Tongue orthotopic xenografts to study fusion-negative rhabdomyosarcoma invasion and metastasis in live animals.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Point mutations in Arf1 reveal cooperative effects of the N-terminal extension and myristate for GTPase-activating protein catalytic activity.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

In vitro Fluorescence Imaging-based Actin Bundling Assay.

Understanding the molecular and structural mechanisms that govern the assembly and organization of higher-order actin architecture requires the use of in vitro actin binding and bundling assays. Crosslinking of actin filaments into bundles can be monitored in vitro via several techniques, including negative staining/electron microscopy, low-speed co-sedimentation assay/SDS-PAGE, and fluorescence staining/confocal microscopy. We and others have previously characterized the N-BAR domain of ASAP1, an ADP-ribosylation factor GTPase-activating protein, as an actin-bundling module; we further identified key lysine residues responsible for actin cross-linking. Here, we use the ASAP1 BAR domain as an example and describe a detailed procedure for observing the actin bundle formation by confocal microscopy. This protocol requires small reaction volumes and takes advantage of bright commercially available fluorescent phalloidins, making it an ideal choice for medium-throughput screening of mutants or domain truncations in their ability to bundle actin. Graphical abstract.

ERK phosphorylation is dependent on cell adhesion in a subset of pediatric sarcoma cell lines.

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.

A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments.

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

In vitro reconstitution reveals cooperative mechanisms of adapter protein-mediated activation of phospholipase C-γ1 in T cells.

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC-γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-γ1 activation are regulated both independently and additively by recruitment of PLC-γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC-γ1 tetramer, and by tyrosine phosphorylation of PLC-γ1. The recently solved structure of PLC-γ1 indicates that, in the resting state, several PLC-γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-γ1, enabling contact with its membrane substrate, disinhibition of PLC-γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.

Control of cell signaling by Arf GTPases and their regulators: Focus on links to cancer and other GTPase families.

The ADP-ribosylation factors (Arfs) comprise a family of regulatory GTP binding proteins. The Arfs regulate membrane trafficking and cytoskeleton remodeling, processes critical for eukaryotes and which have been the focus of most studies on Arfs. A more limited literature describes a role in signaling and in integrating several signaling pathways to bring about specific cell behaviors. Here, we will highlight work describing function of Arf1, Arf6 and several effectors and regulators of Arfs in signaling.

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aß, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling-a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to 'nucleotide empty' or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

Rigosertib Induces Mitotic Arrest and Apoptosis in RAS-Mutated Rhabdomyosarcoma and Neuroblastoma.

Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.

Membrane surface recognition by the ASAP1 PH domain and consequences for interactions with the small GTPase Arf1.

Adenosine diphosphate-ribosylation factor (Arf) guanosine triphosphatase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of guanosine triphosphate (GTP) bound to the small GTP-binding protein Arf. Binding of the pleckstrin homology (PH) domain of the ArfGAP With SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is key for maximum GTP hydrolysis but not fully understood. By combining nuclear magnetic resonance, neutron reflectometry, and molecular dynamics simulation, we show that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with an Arf1 mimic to occur. Our model provides a framework to understand how binding of the ASAP1 PH domain to PI(4,5)P2 at the membrane may play a role in the regulation of ASAP1.

Small GTPase ARF6 Is a Coincidence-Detection Code for RPH3A Polarization in Neutrophil Polarization.

Cell polarization is a key step for leukocytes adhesion and transmigration during leukocytes' inflammatory infiltration. Polarized localization of plasma membrane (PM) phosphatidylinositol-4-phosphate (PtdIns4P) directs the polarization of RPH3A, which contains a PtdIns4P binding site. Consequently, RPH3A mediates the RAB21 and PIP5K1C90 polarization, which is important for neutrophil adhesion to endothelia during inflammation. However, the mechanism by which RPH3A is recruited only to PM PtdIns4P rather than Golgi PtdIns4P remains unclear. By using ADP-ribosylation factor 6 (ARF6) small interfering RNA, ARF6 dominant-negative mutant ARF6(T27N), and ARF6 activation inhibitor SecinH3, we demonstrate that ARF6 plays an important role in the polarization of RPH3A, RAB21, and PIP5K1C90 in murine neutrophils. PM ARF6 is polarized and colocalized with RPH3A, RAB21, PIP5K1C90, and PM PtdIns4P in mouse and human neutrophils upon integrin stimulation. Additionally, ARF6 binds to RPH3A and enhances the interaction between the PM PtdIns4P and RPH3A. Consistent with functional roles of polarization of RPH3A, Rab21, and PIP5K1C90, ARF6 is also required for neutrophil adhesion on the inflamed endothelial layer. Our study reveals a previously unknown role of ARF6 in neutrophil polarization as being the coincidence-detection code with PM PtdIns4P. Cooperation of ARF6 and PM PtdIns4P direct RPH3A polarization, which is important for neutrophil firm adhesion to endothelia.

