Abstract
BackgroundThe regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.Principal FindingsAs cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.ConclusionsARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.
Highlights
The small GTPase ARF serves as a key regulator of a number of cellular processes including vesicle trafficking, signal transduction and regulation of the actin cytoskeleton [1]
We have previously generated Chinese Hamster Ovary (CHO) cell lines over-expressing a fusion protein consisting of the green fluorescent protein (GFP) at the amino terminus and a 273 amino acid, non-catalytic domain of ARFGAP1, called GAP273
In CHO cells stably transfected with GFP-GAP273, the fluorescent fusion protein was observed primarily on Golgi membranes in cells that had spread on their glass substrate [14,17,32]
Summary
The small GTPase ARF serves as a key regulator of a number of cellular processes including vesicle trafficking, signal transduction and regulation of the actin cytoskeleton [1]. ARF6 functions in the endocytic pathway and regulates actin cytoskeleton [2] Because of their diverse and complex cellular functions, the activity of ARF proteins is highly regulated. Other than the ARF GAP catalytic domain, these genes are vastly different in terms of their size, the identifiable features that they contain and their subcellular localization [9]. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 serves as a GAP for ARF1, and can affect the actin cytoskeleton
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