During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell, mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced mechanical force culminates in the formation of structures that anchor monocyte adhesion. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4 expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form at the sites of anchor formation. A key mediator of force-induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.

Tip growth is the primary form of growth in hyphal organisms and some plant cells. Tip growth in hyphae is highly dependent on F-actin, which acts to regulate and support growth. One of the models suggested for tip growth, the amebae model of tip growth, suggests that F-actin may also be the primary source of protrusive force for tip growth in some conditions, and that proteins with a similar function to animal integrins would be present an involved in tip growth (Heath and Steinberg 1999). In this thesis we examine the role of F-actin in the growth of the oomycete Phytophthora cinnamomi and the effects on growth of the F-actin disrupting compound Latrunculin B. We demonstrate that F-actin plays a critical role in the tip growth of Phytophthora cinnamomi with it's disruption causing rapid cessation in directional growth, followed by significant subapical swelling. Further more we examine Phytophthora cinnamomi for the presence of an B4 integrin like protein that has been previously reported in the oomycete Achlya bisexualis (Chitcholtan & Garrill 2005) and show that the B4 integrin like protein is not present in Phytophthora cinnamomi. These experiments help further our understanding of tip growth in Phytophthora cinnamomi an economically important plant pathogen.

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

Hannover, Univ., Diss., 2007

Regensburg, Univ., Diss., 2008

Univ., Diss., 2010--Würzburg.

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.

Le cytosquelette d’actine régule la forme de la cellule au cours du temps. Pour lecomprendre, il faut étudier les mécanismes moléculaires qui le constituent. In vivo,ces mécanismes sont masqués par la complexité du vivant. Si nous parvenons àreproduire pièce par pièce le cytosquelette d’actine in vitro et si nous pouvons lecontrôler, aussi bien dans l’espace et dans le temps, alors nous pourrons élucider lessecrets de son fonctionnement. Cette thèse montre que nous avons maintenantdéveloppé la technologie qui nous permet de le faire pour certaines architectures ducytosquelette d’actine.Nous avons développé de nouvelles méthodes de « micropatterning » pour contrôlerla nucléation des monomères d’actine dans l’espace en deux dimensions. Ceci nous aamenés à la reconstitution et au guidage de réseaux de filaments d’actine parallèles àpolarité identique et à la reconstitution de l’anneau de cytocinèse.J’ai crée une puce microfluidique innovante pour contrôler la compositionbiochimique des systèmes d’actine reconstitués au cours du temps. Ceci nous a permisde contrôler la cinétique de polymérisation du filament d’actine individuel libre, et decontrôler la séquence d’intervention des protéines sur les réseaux de filamentsd’actine parallèles à polarité identique.Enfin, nous avons utilisé cette même puce microfluidique pour étudier ladifférentiation des cellules souches hématopoïétiques. / Actin cytoskeleton regulate cell shape over time. To understand that, we have to studymolecular mechanisms that constitute actin cytoskeleton. In vivo, those mechanismsare hidden by cellular complexity. If we achieve to reproduce piece by piece actincytoskeleton in vitro and if we can control it in space and time, then we are able toelucidate the secrets of it operate. This thesis show that we have developed thetechnology that allow us to do it for a few actin cytoskeleton architectures.We have developed new micro-patterning methods to control actin monomersnucleation into two-dimensional space. This led us to the reconstitution and guidanceof parallel actin filament networks with same polarity and allowed us to reconstituteactin contractil ring.I created an innovating microfluidic chip to control biochemical composition ofreconstituted actin systems over time. This allowed us to control kinetics of freeindividual actin filament polymerisation and to control the intervention sequence ofproteins on parallel actin filament networks.Finaly, we used the microfluidic chip to study hematopoïetic stem cellsdifferentiation.