Browsing by Subject "Chemotaxis"

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Eliminierung apoptotischer Zellen durch professionelle Phagozyten: Generierung, Freisetzung und Erkennung des monozytären Attraktionssignals Lysophosphatidylcholin und Bedeutung von Annexin I als Brückenprotein in der phagozytotischen Synapse
(2007) Waibel, Michaela; Graeve, Lutz
The efficient elimination of apoptotic cells by neighbouring cells or professional phagocytes is essential for tissue homeostasis in multicellular organisms. Therefore, the apoptotic cell displays different so-called ?eat-me?-signals on its cell surface that are crucial for its recognition and engulfment. Especially in higher organisms, where the dying cell and the phagocyte are usually not located in immediate proximity, the release of soluble attraction signals is of special importance. Only recently, the phospholipid lysophosphatidylcholine (LPC) could be identified as a central ?find-me?-signal that is generated by the calcium-independent phospholipase A2 (iPLA2)-mediated hydrolysis of phosphatidylcholine. During apoptosis iPLA2 is processed in a caspase-3-dependent fashion. In the present thesis it could be demonstrated that iPLA2 is cleaved directly by caspase-3 and that this processing leads to its activation. The active iPLA2 is essential for the production of the phospholipid-?find-me?-signal LPC in apoptotic cells. However, the observation that overexpression of the active form of iPLA2 alone was not sufficient for the release of the attraction signal from vital cells implied that other apoptotic events might contribute to the generation and export of the ?find-me?-signal LPC. It turned out that the reactive oxygen species-driven oxidation of membrane lipids like phosphatidylcholine is an additional factor that leads to the enhanced production of LPC, probably because oxidized lipids are more susceptible for PLA2-mediated hydrolysis than non-oxidized lipids. Further studies about the detailed export mechanism of LPC revealed that the ATP-binding cassette transporter (ABC)-family member ABCA1 is essential for the release of the attraction signal during apoptosis. Thus, the oxidation of membrane lipids and the ABCA1-driven export of LPC could be identified as important elements of LPC-generation and its subsequent release during apoptosis. After the generation and the release of the attraction signal LPC could be demonstrated in more detail the consequent question arose which receptors might mediate the effects of LPC on the phagocytes. In the present thesis it could be demonstrated that the G-protein-coupled receptor G2A is responsible for the LPC-stimulated migration of monocytic cells. However, the molecular mechanisms that ultimately lead to the LPC-driven, G2A-mediated migration, are not known so far. Accordingly, a participation of other receptors or the existence of further chemotactic signals cannot be ruled out at this point. Moreover, there are some hints for chemotactically active proteins in literature. If these or other factors contribute to the LPC-mediated chemotaxis of monocytic cells is completely unknown and needs to be clarified in future studies. The recognition and internalization of dying cells is mediated by the interaction between different ?eat-me?-signals that are displayed on apoptotic cells, and specific surface receptors on phagocytes. In this scenario, the interaction can be of a direct nature ore rather indirect via bridging molecules. In this context, here it could be demonstrated that the calcium- and phospholipid-binding protein annexin I gets externalized by dying cells independently of the apoptotic stimulus applied, but dependent on the cell type. On the surface of the apoptotic cell, annexin I binds in a calcium-dependent fashion via its annexin-boxes to externalized phosphatidylserine, which represents a central ?eat-me?-signal. Thereby, annexin I is able to stimulate the elimination of these cells by professional phagocytes and thus fulfills the function of a bridging molecule in the phagocytic synapse. In contrast, the receptors that are responsible for the binding of annexin I to phagocytes are not known so far. As a conclusion it can be stated that the phenomena studied in this thesis represent important steps in the process of apoptotic cell elimination. The physiological relevance of apoptotic cell clearance is the fact that apoptosis, in contrast to necrotic cell death, is highly regulated at all stages and usually turns out without any harmful consequences to the organism. If this complex, multistep process is disturbed, non-cleared apoptotic cells can become a source for inflammatory reactions. In different animal models it could be demonstrated that defects in the attraction of phagocytes as well as deficiencies in the recognition and internalization via ?eat-me?-signals and the subsequent degradation of the apoptotic prey can be a reason for the onset of severe autoimmune disorders. In this context, the development of human systemic lupus erythematosus and of chronic arthritis is discussed to be initiated by the inefficient elimination of apoptotic cells.
