Members of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family are important regulators of the actin cytoskeleton dynamics. VASP functions as well as its interactions with other proteins are regulated by phosphorylation at three sites - serine157 (S157), serine239 (S239), and threonine278 (T278) in humans. cAMP- and cGMP- dependent protein kinases phosphorylate S157 and S239, respectively. In contrast, the kinase responsible for T278 was as yet unknown and identified in the first part of this thesis. In a screen for T278 phosphorylating kinases using a phospho-specific antibody against phosphorylated T278 AMP-activated protein kinase (AMPK) was identified in endothelial cells. Mutants of AMPK with altered kinase-activity modulate T278-phosphorylation levels in cells. AMPK-driven T278-phosphorylation impaired stress fiber formation and changed cell morphology in living cells. AMPK is a fundamental sensor of cellular and whole body energy homeostasis. Zucker Diabetic Fatty (ZDF) rats, which are an animal model for type II diabetes mellitus, were used to analyze the impact of phosphorylated T278 in vivo. AMPK-activity and T278-phosphorylation were substantially reduced in arterial vessel walls of ZDF rats in comparison to control animals. These findings demonstrate that VASP is a new AMPK substrate, that VASP phosphorylation mediates the effects of metabolic regulation on actin cytoskeleton rearrangements, and that this signaling system becomes down-regulated in diabetic vessel disorders in rats. In the second part of this thesis, a functional analysis of differential VASP phosphorylations was performed. To systematically address VASP phosphorylation patterns, a set of VASP phosphomimetic mutants was cloned. These mutants enable the mimicking of defined phosphorylation patterns and the specific analysis of single kinase-mediated phosphorylations. VASP localization to the cell periphery was increased by S157- phosphorylation and modulated by phosphorylation at S239 and T278. Latter phosphorylations synergistically reduced actin polymerization. In contrast, S157- phosphorylation had no effect on actin-dynamics. Taken together, the results of the second part show that phosphorylation of VASP serves as a fine regulator of localization and actin polymerization activity. In summary, this study revealed the functions of VASP phosphorylations and established novel links between signaling pathways and actin cytoskeleton rearrangement. / Die Mitglieder der Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) Familie sind bedeutende Regulatoren der Aktinzytoskelettdynamik. Die Funktionen und die Protein-Protein-Wechselwirkungen von VASP werden durch Phosphorylierungen an drei Aminosäureresten reguliert. Im Fall von humanem VASP sind dies Serin157 (S157), Serin239 (S239) und Threonin278 (T278). S157 und S239 sind Substrate der cAMP- und cGMP-abhängigen Proteinkinasen. Die Kinase, die T278-Phosphorylierung vermittelt, ist nicht bekannt. Der erste Teil der Arbeit beschäftigt sich mit der Identifizierung der T278-phosphorylierenden Kinase. Mit Hilfe eines phospho-spezifischen Antikörpers gegen das phosphorylierte T278 (pT278) wurde in Endothelzellen eine systematischen Suche nach T278-phosphorylierenden Kinasen durchgeführt. Dabei wurde die AMP-aktivierte Proteinkinase (AMPK) entdeckt. Mutanten der AMPK, welche eine veränderte Kinaseaktivität besitzen, erhöhten bzw. reduzierenten das Niveau der pT278. Die T278-Phosphorylierung durch die AMPK reduzierte die Stressfaserbildung und führt zu einer veränderten Zellmorphologie. Die AMPK ist ein fundamentaler Sensor des zellulären und Organismus-umfassenden Energiehaushalts. Zur Analyse der Funktion der pT278 in vivo wurden Zucker Diabetic Fatty (ZDF) Ratten, ein Tiermodell für den Diabetes mellitus Typ II, verwendet. Die AMPK-Aktivität und die pT278 waren in arteriellen Gefäßwänden von ZDF-Ratten im Vergleich zu Kontrolltieren deutlich reduziert. Diese Ergebnisse zeigen, dass VASP ein neues Substrat der AMPK ist, dass die T278-Phosphorylierung metabolische Signale an das Aktin-Zytoskelett koppelt und, dass bei diabetischen Ratten dieser Signaltransduktionsweg supprimiert ist. Im zweiten Teil wurde die Bedeutung der VASP-Phosphorylierungsmuster für die Aktin- bildung und die VASP-Lokalisation untersucht. Hierzu wurden systematisch VASP- Phoshorylierungsmutanten generiert. Diese Mutanten imitieren fixierte Phosphorylierungen oder erlauben einzelne Phosphorylierungen durch die jeweilige Kinase. Die Untersuchungen zeigten, dass S157-phosphoryliertes VASP (pS157) sich an der Zellperipherie anreichert, wobei die S239- und T278-Phosphorylierungen diesen Lokalisationseffekt modulieren. Phosphoryliertes S239 und T278 reduzierten synergistisch die Aktinpolymerisation. Im Gegensatz hierzu beeinflusste pS157 die Aktindynamik nicht. Dies zeigt, dass die VASP- Phosphorylierungen als Feinregulator für die Lokalisation und die Aktinpolymerisationsak- tivität fungierten. Zusammenfassend identifiziert diese Studie die Funktionen der einzelnen VASP- Phosphorylierungen und deckt neue Verbindungen von Signalwegen zur Aktinzytoskelett- Reorganisation auf.

