Yes / As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved fromsponges to humans, and play vital roles inmany physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including sixα and five β subunitswere cloned and characterized fromsilkworm. Our results showed that integrins fromsilkwormown more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins performdiverse functions in hemocytes of silkworm. Overall, our results provide a newinsight into the functional and evolutionary features of integrins. / National Basic Research Programof China (No. 2012cb114603), the Research Fund for the Doctoral Program of Higher Education of China (20130182110003), the Natural Science Foundation of Chongqing (cstc2013jcyjys0007), and the Fundamental Research Funds for the Central Universities (SWU111014).

Das Dorsalgefäß von Drosophila ist ein wertvolles Modellsystem für die Untersuchung der genetischen und molekularen Mechanismen der Kardiogenese. Ein Schlüsselereignis der Kardiogenese ist die Bildung eines Herzlumens, durch das die Hämolymphe gepumpt wird, um Nährstoffe und Zellen des angeborenen Immunsystems zu zirkulieren. Ein Schwerpunkt meiner Arbeit umfasste die Identifizierung neuer Gene, die im embryonalen Dorsalgefäß von Drosophila exprimiert sind. Dafür habe ich die Expression von 101 Genen, deren Orthologe spezifisch im Herzen von Zebrafisch exprimiert sind, in Drosophila untersucht. Ich identifizierte ein Gen, das für das Cbl-assoziierte Protein (CAP) kodiert. Durch Herstellung eines anti-CAP Antikörpers konnte ich erstmals eine detaillierte Lokalisation des CAP-Proteins im Dorsalgefäß beschreiben. Interessanterweise stellte sich dabei heraus, dass CAP ähnlich wie die homologen Vertebraten Proteine embryonal an den fokalen Adhäsionskontakten der Kardioblasten und im adulten Dorsalgefäß auch an den Z-Scheiben und den Zell-Zell-Kontaktstellen der Kardiomyozyten lokalisiert ist. Des Weiteren untersuchte ich, welche Auswirkungen der Verlust der CAP Funktion auf die Herzentwicklung hat. Für die Analyse der CAP-Mutanten nutzte ich neben Immunhistochemischen Methoden auch ultrastrukturelle Analysen mittels TEM-Mikroskopie. So konnte ich zeigen, dass embryonale Dorsalgefäße von CAP-Mutanten eine fehlerhafte Anzahl sowie Anordnung der Kardioblasten und Lumendefekte aufweisen. Ein genetischer Interaktionstest untermauerte meine Vermutung, dass CAP mit dem Integrinsignalweg während der embryonalen Dorsalgefäßentwicklung interagiert. Live-Aufnahmen des pumpenden Dorsalgefäßes von Drosophila L3-Larven und Injektionstests an späten Puppen zeigten zudem, dass der Verlust der CAP Funktion auch zu starken Defekten in der Funktionalität des larvalen und adulten Dorsalgefäßes führt. / The heart of Drosophila provides a valuable model system for the examination of the genetic and molecular mechanisms that guide cardiogenesis. A key event of cardiogenesis is the formation of a heart lumen through which the hemolymph is pumped to circulate nutrients and cells of the innate immune system. A main focus of my work was the identification of new genes that are expressed in the embryonic heart of Drosophila. Therefore I studied the expression of 101 genes, whose orthologues are expressed specifically in the heart of zebrafish. I identified a gene that encodes for the Cbl-associated protein (CAP). By generating an anti-CAP antibody I could describe the localization of the CAP protein in the heart for the first time in detail. Interestingly, it turned out that CAP is located similar to the homologous vertebrate proteins at the focal adhesion contacts of cardioblasts in the embryo and at the Z-discs and the cell-cell contact sites of cardiomyocytes in the adult heart. I also examined the consequences of the loss of CAP function on heart development. For the analysis of the CAP mutants I used immunohistochemical and ultrastructural analysis by TEM microscopy. So I was able to demonstrate that embryonic hearts of CAP mutants show a defective number and arrangement of cardioblasts and lumen defects. A genetic interaction test substantiated my guess that CAP interacts with the Integrin signaling pathway during embryonic heart development. Live recordings of the pumping heart of Drosophila L3 larvae and injection tests of late pupae also showed that the loss of CAP function leads to severe defects in the functionality of the larval and adult heart.