Interaktion des bakteriellen Quorum Sensing Moleküls N-(3-oxododecanoyl)-L-Homoserin-Lacton (AHL-12) mit humanen neutrophilen Granulozyten
(2013) Kahle, Nadine; Hänsch, Gertrud Maria
To evade immune-defense mechanisms of the host, Pseudomonas aeruginosa form so called biofilms. To coordinate this complex process, the bacteria communicate via quorum sensing signals, such as N-(3-oxododecanoyl)-L-homoserine lactone (AHL-12). AHL-12 is also recognized by human polymorphonuclear neutrophils (PMN). It is chemotactic for PMN and thus might cause their directed migration to the site of infection in order to eliminate infiltrated microorganisms. Through different approaches, the aim of this work was to find out in which way AHL-12 interacts with PMN. As a first step, an AHL-12 induced signaling-pathway should be elucidated in in vitro experiments using primary, human PMN. In a further approach using radioactive- (3H-AHL-12) and fluorescence-labeled (AHL-12-FITC) AHL-molecules, the interaction of AHL-12 with PMN should be examined, especially with regard to the expression of a specific receptor for AHL-12 on PMN. In this work it was confirmed that AHL-12 induced chemotaxis of human PMN. After stimulation of PMN with AHL-12, MAPK-p38 was phosphorylated and hence activated. Furthermore, it was shown that MAPK-p38 phosphorylated MAPKAP-Kinase 2, which in turn phosphorylated and hence activated leukocyte specific protein 1 (LSP1). LSP1 finally affected chemotaxis via binding to F-Actin and by modulating the intensity of integrin-mediated adherence. The AHL-12 induced chemotaxis was inhibited by SB203580, an inhibitor of MAPK-p38. The question, whether there is a specific receptor for AHL-12 in PMN, could not be answered conclusively, because it was not possible to isolate a protein with receptor function using conventional methods such as immunoprecipitation and crosslinking. In uptake studies with 3H-AHL-12 and AHL-12-FITC, it was shown, that the major portion of AHL-12-FITC bound to the cell membrane. However, another portion got into the cells, which raises the possibility that an intracellular receptor or an intracellular binding protein for AHL-12, for example IQGAP1, exists. Taken together these data provide evidence that AHL-12 is chemotactic for PMN. Assuming that AHL-12 is released in the early phase of biofilm formation, before bacteria are embedded in the EPS, PMN have the chance to kill bacteria and thus could contribute to prevent biofilm formation.
Interaktion des Photosensors Ppr aus Rhodocista centenaria mit Proteinkomponenten der chemotaktischen Signaltransduktion
(2008) Kreutel, Sven; Kuhn, Andreas
Rhodocista centenaria (previously known as Rhodospirillum centenum) is a photosynthetic alpha-proteobacterium which exhibits a unique phototactic response in respect of the direction of light. In this work, the focus is on the potential photoreceptor Ppr and its C-terminal histidine kinase Pph to identify putative binding partners in the signal transduction pathway. The results of overexpression experiments with the Ppr-receptor or the Pph-domain in E. coli indicated that there may be an interaction between the photosensor and the chemotactic signalling pathway. Even cells expressing only small amounts of the R. centenaria proteins showed no chemotactic response at all, whereas uninduced cells exhibited normal chemotactic response on swarm plates as well as in capillary assays. To investigate whether the receptor interacts with components of the chemotactic pathway, the Ppr-protein and the Pph-domain as well as the chemotactic proteins CheW and CheAY of R. centenaria were heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. Binding experiments were carried out by using an IAsys biosensor cuvette system. The results indicated that the kinase domain Pph binds to the chemotactic linker protein CheW with a dissociation constant of 0.13 ± 0.026 μM. Pull-down experiments were made to verify this finding and to investigate the role of ATP in the binding process. The results confirmed our previous observations but in contrast to the complex formation in the E. coli chemotactic pathway, the binding of the C-terminal histidine kinase to CheW was ATP-dependent. To study whether the kinase domain also binds CheW in vivo, expression and coelution experiments with tagged Pph-protein were carried out in R. centenaria. The findings suggest that the complex formation occurs in vivo as well as in vitro. From the data of the E. coli chemotactic complex formation it is well known that CheA is part of the trimeric complex which consists of MCP-receptors, CheW and CheA. To analyse this, pulldown experiments with all three proteins (Pph, CheW and CheAY) were performed. The results clearly showed a participation of CheAY in the formation of the complex with CheW and the kinase Pph. It is known that the photoreceptor Ppr is autophosphorylated during its light induced photocycle. We therefore examined whether the kinase domain is sufficient for this autophosphorylation reaction and whether CheAY could function as a phosphate acceptor. Our results confirmed the hypothesis that the kinase domain is sufficient for autophosphorylation and that the Pph-protein assists CheAY to take over phosphate groups. Taken together, the results in combination with data from the literature lead to a detailed working model for the function of the photoreceptor Ppr and the signal transduction pathway.
NOD1 cooperates with HAX‐1 to promote cell migration in a RIPK2‐ and NF‐ĸB‐independent manner
(2023) Hezinger, Lucy; Bauer, Sarah; Ellwanger, Kornelia; Piotrowsky, Alban; Biber, Felix; Venturelli, Sascha; Kufer, Thomas A.
The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.
Untersuchung einer Methode zur spezifischen Fluoreszenz – Markierung von Signalproteinen und deren Beobachtung in lebenden Escherichia coli (Einzelmolekültechnik & Perspektiven)
(2016) Ehrhard, Tanja Margret; Herten, Dirk-Peter
Malfunctions in signal transduction often cause diseases such as cancer and metabolic disorders. A thorough understanding of the relevant mechanisms of signal transduction is therefore an important requirement for the development of therapies and pharmaceuticals. In this thesis, a method was developed, which allows the observation of individual signaling proteins and their interactions in living cells. Therefore this method has advantages compared to molecular detection methods which are based on ensemble averages. As a model system for signal transduction, the bacterial chemotaxis with its regulator protein CheY was selected. The experimental studies were carried out with total internal reflection fluorescence microscopy (TIRFM), which requires a fluorescent labeling of the examined molecules. To ensure a specific and background reduced labeling, bright and photostable fluorescent ,tags are needed. In this work, the SNAP-tag system was used, which allows the use of different dyes. An advantage of this system is the possibility of using fluorescence-quenched benzyl guanine (BG)-dyes, which show a strong fluorescence only after binding to SNAP-tag. For development of the labeling method, the dyes Atto 620, Atto 633, Atto 655 and Atto 680 were analyzed in preliminary experiments regarding their fluorescence, photostability and blinking behavior. The thorough knowledge of these properties is essential for the correct interpretation of the experimental results. Dyes which are ideal for the method have a high fluorescent signal over a long observation time, and they are stable and do not interfere with the function of the target molecule. The preliminary investigations have shown that among the dyes tested, Atto 633 had the best photophysical properties for labeling with the SNAP-tag system and also the best cell permeability. This allows, under continuous laser excitation, to observe individual molecules for several seconds. In addition, the labeling efficiency was controlled by the protein expression, the dye concentration, and the incubation time of the dye. For single-molecule detection, a low labeling efficiency is of advantage since too high density of fluorescently labeled molecules makes the identification of individual molecules difficult. Subsequently, a labeling protocol was established which allows a specific, background- reduced fluorescence labeling of individual CheY proteins in living E. coli cells, without impairment of the protein’s functionality. Real-time detection with a time resolution of 30 milliseconds showed that it is possible to observe individual CheY molecules as a fluorescent point during the state of binding to an interaction partner. By means of numerical methods, the state of binding can be extracted from the fluorescence intensity traces as on/ off and their probability distribution can be determined. These quantitative studies gave indications on specific protein interactions, but no detailed information on binding times could be found. Different interactions of the protein, both specific and non-specific nature, could be the reason. Therefore, another important development of this labeling system would be the opportunity of simultaneous staining of two or more proteins with spectroscopically distinguishable fluorescent tags (e.g. CLIP-tag) to perform colocalization with alternating laser excitation. Another cause might be found in the nature of the dye itself. Laser- and temperature-dependent studies could provide further information concerning the behavior of the dyes. Thus, the described fluorescence labeling method provides a new approach for quantitative studies of protein interactions in living cells.