Listeria monocytogenes, ein fakultativ intrazellulärer Krankheitserreger, besitzt die Fähigkeit, Wirtszellen zu penetrieren, sich in ihnen zu vermehren, sich intrazellulär zu bewegen und auch benachbarte Zellen direkt zu infizieren. Die intrazelluläre Fortbewegung erfolgt durch Polymerisation von zellulärem Aktin, wodurch charakteristische Aktinschweife an einem Pol der Bakterien entstehen. Der einzige bakterielle Faktor, der für die Aktinpolymerisation notwendig ist, ist das Oberflächenprotein ActA. ActA allein ist aber nicht in der Lage, Aktin zu polymerisieren, sondern kann dies nur in Assoziation mit Proteinen der Wirtszelle. Die einzigen bisher bekannten Wirtszellproteine, die direkt mit ActA interagieren, sind das Phosphoprotein VASP und der Arp2/3-Komplex. VASP bindet an den zentralen prolinreichen Bereich von ActA und beschleunigt durch die Rekrutierung von Profilin den Prozeß der Aktinpolymerisation. Der Arp2/3-Komplex interagiert mit dem N-terminalen Bereich von ActA und initiiert die eigentliche Aktin-Polymerisation. Um weitere eukaryotische, mit ActA interagierende Proteine (AIPs) zu isolieren, wurde über einen "Yeast Two-Hybrid"-Test mit ActA als Köder eine embryonale Maus-cDNA-Genbank getestet. Dabei wurden drei verschiedene AIPs identifiziert, von denen eines identisch mit dem humanen Protein LaXp180 (auch "CC1" genannt) ist. LaXp180 ist ein 180 kDa Protein mit über 50 theoretischen Phosphorylierungsstellen in der N-terminalen Hälfte, während die C-terminale Hälfte "coiled-coil"-Strukturen ausbilden kann. Darüberhinaus enthält LaXp180 eine Kern-Lokalisations-Sequenz und ein Leucin-Zipper-Motiv. Die Bindung von LaXp180 an ActA wurde in vitro unter Verwendung von rekombinantem His6-Tag-LaXp180 und rekombinantem ActA bestätigt, da rekombinantes ActA nur an einer Ni-Agarose-Säule gebunden wurde, wenn diese vorher mit His6-Tag-LaXp180 beladen war. Über RT-PCR konnte zum ersten Mal die Expression LaXp180-spezifischer mRNA in verschiedenen Säugerzellen nachgewiesen und mit einem polyklonalen anti-LaXp180-Serum durch Immunopräzipitation erstmals ein 194 kDa großes Protein in Säugerzellextrakten detektiert werden. Die intrazelluläre Lokalisation von LaXp180 wurde über Immunfluoreszenzmikroskopie untersucht. Immunfluoreszenzfärbungen von Fibroblasten mit dem anti-LaXp180-Serum zeigten eine starke Färbung der Zellkerne und definierter Bereiche direkt neben den Kernen, während das restliche Zytoplasma schwach gefärbt war. Über Immunfluoreszenzmikroskopie mit dem anti-LaXp180-Serum an mit L. monocytogenes infizierten Zellen konnte gezeigt werden, daß LaXp180 mit der Oberfläche vieler, aber nicht aller intrazellulärer, ActA-exprimierender Listerien kolokalisiert. Dagegen wurde nie eine Kolokalisation mit intrazellulären, aber ActA-defizienten Mutanten beobachtet. Darüberhinaus ist LaXp180 asymmetrisch auf der Bakterienoberfläche verteilt und schließt sich gegenseitig mit der F-Aktin-Polymerisation aus. LaXp180 ist ein putativer Bindungspartner von Stathmin, einem 19 