Dokument

Summary:
Cell migration is highly dependent on the precise orchestration of the assembly and disassembly of filamentous actin at their leading edge. Therefore, the temporal and spatial regulation of WAVE activity as the main regulator of Apr2/3 for branched actin polymerization is crucial for efficient lamellipodia-driven cell motility. This work shows that wave mutant Drosophila macrophages completely lack lamellipodia that causes severe migration defects. However, they still respond to wound signals by relying on rudimentary filopodia based migration. Furthermore, it could be shown that the disruption of the WIRS receptor binding site within the WRC does not interfere with wound response of macrophages. The non-receptor tyrosine kinase Abl is assumed to be a key regulator of WAVE, altering its activity via phosphorylation. The results in this study confirm that the active form of Abl is an effector of WAVE. The loss of abl and the overexpression of Abl kinase dead transgene leads to an increase of cell spreading. Further, WAVE localization at the membrane is elevated in the absence of Abl. This alteration seems to influence random migration by reducing the explorative behavior of macrophages. However, loss of abl did not affect the responsiveness of macrophages to external damage signals. WAVE possesses four tyrosine residues within the WHD: Y127 is a Src kinase target and Y153 of the Abl kinase. The WAVE phospho-mutant Y153F is still phosphorylated via Abl. This indicates that additional tyrosine sites are targeted by Abl. Nevertheless, the phosphorylation of Y153 results in an elevated F-actin level that indicates the physiological relevance of this tyrosine residue. This suggests that phosphorylation of Y153 activates WAVE and the WRC, which is consistent with the results of previous studies. The analysis of random migrating macrophages revealed that this leads to a reduction of cell speed. In contrast, the phosphorylation of Y127 in the WHD showed neither an impact on the F-actin level nor on the migratory behavior of macrophages. Interestingly, the simultaneous phosphorylation of both residues leads to a drastic reduction of lamellipodial structures in macrophages. These cells exhibited extended filopodia and showed a stellar cell shape comparable to wave mutant macrophages. Consequently, in vivo migrating macrophages show a reduction of cell speed and a negative impact on the persistence of these cells. Individual phospho-mimicking mutation of Y127 or Y153 also do not interfere with the viability of the flies, whereas the simultaneous phosphorylation of both sites lead to late pupal lethality. These results indicate that phosphorylation of multiple tyrosine residues has a negative regulatory effect on WRC function. In conclusion, it can be shown that phosphorylation state of WAVE finetunes WRC activity.

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