif et qu'un axe de polarité dynamique peut être établi en l'absence de croissance directionnelle. / Candida albicans is a fungal human pathogen that can cause life-threatening infections in immunocompromised patients, in part, due to its ability to switch between an oval budding form and a filamentous hyphal form. The small-Rho GTPase Cdc42 is crucial for filamentous growth and, in its active form, localizes as a tight cluster at the tips of growing hyphae. I have used a light-activated membrane recruitment system comprised of the Arabidopsis thaliana Cry2PHR-CibN domains to control the recruitment of constitutively active Cdc42 to the plasma membrane. I have determined how photorecruitment of constitutively active Cdc42 perturbs filamentous growth and where, when and how new filamentous growth is subsequently initiated. My results demonstrate that, upon photorecruitment of constitutively active Cdc42, filament extension is abrogated and a new growth site can be established in the cell. Location of a new filamentous growth site correlates with the length of the initial filament. I have investigated the molecular mechanisms that underlie the disassembly of an initial growth site and the specific location of the new filamentous growth site. In growing hyphae a cluster of vesicles, referred to as a Spitzenkörper, is localized at the tip of the filament. Upon photorecruitment of constitutively active Cdc42, a new cluster of vesicles, with a composition similar to that of the initial Spitzenkörper, appears in the mother cell. I have followed the dynamics of the Spitzenkörper, active Cdc42, sites of endocytosis, secretory vesicles and actin cables subsequent to disruption of the initial growth site in the filament. Taken together, my results suggest that there is competition for growth between the Spitzenkörper and the cluster of vesicles that forms immediately after the photorecruitment of constitutively active Cdc42 and that a dynamic polarity axis can be established in the absence of directional growth.

Parmi les régulateurs nucléiques, les petits ARN régulateurs sont un moyen rapide d’adaptation. On les retrouve dans tous les domaines du vivant. Chez les bactéries, l’action d’un grand nombre d’entre eux dépend de la protéine chaperonne d’ARN Hfq. Leur mode d’action consiste à d’apparier directement à l’ARNm cible par complémentarité imparfaite de séquence. Il en résulte une régulation positive ou négative, de la traduction et/ou de la stabilité de l’ARNm cible.Au cours de la première partie cette thèse, nous avons essayé de caractériser la régulation des ARNm par les petits ARN régulateurs. Pour ce faire, nous avons étudiés les différents composants de cette régulation et nous avons pu montrer que les sites de fixation à la protéine Hfq sur les ARN devaient être compatibles pour obtenir une régulation optimale des ARNm par les petits ARN régulateurs.Lors de la seconde partie de la thèse, nous avons pu identifier un nouveau type de régulation des ARNm par les petits ARN régulateurs : la polarité transcriptionnelle. Nous avons pu montrer que la fixation du petit ARN ChiX sur la région 5’UTR de l’ARNm chiPQ entrainer l’inhibition de la traduction de chiP mais également une terminaison prématurée rho-dépendante de la transcription de l’ARNm.Nous avons par la suite cherché à savoir si ce type de régulation pouvait se retrouver dans la régulation d’autre ARNm comme l’opéron dppABCDF et l’opéron pgaABCD. En parallèle de ces études, nous avons également essayé de caractérisé la terminaison Rho-dépendante entre S. Typhimurium et E.coli au sein de l’ARNm chiPQ. / Among the nucleic regulators, small regulatory RNAs are a quick way to adapt. They are found in all areas of life. In bacteria, the action of a large number of them depends on the chaperone protein of Hfq RNA. Their mode of action consists of matching directly to the target mRNA by imperfect complementary sequence. This results in a positive or negative control, translation and/or stability of the target mRNA.During the first part of this thesis, we have tried to characterize the regulation of mRNA by small regulatory RNAs. To do this, we studied the different components of this regulation and we have shown that the binding sites on protein Hfq on RNA should be consistent for optimal regulation of mRNA by small regulatory RNAs.In the second part of the thesis, we identified a new type of mRNA regulation by small RNA regulators: transcriptional polarity. We could show that the fixing of small RNA ChiX on the 5 'UTR region of the mRNA chiPQ cause inhibition of translation of chiP but also a Rho-dependent premature termination of mRNA transcription.We subsequently investigated whether this type of regulation could be in the control of other mRNAs as dppABCDF’s operon and pgaABCD’s operon. In parallel with these studies, we have also tried characterized Rho-dependent termination between S. Typhimurium and E. coli in mRNA chiPQ.