The ArfGAP ASAP1 Controls Actin Stress Fiber Organization via Its N-BAR Domain.

ASAP1 is a multi-domain ArfGAP that controls cell migration, spreading, and focal adhesion dynamics. Although its GAP activity contributes to remodeling of the actin cytoskeleton, it does not fully explain all cellular functions of ASAP1. Here we find that ASAP1 regulates actin filament assembly directly through its N-BAR domain and controls stress fiber maintenance. ASAP1 depletion caused defects in stress fiber organization. Conversely, overexpression of ASAP1 enhanced actin remodeling. The BAR-PH fragment was sufficient to affect actin. ASAP1 with the BAR domain replaced with the BAR domain of the related ACAP1 did not affect actin. The BAR-PH tandem of ASAP1 bound and bundled actin filaments directly, whereas the presence of the ArfGAP and the C-terminal linker/SH3 domain reduced binding and bundling of filaments by BAR-PH. Together these data provide evidence that ASAP1 may regulate the actin cytoskeleton through direct interaction of the BAR-PH domain with actin filaments.

Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first ß-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

ARF GTPases and their GEFs and GAPs: concepts and challenges.

Detailed structural, biochemical, cell biological, and genetic studies of any gene/protein are required to develop models of its actions in cells. Studying a protein family in the aggregate yields additional information, as one can include analyses of their coevolution, acquisition or loss of functionalities, structural pliability, and the emergence of shared or variations in molecular mechanisms. An even richer understanding of cell biology can be achieved through evaluating functionally linked protein families. In this review, we summarize current knowledge of three protein families: the ARF GTPases, the guanine nucleotide exchange factors (ARF GEFs) that activate them, and the GTPase-activating proteins (ARF GAPs) that have the ability to both propagate and terminate signaling. However, despite decades of scrutiny, our understanding of how these essential proteins function in cells remains fragmentary. We believe that the inherent complexity of ARF signaling and its regulation by GEFs and GAPs will require the concerted effort of many laboratories working together, ideally within a consortium to optimally pool information and resources. The collaborative study of these three functionally connected families (≥70 mammalian genes) will yield transformative insights into regulation of cell signaling.

Functional Expression and Characterization of Human Myristoylated-Arf1 in Nanodisc Membrane Mimetics.

Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers. Here, we report on the production of a functional human Arf1 membrane platform on nanodiscs for biophysical studies. Large scale bacterial production of highly pure, N-myristoylated human Arf1 has been achieved, including complex isotopic labeling for nuclear magnetic resonance (NMR) studies, and the myr-Arf1 can be readily assembled in small nanoscale lipid bilayers (nanodiscs, NDs). It is determined that myr-Arf1 requires a minimum binding surface in the NDs of ∼20 lipids. Fluorescence and NMR were used to establish nucleotide exchange and ArfGAP-stimulated GTP hydrolysis at the membrane, indicating that phophoinositide stimulation of the activity of the ArfGAP ASAP1 is ≥2000-fold. Differences in nonhydrolyzable GTP analogues are observed, and GMPPCP is found to be the most stable. Combined, these observations establish a functional environment for biophysical studies of Arf1 effectors and interactions at the membrane.

Arf GAPs: A family of proteins with disparate functions that converge on a common structure, the integrin adhesion complex.

ADP-ribosylation factors (Arfs) are members of the Ras GTPase superfamily. The function of Arfs is dependent on GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), which allow Arfs to cycle between the GDP-bound and GTP-bound forms. Arf GAPs have been shown to be present in integrin adhesion complexes, which include focal adhesions. Integrin adhesion complexes are composed of integrins, scaffolding proteins and signaling proteins and regulate cell proliferation, survival, differentiation and migration. Understanding the role of Arf GAPs in the regulation of integrin adhesion complexes is relevant to understanding normal physiology and cancer. In this review, we will discuss the contribution of the Arf GAP family members to the regulation of integrin adhesion complexes, examining the diverse mechanisms by which they control integrin adhesion complex formation, maturation and dissolution. GIT1 and ARAP2 serve as GAPs for Arf6, regulating Rac1 and other effectors by mechanisms still being defined. In contrast, GIT2 regulates Rac1 independent of Arf6. AGAP2 binds to and regulates focal adhesion kinase (FAK). ARAP2 and ACAP1, both Arf6 GAPs, regulate membrane trafficking of integrins through different endocytic pathways, exerting opposite effects on focal adhesions. ASAP1 not only regulates actin cytoskeleton remodeling through its interaction with nonmuscle myosin 2A, but is also important in integrin recycling. These examples illustrate the diversity and versatility of Arf GAPs as regulators of integrin adhesion complex structure and function.

Arf GAPs and molecular motors.

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling.