Einfluss der ERM-Proteine auf die Protrusion-Ausbildung und Zell-Zell-Ausbreitung von Listeria monocytogenes

Seit der Entdeckung der ERM-Proteinfamilie sind zahlreiche Funktionen für diese Proteine gefunden wurden. Im Rahmen dieser Arbeit konnte eine neue Funktion der ERM-Proteine bei der Ausbildung von Listerien-induzierten Protrusions nachgewiesen werden. Es wurden verschiedene Methoden zur funktionellen Inhibierung der ERM-Proteine angewandt: i) Blockierung der Membranliganden bzw. des Aktinzytoskeletts, ii) Delokalisation der ERM-Proteine von der Plasmamembran bzw. vom CD44-Rezeptor und iii) Blockierung der ERM-CD44-Interaktion. Zur Analyse der Rolle von ERM-Proteinen bei der Protrusion-Formation wurden drei Parameter erhoben, die Dynamik der Ausbildung, die durchschnittliche Länge und die Anzahl ausgebildeter Protrusions pro infizierter Zelle. Die Untersuchungen zeigten, dass die funktionelle Inhibierung der ERM-Proteine mit einer signifikanten Reduzierung der Protrusionzahl korrelierte. Insbesondere die Interaktion zwischen ERM-Proteinen und CD44 oder analogen Rezeptoren scheint eine wichtige Rolle zu spielen. Erste Untersuchungen deuten darauf hin, dass für die Aktivierung der ERM-Proteine und ihre daraus resultierenden Rolle bei der Protrusion-Ausbildung die Phosphorylierung eines konservierten carboxyterminalen Threonins signifikant ist. Insgesamt führt die Inhibierung der ERM-Proteinfamilie zu einer verminderten Protrusion-Ausbildung, was eine verminderte Zell-Zell-Ausbreitung von Listeria monocytogenes zur Folge hat.Since the ERM proteinfamily were discovered many functions for these proteins have been found.In the course of this work it could be shown that the ERM proteins are also involved in the Listeria-induced protrusion-formation. To inhibit the function of the ERM proteins several methods were applied: i) blocking of the membran ligands and the actin cytoskeleton ii) delocalization of the ERM proteins from the membran and the CD44 receptor, respectively and iii) blocking of the ERM-CD44 interaction. To analyse the role of the ERM proteins in the process of the protrusion formation three parameters were measured, the dynamik of the protrusion-formation, the mean length of the protrusions and the number of formed protrusions per cell. The investigations showed that the inhibition of the ERM proteins correlates directly with a decrease in the protrusion-number. In particular the interaction between the ERM proteins and the CD44 receptor seems to play a significant role in the protrusion-formation. Furthermore, there are first hints that a phosphorylation of a conserved threonin-residue is significant for the activation of the ERM proteins and the resulting role in the protrusion-formation. Altogether, the ERM proteins are significant for an efficient listerial cell-to-cell spreading and an inhibition of these proteins leads to a decreased protrusion-formation

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin

Clathrin- and Dynamin-Independent Endocytosis of FGFR3 – Implications for Signalling

Endocytosis of tyrosine kinase receptors can influence both the duration and the specificity of the signal emitted. We have investigated the mechanisms of internalization of fibroblast growth factor receptor 3 (FGFR3) and compared it to that of FGFR1 which is internalized predominantly through clathrin-mediated endocytosis. Interestingly, we observed that FGFR3 was internalized at a slower rate than FGFR1 indicating that it may use a different endocytic mechanism than FGFR1. Indeed, after depletion of cells for clathrin, internalization of FGFR3 was only partly inhibited while endocytosis of FGFR1 was almost completely abolished. Similarly, expression of dominant negative mutants of dynamin resulted in partial inhibition of the endocytosis of FGFR3 whereas internalization of FGFR1 was blocked. Interfering with proposed regulators of clathrin-independent endocytosis such as Arf6, flotillin 1 and 2 and Cdc42 did not affect the endocytosis of FGFR1 or FGFR3. Furthermore, depletion of clathrin decreased the degradation of FGFR1 resulting in sustained signalling. In the case of FGFR3, both the degradation and the signalling were only slightly affected by clathrin depletion. The data indicate that clathrin-mediated endocytosis is required for efficient internalization and downregulation of FGFR1 while FGFR3, however, is internalized by both clathrin-dependent and clathrin-independent mechanisms

Der Wissenschaftlich-Technische Rat - Wer ist das, was macht er, und warum eigentlich?

Festschrift zum fünfzigjährigen Bestehen der Hauptkommission des Wissenschaftlich-Technischen Rate

Multidimensional electrochemical imaging in materials science

International audienceIn the past 20 years the characterization of electroactive surfaces and electrode reactions by scanning probe techniques has advanced significantly, benefiting from instrumental and methodological developments in the field. Electrochemical and electrical analysis instruments are attractive tools for identifying regions of different electrochemical properties and chemical reactivity and contribute to the advancement of molecular electronics. Besides their function as a surface analytical device, they have proved to be unique tools for local synthesis of polymers, metal depots, clusters, etc. This review will focus primarily on progress made by use of scanning electrochemical microscopy (SECM), conductive AFM (C-AFM), electrochemical scanning tunneling microscopy (EC-STM), and surface potential measurements, for example Kelvin probe force microscopy (KFM), for multidimensional imaging of potential-dependent processes on metals and electrified surfaces modified with polymers and self assembled monolayers