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Bakterien der Gattung Shigella sind die Erreger der bakteriellen Ruhr beim Menschen. Der wesent-liche Virulenzfaktor der Shigellen ist ihre Fähigkeit zur Invasion. Die Invasion in Epithel-zellen ist Ausdruck des erregerspezifischen Infektionsprozesses, der gekennzeichnet ist durch eine vom Bakterium induzierte zelluläre Aufnahme über einen Phagozytose-ähnlichen Mechanismus. In dessen Verlauf führt die charakteristische Reorganisation des Zytoskeletts der Zelle zur Ausbildung einer blütenartigen Membranstruktur an der bakteriellen Eintrittsstelle. Als essentielles Glied der Signalisationskaskade vom Bakterium zum zellulären Zytoskelett erwies sich das kleine G-Protein Rho. Es wurde gezeigt, daß Rho als Regulator der Veränderungen des Zytoskeletts selbst an den bakteriellen Invasionslocus rekrutiert wird. Diese Rekrutierung umfaßt drei Isoformen von Rho: RhoA, RhoB und RhoC. Trotz hoher Sequenzhomologie der Isoformen untereinander ( 85% ) existiert ein unterschiedliches Rekrutierungsmuster dieser Isoproteine an der Eintrittsstelle von Shigella flexneri. RhoA akkumuliert vorwiegend um die eindringenden Bakterien herum. Demgegenüber werden RhoB und RhoC in die bakterieninduzierten zellulären Protrusionen rekrutiert. Der Mechanismus dieser isoformspezifischen Rekrutierung ist nicht bekannt. Anhand unserer Experimente konnten wir zeigen, daß das allen kleinen G-Proteinen gemeinsame C-terminale Peptid-Motiv CAAX ( C = Cystein, A = aliphatische Aminosäure, X = Leucin ) eine wesentliche Voraussetzung für die Rekrutierung von Rho darstellt, sich die isoformspezifische Rekrutierung jedoch nicht anhand des CAAX-Motivs erklären läßt. Bedeutsam für die differentielle Rekrutierung der Isoproteine ist vielmehr die präterminale Region des Moleküls. Diese Beobachtung hat weitreichende Konsequenzen für die funktionelle Rolle von Rho bei der Epithelzellinvasion durch Shigella. / Shigella is the etiologic agent of human bacillary, an infectious large bowel disease. A major feature of Shigella's pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization requiring cytosceletal rearrangements; Shigella entry induces a blossom-like membrane structure at the bacterial entry site. This membrane folding process is dependent on the small GTPase rho. It has been shown that three rho isoforms rhoA, rhoB and rhoC are recruited into bacterial entry sites with different localization relative to the membrane structures. While rhoA preferentially accumulates in close vicinity to entering bacteria, rhoB and rhoC are recruited into the tips of Shigella-induced cellular protrusions. We could show that the C-terminal CAAX-region of rho is a prerequisite for recruitment, but not sufficient for different recruitment of rho isoforms. Additional information for differential recruitment patterns seems to come from the preterminal region of rho proteins. This result is of great importance for the functional role of rho during Shigella-invasion into epithelial cells.

In einem RT-PCR Ansatz aus neuronalen post mortem Gewebe des Menschen konnten EPB41 (Erythrozytäres Protein Bande 4.1) Spleißformen in verschiedenen Hirnregionen nachgewiesen werden. In einem weiteren RT-PCR Ansatz wurden höhermolekulare p4.1R-Spleißormen generiert, kloniert und zwei der erhaltenen Spleißformen (Klon 9 und Klon 13) charakterisiert. In einer In-situ-Hybridisierungsstudie an humanen Temporalkortex und Hippocampus konnten EPB41-Isoformen in fast allen Neuronen nachgewiesen werden. In immunhistochemischen Untersuchungen mit selbstgenerierten p4.1R spezifischen Antikörper wurden ebenfalls ausschließlich Neurone markiert. In proteinbiochemischen Untersuchungen konnte in verschiedenen humanen Hirnareale mit den p4.1R spezifischen Antikörpern eine 110 kDa und 120 kDa immunreaktive Bande nachgewiesen werden. In Experimenten an Primärkulturen von Rattenneuronen konnte eine Herunterregulation der p4.1R Proteine sowie der mRNA von p4.1R durch Verarmung des funktionellen Pools an G-Proteinen der Rho-Familie in der Zelle gezeigt werden. Die GTPasen der Rho-Familie regulieren unter anderem die Plastizität des Dendritenbaumes von Neuronen. / In a RT-PCR approach using human postmortem cerebral tissue from different brain regions several EPB41 (erythrocyte protein band 4.1) spliceforms could be generated. The amplificates were cloned and two of the highmolecular EPB41 spliceforms Klon 9 and Klon 13 were characterized. Klon 9 is a new spliceform, Klon13 is identical with EPB41 (accesion number AF156225). In an in situ hybridization study the EPB41 spliceforms were detected in almost all neurons of the temporal cortex and the hippocampus. Immunhistochemical localization of the p4.1R immunreactive proteins in human temporal cortex using p4.1R specific peptide antibodies, confirmed these results. The stning pattern of soma and dendrites of the neurones was punctuated. In Western Blot experiments a 110 kDa and 120 kDa p4.1R immunreactive proteinband was detected. A regulation of the protein 4.1R immunreactive proteins as well as the mRNA of protein 4.1 was found in experiments in which the functional pool of Rho GTPases in hippocampal primary neurones of the rat was manipulated.