kDa Phosphoprotein, das die Mikrotubuli-Dynamik reguliert. Über Immunfluoreszenz konnte gezeigt werden, daß auch Stathmin mit intrazellulären, ActA-exprimierenden L. monocytogenes kolokalisiert. / Listeria monocytogenes, a facultative intracellular pathogen, is able to penetrate and to multiply in host cells, to move intracellularly and to infect neighbouring cells directly. Intracellular movement is caused by the polymerisation of host cell actin leading to the generation of characteristic actin tails at one pole of the bacterium. The only bacterial factor necessary for actin polymerisation is the surface protein ActA. However, ActA itself is unable to polymerize actin, and can only do so in association with host cell proteins. So far the only known host cell proteins directly interacting with ActA are the phosphoprotein VASP and the Arp2/3 complex. VASP binds to the central proline-rich repeat region of ActA and accelerates actin polymerisation by the recruitment of profilin. The Arp2/3 complex interacts with the N-terminal domain of ActA and initiates the actin polymerisation process. To identify additional eukaryotic ActA-interacting proteins (AIPs), an embryonic mouse cDNA library was screened in a yeast two-hybrid approach using ActA as bait. Three different AIPs were isolated, one of which was identical to the human protein LaXp180 (also called "CC1"). LaXp180 is a 180 kDa protein with more than 50 theoretical phosphorylation sites in the N-terminal part. The C-terminal part is able to form coiled-coil structures. Furthermore, LaXp180 contains a nuclear localisation sequence and a leucine-zipper motif. Binding of LaXp180 to ActA was demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Recombinant ActA was retained on a Ni-agarose column after prior loading of the column with histidine-tagged LaXp180. Using RT-PCR, the expression of LaXp180 specific mRNA in different mammalian cells was demonstrated for the first time. A protein with a molecular weight of 194 kDa was detected in cell extracts by immunoprecipitation with a polyclonal anti-LaXp180 antiserum. The intracellular localisation of LaXp180 was analysed by immunofluorescence microscopy. Immunofluorescence staining of fibroblasts with the anti-LaXp180 serum showed a strong staining of the nuclei and of defined regions next to the nuclei as well as a weak staining of the rest of the cytoplasm. Fluorescence microscopy with the anti-LaXp180 antiserum of cells infected with L. monocytogenes revealed colocalisation of LaXp180 with the bacterial surface of a subset of intracellular, ActA-expressing listeriae. In contrast, colocalisation with intracellularly growing but ActA-deficient mutants was never observed. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerisation on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a 19 kDa phosphoprotein regulating microtubule dynamics. Using immunofluorescence, colocalisation of stathmin with intracellular, ActA-expressing L. monocytogenes was also demonstrated.

Neospora caninum é um protozoário pertencente ao filo Apicomplexa que atinge, dentre diversos hospedeiros intermediários, principalmente bovinos e tem emergido como um importante causador de problemas reprodutivos e abortos em rebanhos de corte e leiteiro. Organismos do filo Apicomplexa são parasitas intracelulares obrigatórios que, para locomoverem-se e realizarem a invasão das células hospedeiras, utilizam um mecanismo próprio de locomoção ativa impulsionada pelo motor actina/miosina denominado motilidade por deslizamento (gliding motility), cujo complexo motor localiza-se entre a membrana plasmática e o complexo de membrana interno do parasita. A investigação a respeito do funcionamento desse mecanismo de locomoção e invasão vem sendo realizada principalmente em Toxoplasma gondii e Plasmodium spp., entretanto não há nenhuma publicação envolvendo actina em N. caninum. Esse trabalho envolveu a clonagem e expressão da sequência NcAct201-310 e deu início a caracterização da actina de N. caninum (NcAct). A sequência NcAct foi obtida em banco de dados ToxoDB, e uma comparação por alinhamento entre as actinas de Apicomplexas relacionados revelou que NcAct é idêntica à TgACT1 (100% identidade). Com outras espécies, a NcAct tem maior identidade/similaridade com a actina de Eimeria tenella (97%/99%), seguida da actina de Plasmodium falciparum PfACT1 (93%/97%), da actina de Babesia bovis (86%/94%) e PfACT2 (80%/92%). Quando localizada com anticorpo anti-?-actina C4, NcAct apresenta-se em duas bandas de 43 e 45 kDa em gel de acrilamida 1D e em nove isoformas em gel de acrilamida 2D. Todas as identidades das bandas e spots foram confirmados por espectrometria de massas (MS/MS). Além disso, NcAct localiza-se, em sua maioria, na região periférica do taquizoíta de N. caninum e sua distribuição é alterada após incubação dos taquizoítas com 5 ?M de jasplakinolida (JAS) ou 2 ?M de citocalasina D (CytD). Por fim, por meio de ensaio de fracionamento de actina monomérica (actina-G) e filamentosa (actina-F), demonstramos que a JAS é capaz de aumentar a quantidade de actina-F em taquizoítas de N. caninum. / Neospora caninum is an Apicomplexan protozoan that infects, among a whole range of intermediate hosts, bovine where it is emerging as a relevant cause of reproductive problems and abortion in dairy and beef cattle. As obligatory intracellular organisms, parasites from Apicomplexa Phylum use their own active locomotion system to move and invade host cells. This mechanism is driven by the actin/myosin motor known as gliding motility, localized between the plasma and the inner membrane complex. Studies involving this locomotion and invasion system have been conducted mainly in Toxoplasma gondii and Plasmodium spp. To our knowledge there is no publication involving actin in N. caninum, so this work was outlined and involved the cloning and expression of the sequence NcAct201-310, initiating the characterization of actin of N. caninum (NcAct). The sequence NcAct was obtained from the Database ToxoDB, and a comparison of actins from Apicomplexa-related revealed total identity of NcAct with TgACT1 (100% identity). With other species, NcAct has higher identity/similarity with Eimeria tenella actin (97%/99%), followed by Plasmodium falciparum actin PfACT1 (93%/97%), Babesia bovis actin (86%/94%) and PfACT2 (80%/92%). When localized with the antibody anti-?-actin C4, NcAct is presented as two bands of 43 and 45 kDa in 1D acrylamide gel and as nine isoforms in 2D acrylamide gel. All these findings were confirmed by mass spectrometry (MS/MS). Moreover, NcAct localizes predominantly in the peripheric region of N. caninum tachyzoites. This distribution is altered after incubation of the tachyzoites with 5 ?M of jasplakinolide (JAS) or 2 ?M of cytochalasin D (CytD). Finally through fractionating assay of monomeric (actin-G) and filamentous (actin-F), we demonstrated that JAS is capable of increasing the quantity of actin-F in N. caninum tachyzoites.

Accumulating evidence highlights the importance of studying the migration of cells within the context of their natural environment as manipulating the substrate on which a cell is migrating can have a dramatic impact on the mode/mechanisms employed by cells during migration. Central to this phenomenon is the requirement of adhesion to the ECM in order to gain traction during migration. Integrins constitute the main family of cell receptors involved in mediating cell-ECM interactions during motility. Whilst traditionally two-dimensional cell culture studies have placed emphasis on the importance of these receptors for spreading and migration, it has become evident that within more confined environments these receptors, at least for some cell types, are less crucial. In this research we utilise Drosophila embryonic hemocytes as an in vivo model for cell migration. We show that whilst hemocytes migrate within confined environments in vivo, these cells depend on integrins for powering both developmental and inflammatory migrations. Given the close association between these receptors and the actin cytoskeleton we were surprised to discover that removal of the main β integrin subunit, Myospheroid, did not affect cell spreading in vivo and had only a small impact on lamellipodial structure and dynamics. Furthermore we discovered that, in contrast to other cell types previously analysed, removal of this integrin subunit in hemocytes was not accompanied by an increase in the rate of actin retrograde flow within the protrusions, which we believe could reflect abrogation of a positive feedback between Rho, ROCK and Myosin II contraction. Instead, we discover a key role for integrins in regulating the microtubule cytoskeleton, in the maintenance of a polarised microtubule bundle, termed a ‘microtubule-arm’. Although the molecular mechanisms by which this stabilisation is coordinated have yet to be identified, this provides important insight into the co-regulation of adhesion and microtubule cytoskeleton important for the migratory behaviour of these cells. Cell migration reflects the complex and integrated regulation of the actin cytoskeleton by diverse families of actin regulatory proteins. Using hemocytes as a model system, we also explore the regulatory interactions between two main actin regulatory proteins, Diaphanous and Enabled, in vivo. Whilst the function of these proteins in the formation of filopodial protrusions is overlapping, recent research has highlighted the ability of these proteins to regulate the activity of one another. We find that co-expression of Enabled in hemocytes is able to rescue the morphological and migratory defects resulting from overexpression of active Diaphanous. Thus, data here presents Enabled as a negative regulator of Diaphanous, which may play an important role in the migration of hemocytes in vivo.

