Abstract
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.
Highlights
Coordinated changes in cell morphology accompany an array of cellular activities, such as the acquisition of a migratory phenotype and regulated progression throughout the cell cycle [1]
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
Endogenous ARF6 concentrates near the cleavage furrow after anaphase onset, and constitutively active and dominant negative ARF6 displays distinct distributions in mitotic cells, supporting a physiological role for ARF6 activity during mitosis
Summary
Cell Culture, Transfection, and Retroviral Transduction—HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin, and streptomycin (complete Dulbecco’s modified Eagle’s medium). The rabbit polyclonal antibody against ARF6 was generated as described previously [3], except that the collection was performed at the Freimann Center at the University of Notre Dame. The beads were collected and washed three times with 50 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 2 mM ZnCl2, and 0.01% mammalian protease inhibitor mixture (Sigma) (wash buffer). Mitotic Synchronization—For preparation of mitotic HeLa cell lysates, cells were synchronized using a double-thymidine block followed by nocodazole arrest, essentially as described previously [14]. Each containing 2–5 ϫ 105 cells, were cultured until the appropriate time point, and lysates were prepared and subjected to the MT-2 pulldown assay as described above. Mitotic cells were collected, seeded on poly-L-lysine-coated coverslips and fixed at various time points following release from nocodazole arrest
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