Shigellen sind Erreger der bakteriellen Dysenterie beim Menschen. Ein notwendiger Schritt bei der Pathogenese der Shigellose ist die Invasion von Darmepithelzellen durch das Bakterium. Der Mikroorganismus löst dabei in der Wirtszelle Veränderungen des Aktinzytoskeletts aus, die zur Bildung einer blütenähnlichen Membranstruktur und schließlich zur Internalisierung des Pathogens führen. Diese Umbauvorgänge am Zytoskelett sind abhängig von einem Wirtszellprotein, der kleinen GTPase Rho. Drei Isoformen von Rho (A, B und C) sind beschrieben, deren Aminosäuresequenzen zu etwa 90% identisch sind. Während der Zellinvasion durch Shigella akkumulieren verschiedene Rho-Isoformen an unterschiedlichen Lokalisationen des Invasionskomplexes. Dabei werden RhoA vorwiegend um die eindringenden Bakterienherum, RhoB und RhoC hingegen hauptsächlich in die bakterieninduzierten zellulären Protrusionen rekrutiert. Durch Untersuchung von Rho-Hybridkonstrukten konnte gezeigt werden, daß ein prä-C-terminales, acht Aminosäuren umfassendes Modul die Rekrutierungsmuster von RhoA bzw. RhoC bestimmt. Der Austausch zweier Aminosäuren innerhalb des Moduls führte zu einer Konversion des Rekrutierungsmusters von RhoA. Wir konnten zeigen, daß die Rekrutierung von RhoA vom Funktionszustand der GTPase (Bindung von GTP/GDP) sowie von der Phosphorylierung durch die Proteinkinase A unabhängig ist. Schließlich wurde hier nachgewiesen, daß auch RhoD, das zu RhoA, B und C auf der Primärstrukturebene nur zu etwa 50% homolog ist, an die Bakterieneintrittsstelle rekrutiert werden kann. RhoD folgt dabei dem Rekrutierungsmuster von RhoB und RhoC. / Shigella causes bacillary dysentery in humans. Bacterial invasion of enterocytes is an essential step in the pathogenesis of shigellosis. Pathogen-triggered rearrangements of the host cell actin cytoskeleton induce a blossom-like membrane structure for internalisation of the microorganism. Actin remodeling requires activity of the host cell small GTPase rho. Three highly homologous rho isoforms (A, B and C) have been described with amino acid identities of about 90%. During Shigella invasion these rho isoforms accumulate at different sites of the invasion complex. While rhoA is chiefly recruited around entering bacteria, rhoB and rhoC are essentially translocated to the bacteria-induced cellular protrusions. Using a variety of rho hybrid constructs in a HeLa cell transfection-infection assay we were able to show that a pre-C-terminal stretch of eight amino acids determines the recruitment patterns of rhoA and rhoC. Exchange of two amino acids was sufficient for conversion of the rhoA recruitment pattern into a rhoC-like pattern. We could demonstrate that rhoA recruitment is independent of its functional state (GDP- or GTP-bound) or phosphorylation by the proteinkinase A (PKA). Finally, we have shown that rhoD, another member of the rho family which shares only 50% of its primary structure with rhoA, B or C, is also recruited to the bacterial entry site exhibiting a rhoB/C-like pattern of distribution.