Orientador: José Ricardo Paciência Rodrigues / Banca: Afonso Mazario / Banca: Henrique Benedito Brenelli / Banca: Cleverson Teixeira / Banca: Gilberto Uemura / Resumo: Estudos de expressão gênica têm identificado vários grupos moleculares de carcinoma de mama, com diferentes comportamentos clínico e biológico. A correlação entre "cDNA microarray" e imunoistoquímica(IQ) com marcadores para citoceratinas, Her2/neu, receptor de estrógeno(RE) e de células basais mioepiteliais (1A4, S-100 e p63), identificaram cinco grupos: (1) luminal A (RE+; Her2/neu-), (2) luminal B (RE+; Her2/neu+), (3) superexpressão de Her2/neu (RE-; Her2/neu+), (4) tipo basal (RE-; Her2/neu-; Ck 5/6 +) e (5) nenhum destes ("null"). Os de tipo luminal expressam citoceratinas de padrão luminal (Ck8/18) e os de tipo basal expressam citoceratinas 5/6 e 14 ou marcadores de células basais mioepiteliais. Avaliamos a expressão de Ck5/6, Ck8/18 e 1A4 em material de citoinclusão, comparando-a ao espécime cirúrgico. Material e Métodos: Foram selecionados 62 casos, seqüenciais, de carcinoma de mama diagnosticados por PAAF, com citoinclusão e espécime cirúrgico. Cortes de citoinclusão e do espécime cirúrgico foram imunocorados para Ck 5/6, Ck 8/18 e 1A4. Resultados e Conclusão: Os valores, em porcentagem, de sensibilidade, especificidade, valor preditivo positivo(VPP), valor preditivo negativo(VPN) e acurácia foram, respectivamente: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 ( 98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Portanto, a identificação de Ck5/6, Ck8/18 e 1A4 por IQ em material de citoinclusão é método confiável, com resultados muito próximos aos obtidos no espécime cirúrgico e pode contribuir para a classificação dos carcinomas mamários de expressão luminal e basal, fornecendo informações importantes que possam orientar na escolha do tratamento, bem como na avaliação de fatores prognósticos e preditivos. A importância da obtenção de dados morfológicos e imunoistoquímicos sobre os carcinomas mamários através do material... (Rewsumo completo, clicar acesso eletrônico abaixo) / Abstract: Genetic expression studies have identified many molecular groups of breast carcinoma, with different clinical and biological behavior. The correlation between cDNA microarray and immunohistochemistry (IHC) with markers for cytokeratin, Her2/neu, estrogen receptor (ER) and of basal myoepithelial cells (1A4, S-100 e p63), identified five groups: (1) luminal A (ER+; Her2/neu-), (2) luminal B (ER+; Her2/neu+), (3) overexpression of Her2/neu (ER- ; Her2/neu+), (4) basal-like (ER- ; Her2/neu-; Ck 5/6 +) and (5) none of them (null). The luminal-like express cytokeratines of luminal pattern (Ck8/18) and the basal-like express cytokeratines 5/6 and 14 or markers of myoepithelial basal cells. We have evaluated the expression of Ck5/6, Ck8/18 and 1A4 in cell block comparing it to the surgical specimen. Material and Methods: 43 62 cases have been selected, sequencial, of breast carcinoma diagnosed through fine needle aspiration (FNA), with cell block and surgical specimen. Cuts of cell block and from the surgical specimen were immunostained for Ck 5/6, Ck 8/18 and 1A4. The value, in percentage, of sensibility, specificity, positive predictive value, negative predictive value, and accuracy were respectively: Ck5/6 (77, 100, 100, 92 e 94); Ck8/18 (98, 66, 96, 80 e 95) e 1A4 ( 92, 96, 85, 98 e 95). Therefore, the identification of CK5/6, 8/18 and 1A4 for IHC in cell block is a reliable method, with results very close to the ones obtained in the surgical specimen, and it can contribute to the sub classification of the breast carcinomas of luminal and basal expression, providing important information, which can orientate the treatment... (Complete abstract click electronic access below) / Doutor

Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.

