Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis.
The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.
Schweitzer
Jill Kuglin
JK
Department of Biological Sciences, Walther Cancer Institute, Galvin Life Sciences Building, University of Notre Dame, Notre Dame, IN 46556, USA.
D'Souza-Schorey
Crislyn
C
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000096:biosynthesis* / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002455:Cell Division; D003593:Cytoplasm / Q000378:metabolism; D004789:Enzyme Activation; D005799:Genes, Dominant; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008668:Metallothionein / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D010641:Phenotype; D010743:Phospholipids / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000089982:ADP-Ribosylation Factor 6; D000970:Antineoplastic Agents; D010743:Phospholipids; D008668:Metallothionein; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D015739:Nocodazole