The group is focussing on the regulation of actin dynamics in cell motility, phagocytosis, and cytokinesis. In chemotaxis, a new leading edge is induced by actin assembly within 5 seconds of exposure to a gradient of attractant. In phagocytosis, a signal is transmitted to the actin system, resulting in protrusion of a phagocytic cup. In cytokinesis, polar regions rich in actin filaments are distinguished from the midzone, the site of cleavage furrow ingression. Proteins are tagged with GFP to visualize, in Dictyostelium cells, their recruitment from the cytoplasm to active sites in the cell cortex, either in response to external signals or as a result of cell-autonomous local assembly. Proteins include members of the Arp2/3 complex, actin itself, and four actin-binding proteins discovered in our group, coronin, coactosin, cortexillin I and II. The function of particular proteins of interest is studied in vivo by knock-out (if the protein is not essential) or by mutagenesis, and in vitro by analyzing complexes of these proteins with actin. In addition, a role of calreticulin and calnexin in phagocytosis has been found by double knock-out of these ER proteins. An overview on GFP-tagged proteins used to study organelle and cytoskeleton dynamics is given in the figure below.
Localization of GFP-fusion proteins to specific organelles (top panels) or to cytoskeletal structures (bottom panels).
Cells of Dictyostelium discoideum are shown, most of them compressed by agar-overlay. Confocal images of the GFP fluorescence in green are superimposed to phase-contrast images in red or in blue (where a second label is colored in red).
Organelle markers: (1) a binucleate cell with the nuclei labeled with histone 2-GFP (green). Microtubules emanating from centrosomes, one attached to each nucleus, are labeled with anti-a-tubulin antibodies (red) (this cell has been fixed; all other panels show living cells); (2) a cell expressing calnexin-GFP, showing labeling of ER membranes including the nuclear envelope in the left half of the cell; (3) the contractile vacuole complex consisting of bladders and a network of connecting tubules. The cell has been counter-stained with TRITC-dextran (red) to label endosomes, which stay separate from the contractile vacuole compartment; (4) golvesin(C)-GFP (green) provides a highly specific marker of the Golgi apparatus, showing no association with endosomes (red); (5) GFP-(N)golvesin is primarily associated with the membranes of endosomes, the lumen of which is labeled with TRITC-dextran (red); (6) mitochondria labeled with a polypeptide probe carrying an N-terminal targeting sequence and the GFP-tag at its C-terminus.
Cytoskeleton and associated proteins: (7) GFP-actin in a cell growing on bacteria, showing actin accumulation in cell-surface projections and phagocytic cups; (8) GFP-Arp3 representing localization of the Arp2/3 complex, which is recruited to cell-surface extensions and to late, neutral endosomes. The complex is displaced from early, acidic endosomes that are labeled with neutral red; (9) GFP-coronin showing co-localization of coronin, a regulatory protein involved in phagocytosis, cell motility and cytokinesis, with filamentous actin; (10) GFP-cortexillin labeling the entire cell cortex; (11) GFP-Rac1enriched in the cell cortex and causing profuse filopod formation; (12) GFP-a-tubulin visualizing the system of microtubules originating at the centrosome.
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