O carcinoma espinocelular é uma neoplasia maligna que representa aproximadamente 94% de todas as ocorrências presentes em boca e uma das suas principais características celulares é a migração de suas células para formar metástases. A adesão celular é considerada um dos eventos determinantes da migração celular. Para as células formarem uma estrutura tecidual tridimensional as adesões entre células e entre células e matriz extracelular são de grande importância. As junções de adesão celulares surgem, caracteristicamente, pela interação entre receptores adesivos, vias de sinalização e elementos do citoesqueleto. A proteína E-caderina está presente em adesões entre células no tecido epitelial. A proteína FAK está envolvida na maioria dos eventos relacionados à adesão celular estimulada por integrinas. A Vinculina é uma proteína de adesão que se liga ao citoesqueleto de actinomiosina como uma proteína de adesão focal através das integrinas. Estudos recentes sugerem que há alteração na expressão e atividade de proteínas de adesão em tumores malignos. O objetivo deste trabalho foi descrever o padrão de expressão e de regulação da atividade de proteínas de adesão em amostras de tumores de carcinoma espinocelular. Foram realizadas reações de imunoistoquímica para verificar o padrão de distribuição das proteínas E-caderina, Vimentina e FAK-y397 em amostras de tumores de carcinoma espinocelular oral. Verificou-se a diminuição da expressão de E-caderina e de Vinculina em regiões de adesão célula-célula e em contrapartida constatou-se aumento na marcação citoplasmática de Vinculina bem como na marcação de FAK-y397 em todas as amostras de tumores. Apesar dos avanços, ainda são necessários mais estudos observacionais que averiguem não apenas o grau de expressão dessas proteínas de adesão, mas também o seu nível de regulação. A partir dos resultados deste estudo, pode-se sugerir que o controle do nível de expressão e de atividade da adesão celular podem ser considerados como potenciais alvos para a aplicação de terapias coadjuvantes que visam a diminuir ou impedir a progressão tumoral, bem como o desenvolvimento de metástases. / Squamous cell carcinoma is a malignant neoplasm that accounts for approximately 94% of all occurrences present in mouth and one of its main characteristics is the cellular migration of its cells to form metastases. Cell adhesion is considered one of the defining events of cell migration. For a three-dimensional tissue structure, adhesions between cells and between cells and the extracellular matrix is of great importance. Cell adhesion junctions arise characteristically by interaction between adhesive receptors, signaling pathways and cytoskeletal elements. The protein E-cadherin is present in cells in the adhesion between epithelial tissue. The Focal Adhesion Kinase (FAK) protein is involved in most events related to cell adhesion stimulated by integrins. The vinculin is an adhesion protein that binds cytoskeletal protein through integrins activaion. Recent studies suggest that there are alterations in the expression and activity of adhesion proteins in malignant tumors. The aim of this study was to describe the pattern of expression and regulation of the activity of adhesion proteins in tumor samples of squamous cell carcinoma. Immunohistochemical reactions were performed to check the distribution pattern of the protein E-cadherin, vimentin and FAK-y397 in tumor samples of oral squamous cell carcinoma. There was a decrease in the expression of E-cadherin and vinculin in regions of cell-cell adhesion but, on the other hand, it was found to increase in cytoplasmic as well as unscheduled vinculin FAK-y397 in all tumor samples. Despite progress, it is necessary more observational studies that examine not only the degree of expression of these adhesion proteins, but also its level of regulation. From the results of this study it is suggested that the control of the expression level and activity of cell adhesion may be considered as potential targets for application adjuvant therapies that aim to reduce or prevent tumor progression and the development metastases.