Introdução: Caderinas e integrinas são importantes para a manutenção da integridade tecidual e transdução de sinal durante o desenvolvimento da pele. A distribuição destas moléculas no desenvolvimento da pele humana foi investigada e associada com os marcadores de diferenciação, Citoqueratinas (CK) e involucrina (INV). Método: Usando a técnica de imunoistoquímica foram investigadas as proteínas E- e P- caderinas, integrinas beta- 1 e -4, CK 10, CK 14 e INV em fragmentos de pele de várias regiões corpóreas de 7 fetos humanos (semana gestacional de 4 a 24, todos pesando até 500 g). Resultados: Na fase inicial do desenvolvimento, integrinas beta-1 e -4 and E- and P- caderinas estavam presentes na membrana plasmática das células epiteliais em todos as camadas do epitélio. CK14 e CK10 foram observadas em todas as camadas epiteliais e a INV fracamente detectada em células da camada mais superficial. Em estágios mais avançados, integrinas foram detectadas em todas as camadas epiteliais, com expressão polarizada principalmente na camada basal. E- caderina foi detctada em todas as camadas, menos no estrato cornificado e a P- caderina foi observada em camadas mais profundas do epitélio. CK14 estava presente na camada basal, CK 10 no estrato suprabasal e a INV foi observada no estrato cornificado. Conclusão: Caderinas e integrinas são essenciais para o desenvolvimento da pele, sendo espacialmente e temporalmente regulados. Suas expressões são relatas com a expressão da maturação de marcadores da epiderme. / Introduction: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P- cadherins, integrins beta-1 and -4, CK10, CK 14 and INV was assessed in skin fragments of 7 human fetuses (gestacional weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P- cadherins were present on epithelial cell membranes in all layers. CK 14 and CK 10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E- cadherin was detected in all layers, but the cornified stratum and P- cadherin were observed in the lower layers. CK 14 was expressed in layer, CK 10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Introdução: Caderinas e integrinas são importantes para a manutenção da integridade tecidual e transdução de sinal durante o desenvolvimento da pele. A distribuição destas moléculas no desenvolvimento da pele humana foi investigada e associada com os marcadores de diferenciação, Citoqueratinas (CK) e involucrina (INV). Método: Usando a técnica de imunoistoquímica foram investigadas as proteínas E- e P- caderinas, integrinas beta- 1 e -4, CK 10, CK 14 e INV em fragmentos de pele de várias regiões corpóreas de 7 fetos humanos (semana gestacional de 4 a 24, todos pesando até 500 g). Resultados: Na fase inicial do desenvolvimento, integrinas beta-1 e -4 and E- and P- caderinas estavam presentes na membrana plasmática das células epiteliais em todos as camadas do epitélio. CK14 e CK10 foram observadas em todas as camadas epiteliais e a INV fracamente detectada em células da camada mais superficial. Em estágios mais avançados, integrinas foram detectadas em todas as camadas epiteliais, com expressão polarizada principalmente na camada basal. E- caderina foi detctada em todas as camadas, menos no estrato cornificado e a P- caderina foi observada em camadas mais profundas do epitélio. CK14 estava presente na camada basal, CK 10 no estrato suprabasal e a INV foi observada no estrato cornificado. Conclusão: Caderinas e integrinas são essenciais para o desenvolvimento da pele, sendo espacialmente e temporalmente regulados. Suas expressões são relatas com a expressão da maturação de marcadores da epiderme. / Introduction: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P- cadherins, integrins beta-1 and -4, CK10, CK 14 and INV was assessed in skin fragments of 7 human fetuses (gestacional weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P- cadherins were present on epithelial cell membranes in all layers. CK 14 and CK 10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E- cadherin was detected in all layers, but the cornified stratum and P- cadherin were observed in the lower layers. CK 14 was expressed in layer, CK 10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.

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Die Mehrheit chronischer Nierenerkrankungen wird durch glomeruläre Defekte hervorgerufen. In dieser Arbeit wurde deshalb im Mausmodell die Bedeutung der Kollagenrezeptoren DDR1 (Discoidin Domain Rezeptor 1) und ITGA2 (Integrin Alpha 2) in der Pathogenese von glomerulären Nierenerkrankungen untersucht. Von zentralem Interesse waren neben der Betrachtung des renalen Phänotyps, die Analyse der glomerulären Basalmembran sowie die Prüfung auf Vorhandensein nierenschädigender Faktoren. Zur Orientierung angefertigte H.E.-Färbungen waren lichtmikroskopisch unauffällig, jedoch ließ sich mittels Gelelektrophorese eine Mikro-, Makro- und Albuminurie mit einem Maximum zum Zeitpunkt von 100 Lebenstagen nachweisen, die mit 200 Tagen wieder stark sank. Auf dem Boden der nierenschädigenden Proteinurie, zeigten die Western-Blot-Analysen das Vorhandensein der Zytokine TGF-ß und CTGF auf. Die zur Detektion von Narbengewebe durchgeführte Fibronektinfärbung, erbrachte keinerlei weiterführende Anhaltspunkte. In der Elektronenmikroskopie ließ sich vereinzelt eine Mehrschichtung der GBM nachweisen, was als Ausreifungsstörung interpretiert wurde. Der Wegfall der beiden Kollagenrezeptoren ITGA2 und DDR1 scheint somit die Interaktion der Podozyten mit der GBM zu stören. Dies hat eine Proteinurie zur Folge. In Folge dessen werden profibrotische Zytokine sezerniert. Das Fehlen der beiden Kollagenrezeptoren DDR1 und ITGA2 führte jedoch nicht zur Ausbildung einer renalen Fibrose, wie in der Fibronektin-Färbung gezeigt werden konnte. Gross und Girgert zeigten, dass nierenkranke Mäuse nach dem Verlust von DDR1 oder ITGA2 einen verzögerten Verlauf der Nierenfibrose entwickelten. Vielversprechend scheinen Untersuchungen z.B. am Mausmodell Col4A3/DDR1/ITGA2 -/- oder an einer diabetischen ITGA2/DDR1 -/- Maus. Gesetzt dem Fall, dass eine renale Fibrose im Vergleich zum Einzelknockout noch später eintritt, eignen sich diese beiden Kollagenrezeptoren als therapeutisches Ziel. Aktuell stehen nur wenige nephroprotektive Medikamente, wie ACE-Hemmer, zur Verfügung. Anti-Integrine und Inhibitoren gegen Tyrosinkinase-Rezeptoren, wie DDR1, haben bereits Einzug in den klinischen Alltag gehalten und stellen eventuell einen wirksamen Ansatzpunkt zur Verhinderung einer renalen Fibrose dar.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Les intégrines sont des cibles thérapeutiques pertinentes en oncologie. Dans cette thèse, nous avons exploré le rôle de l’intégrine α5β1 dans les gliomes et les tumeurs du colon. Nous nous sommes particulièrement focalisés sur la voie intégrine-protéine p53 et son implication dans la résistance aux thérapies. Dans les gliomes, l’intégrine α5β1 est surexprimée dans les glioblastomes et participe à un mauvais pronostic de survie des patients. Nous avons démontré que l’intégrine confère une résistance à la chimiothérapie par le Temozolomide en régulant négativement l’activité de la protéine suppresseur de tumeurs p53. L’activation directe de p53 par un agent non-génotoxique, la Nutlin-3a, entraine une inhibition de l’expression de l’intégrine suggérant ainsi une réaction croisée négative entre intégrine α5β1 et p53. L’association de la Nutlin-3a avec un antagoniste de l’intégrine α5β1 entraine une mort des cellules par apoptose. Nous avons confirmé l’existence d’une réaction croisée négative entre intégrine α5β1 et protéine p53 dans les tumeurs du colon où l’intégrine représente également une cible thérapeutique. / Integrins seem to be attractive anti-cancer targets. In this work we investigated the role of integrin α5β1 in glioma brain tumors and colon cancer. We were particularly interested in the role of integrin α5β1/p53 pathway in resistance to therapy. We first focused on gliomas and found that α5β1 integrin was overexpressed in aggressive malignant glioma tumors. Moreover, we showed that α5β1 integrin upregulation was associated with a shorter patient survival. We also demonstrated that α5β1 integrin expression in glioblastomas participates to the resistance to the chemotherapeutic agent Temozolomide, through a negative regulation of the tumor suppressor p53. A direct p53-activation by the non-genotoxic agent Nutlin-3a down-regulated α5 integrin subunit and thus sensitized glioblastoma cells to Nutlin-3a. Furthermore, we demonstrated that the inhibition of α5β1 integrin with a concomitant p53-activation enhanced the effects of p53-based therapy. We also confirmed the existence of a negative cross-talk between α5β1 integrin and p53 in colon cancer.

Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins. The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering. To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis. In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction. The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.