Richard Albrecht

Coronin involved in phagocytosis: dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.

Coronin is a protein involved in cell locomotion and cytokinesis of Dictyostelium discoideum. Here we show that coronin is strongly enriched in phagocytic cups formed in response to particle attachment. A fusion of coronin with green fluorescent protein (GFP) accumulates in the cups within less than 1 min upon attachment of a particle and is gradually released from the phagosome within 1 min after engulfment is completed. Phagocytic cup formation competes with leading edge formation and can be interrupted at any stage. When the cup regresses, coronin dissociates from the site of accumulation. TRITC-labeled yeast cells have been used to assay phagocytosis quantitatively in wild-type and coronin-null cells. In the mutant, the rate of uptake is reduced to about one third, which shows that coronin contributes to the efficiency of phagocytosis to about the same extent as it improves the speed of cell locomotion.

Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein–coronin fusion protein

BACKGROUND The highly motile cells of Dictyostelium discoideum rapidly remodel their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a cytoplasmic actin-associated protein that accumulates at the coritcal sites of moving cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins and is known to interact with actin-myosin complexes. In coronin null mutants, cell locomotion is slowed down and cytokinesis is impaired. RESULTS We have visualized the redistribution of coronin by fluorescence imaging of motile cells that have been transfected with an expression plasmid containing the coding sequence of coronin fused to the sequence encoding the green fluorescent protein (GFP). This coronin-GFP fusion protein (GFP). This coronin-GFP fusion protein transiently accumulates in the front regions of growth-phase cells, reflecting the changing positions of leading edges and the competition between them. During the aggregation stage, local accumulation of coronin-GFP is biased by chemotactic orientation of the cells in gradients of cAMP. The impairment of cell motility in coronin null mutants shows that coronin has an important function at the front region of the cells. The mutant cells are distinguished by the formation of extended particle-free zones at their front regions, from where pseudopods often break out as blebs. Cytochalasin A reduces the size of these zones, indicating that actin filaments prevent entry of the particles. CONCLUSIONS These data demonstrate that coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of a nascent leading edge, where it participates in the reorganization of the cytoskeleton. Monitoring the dynamics of protein assembly using GFP fusion proteins and fluorescence microscopy promises to be a generally applicable method for studying the dynamics of cytoskeletal proteins in moving and dividing cells.

Image processing for combined bright-field and reflection interference contrast video microscopy

Image processing algorithms for automatic extraction of cell body contours and cell-substratum contacts from video images, which were obtained by bright-field microscopy and reflection interference contrast microscopy, respectively, are described. Double-view imaging, which combines these two optical techniques, is used to investigate the relationship between cell-to-substratum adhesion and cell shape changes during locomotion of the amoeboid cells of Dictyostelium discoideum. Contact areas of cells are extracted from reflection interference contrast images via a routine which performs binarisation on the basis of threshold estimation, as it is calculated by a histogram minimum method. Boundaries of cells are extracted from bright-field images by utilising and algorithm that includes background subtraction and binarisation based on texture discrimination by means of a rank operator.

Actin and Actin-Binding Proteins in the Motility of Dictyostelium

Dictyostelium discoideum cells are highly motile throughout all stages of development and directed cell motility is essential for morphogenesis to occur (Williams and Jermyn 1991). Cells move by pseudopod extension, and formation and retraction of pseudopods in response to a chemotactic stimulus is critical for directed migration. Folate acts as chemotactic agent during growth, cAMP is the active compound during development. The understanding of chemotactic signaling has advanced considerably (Devreotes 1989), but its linkage to intracellular changes that lead to cell motility is less clear (reviewed in Schleicher and Noegel 1992). Early observations indicated that chemoattractant-induced pseudopod formation correlates temporally and spatially with the polymerization of actin, and peaks of F-actin occur by 15–60 s after stimulation (McRobbie and Newell 1983; Hall et al. 1988). Five different steps in actin changes can be distinguished (Fig. 1), and these steps were found to coincide with retraction of the existing pseudopod and generation of new pseudopods (Dhamaward- hane et al. 1989). Parallel processes are an influx of Ca2+ and an efflux of protons leading to an alkalinization of the cytoplasm. The phosphoinositides are also affected with an increase of IP3 as fast as 6 s after stimulation at the expense of PIP and PIP2 (Europe-Finner et al. 1991).