<i>Legionella pneumophila,</i> the etiological agent of Legionnaires’ disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates over 330 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK (Lpg0969) as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H₉₅E_XXH₉₉ (x, any amino acid) motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage occurs during <i>L. pneumophila </i>infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, <i>L. pneumophila</i> exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

The I-BAR proteins are a family of actin regulators which include IRSp53 and Missing-In-Metastasis (MIM). These proteins possess an N-terminal I-BAR domain which associates with both the actin cytoskeleton and membrane phospholipids and is able to induce membrane curvature. Previous cell culture and in vitro studies have implicated I-BAR proteins in the regulation of actin protrusion formation, however their roles within the organism are poorly understood. We have used Drosophila melanogaster as a model system in which to study I-BAR protein function at the cellular and organismal level. Drosophila possess two I-BAR proteins, one homologous to IRSp53 and one homologous to MIM. Using full- length and truncated splice variants generated for both dMIM and dIRSp53, we have performed structure/function analysis to determine the role of specific domains in the localisation and actin modifying abilities of the proteins in both cell culture and in vivo. We found that dMIM overexpression typically promotes a lamellipodial morphology, with dMIM localising to the edges of extending lamellipodia. dIRSp53 expression induced a more filopodial phenotype in cell culture, which was not as notable in vivo, however expression of a dIRSp53 splice variant with a WH2 domain resulted instead in a predominantly lamellipodial morphology. Similar to dMIM, dIRSp53 localises to the tip of extending protrusions, albeit more transiently. We found that multiple domains contribute to the localisation and activity of dIRSp53 and dMIM. Following overexpression analysis, complementary loss-of-function analysis was performed in vivo using Drosophila mutants lacking dMIM, dIRSp53 or both genes together. Surprisingly these mutants were viable and morphologically normal. Absence of these genes individually or together did not greatly affect cell migration or actin dynamics in haemocytes or epithelial cells undergoing dorsal closure. However, a role for I- BAR proteins in axonal filopodia formation within primary neuronal cultures was apparent, as was a notable role in neuromuscular junction morphology. We have also identified potential redundancy between Drosophila MIM and the Drosophila F-BAR protein Cip4 in actin bundle regulation within embryonic haemocytes, with an additional novel role for Cip4 alone in haemocyte lamellipodial maintenance. Our results suggest that the Drosophila I-BAR proteins contribute to actin cytoskeleton regulation in vitro and in vivo, particularly within the CNS, and with novel shared functions with other BAR domain family proteins contributing to their regulation of actin cytoskeletal organisation and function.

My thesis, written in an alternative format, consists of three manuscripts. The first one is published in Journal of Cell Science and is entitled "GAS2-like proteins mediate communication between microtubules and actin through interaction with end-binding (EB) proteins." This article describes the mechanisms of how members of the GAS2 family of proteins mediate the crosstalk between actin and microtubules (MTs). We show that in particular GAS2-like 1 (G2L1) and GAS2-like 2 (G2L2) coordinate this cross-communication, as their exogenous expression leads to the stabilisation of MTs and guidance along actin stress fibres. We found that the association of GAS2-like members with MTs is mediated through their binding to EB proteins. The second article is a follow up story of the first article, in which we further elucidate the role of GAS2-like proteins during cell division. We show that G2L1 localises to the mitotic spindle and cleavage furrow during cell division. G2L1 knockdown leads to reduced cell division rates, multinucleation and nuclear deformation. As for MT guidance along actin filaments, we demonstrate that the binding of G2L1 to EB proteins plays an important role in cell division. Although overexpression of G2L1 had no effect, the expression of a mutant that blocks the association with EB proteins phenocopies the knockdown effect of G2L1 on cell division. Actin and MTs undergo major reorganisation during cell division. This reorganisation involves the fast remodelling of focal adhesions (FAs) but the mechanisms of this remodelling were not clear. In the third paper we demonstrate that the majority of FAs disassemble shortly before cell division and reassemble in newly formed daughter cells during cytokinesis. Interestingly, our data suggest that the regulation of FA disassembly during cell division differs from the disassembly processes during cell migration. While in migrating cells FAs can be stabilised by the expression of constitutively active vinculin (vinT12, known to circumvent the requirement forces for FA stability), this was not case for FAs during cell division. Further experiments using inhibitors suggested that calpain-driven cleavage of FA components but not endocytosis play a key role in FA disassembly during cell division. Altogether, the three manuscripts provide insight into important molecular aspects involved in the regulation of cell cytoskeletal networks and cell adhesion during cell division.