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.
Gerisch
G
G
Max-Planck-Institut für Biochemie, Martinsried, Germany. gerisch@vms.biochem.mpg.de
Albrecht
R
R
Heizer
C
C
Hodgkinson
S
S
Maniak
M
M
D000818:Animals; D002465:Cell Movement; D002630:Chemotactic Factors / Q000502:physiology*; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism* / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002630:Chemotactic Factors; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; C072061:coronin proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP