610 Medizin und Gesundheit
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This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10.
The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation.
This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation.
Abscission marks the last step of cytokinesis and gives rise to two physically separated daughter cells and a midbody remnant. This work studies abscission by examining the extent of the abscission failure in C. elegans septin and ESCRT mutants with the help of the ZF1-degradation technique. The ZF1 technique is also applied to discern a possible role for PI3K during abscission. Lastly, we test the role of proteins required for macroautophagy but not for LC3-associated phagocytosis (LAP) and show that after release into the extracellular space, the midbody is resolved via LAP.
Bei der Teilung einer Zelle werden das Genom und die Zellbestandteile zwischen zwei Tochterzellen aufgeteilt. Dies erfordert verschiedene fein aufeinander abgestimmte Vorgänge. Unter anderem ist eine proteinreiche Struktur beteiligt, die 1891 entdeckt wurde: der Mittelkörper. In vorliegender Arbeit wurden gezielt gekennzeichnete Mittelkörperproteine analysiert und verschiedene Phasen des Transports unterschieden. Es erfolgten erstmals Messungen unter Nutzung der ZF1-Methode. Zudem wird anhand der ZF1-Technik nachgewiesen, dass im Rahmen der Zellteilung die Trennung der interzellulären Brücke zu beiden Seiten des Mittelkörpers stattfindet, woraufhin dieser nach extrazellulär abgegeben wird und über einen der Phagozytose ähnlichen und von Aktin abhängigen Mechanismus von einer Tochterzelle oder unverwandten Nachbarzelle aufgenommen wird.
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.