Differential Membrane Association Properties and Regulation of Class I and Class II Arfs

ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.     

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.     

Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors

The ADP ribosylation factors (Arfs) are a family of small, ubiquitously expressed and evolutionarily conserved guanosine triphosphatases that are key regulators of vesicular transport in eukaryotic cells (D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 2006;7:347-358). Although Arfs are best known for their role in the nucleation of coat protein assembly at a variety of intracellular locations, it is increasingly apparent that they are also integral components in a number of important signaling pathways that are regulated by extracellular cues. The activation of Arfs is catalyzed by a family of guanine nucleotide exchange factors (GEFs), referred to as the Sec7 family, based on homology of their catalytic domains to the yeast Arf GEF, sec7p. While there are only six mammalian Arfs, the human genome encodes 15 Sec7 family members, which can be divided into five classes based on related domain organization. Some of this diversity arises from the tissue-specific expression of certain isoforms, but all mammalian cells appear to express at least six Arf GEFs, suggesting that Arf activation is under extensive regulatory control. Here we review recent progress in our understanding of the structure, localization and biology of the different classes of Arf GEFs.     

The protein tyrosine phosphatase PTP-Basophil/Basophil-like. Interacting proteins and molecular functions.

The protein tyrosine phosphatase PTP-Basophil (PTP-Bas) and its mouse homologue, PTP-Basophil-like (PTP-BL), are high molecular mass protein phosphatases consisting of a number of diverse protein-protein interaction modules. Several splicing variants of these phosphatases are known to exist thus demonstrating the complexity of these molecules. PTP-Bas/BL serves as a central scaffolding protein facilitating the assembly of a multiplicity of different proteins mainly via five different PDZ domains. Many of these interacting proteins are implicated in the regulation of the actin cytoskeleton. However, some proteins demonstrate a nuclear function of this protein tyrosine phosphatase. PTP-Bas is involved in the regulation of cell surface expression of the cell death receptor, Fas. Moreover, it is a negative regulator of ephrinB phosphorylation, a receptor playing an important role during development. The phosphorylation status of other proteins such as RIL, IkappaBalpha and beta-catenin can also be regulated by this phosphatase. Finally, PTP-BL has been shown to be involved in the regulation of cytokinesis, the last step in cell division. Although the precise molecular function of PTP-Bas/BL is still elusive, current data suggest clearly that PTP-Bas/BL belongs to the family of PDZ domain containing proteins involved in the regulation of the cytoskeleton and of intracellular vesicular transport processes.     

BRAG2/GEP100/IQSec1 Interacts with Clathrin and Regulates α5β1 Integrin Endocytosis through Activation of ADP Ribosylation Factor 5 (Arf5)

ADP ribosylation factors (Arfs) are small GTP-binding proteins known for their role in vesicular transport, where they nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. Similar to other GTPases, Arfs require guanine nucleotide exchange factors to catalyze GTP loading and activation. One subfamily of ArfGEFs, the BRAGs, has been shown to activate Arf6, which acts in the endocytic pathway to control the trafficking of a subset of cargo proteins including integrins. We have previously shown that BRAG2 modulates cell adhesion by regulating integrin surface expression. Here, we show that, in addition to Arf6, endogenous BRAG2 also activates the class II Arfs, Arf4 and Arf5, and that surprisingly, it is Arf5 that mediates integrin internalization. We observed that cell spreading on fibronectin is enhanced upon inhibition of BRAG2 or Arf5 but not Arf6. Similarly, spreading in BRAG2-depleted cells is reverted by expression of a rapid cycling Arf5 mutant (T161A) but not by a corresponding Arf6 construct (T157A). We also show that BRAG2 binds clathrin and the AP-2 adaptor complex and that both BRAG2 and Arf5 localize to clathrin-coated pits at the plasma membrane. Consistent with these observations, depletion of Arf5, but not Arf6 or Arf4, slows internalization of β1 integrins without affecting transferrin receptor uptake. Together, these findings indicate that BRAG2 acts at clathrin-coated pits to promote integrin internalization by activating Arf5 and suggest a previously unrecognized role for Arf5 in clathrin-mediated endocytosis of specific cargoes.     

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.     

Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly

Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.     

Cell-cell adhesion and signalling

Signalling pathways activated by Rho small GTPases have recently been identified that coordinate junction assembly, stability and function, as well as interactions of adhesive complexes with the underlying cortical cytoskeleton. Particularly exciting is the interplay between adherens junctions, activation of Rho proteins and the dynamics of microtubule, actin and intermediate filaments. This interplay has important implications for functional regulation of cell-cell adhesion, and points to a more integrated view of signalling processes.     

The structural GDP/GTP cycle of human Arf6

The small GTP-binding protein Arf6 coordinates membrane traffic at the plasma membrane with aspects of cytoskeleton organization. This function does not overlap with that of other members of the ADP-ribosylation factor (Arf) family, although their switch regions, which are their major sites of interaction with regulators and effectors, have virtually identical sequences. Here we report the crystal structure of full-length, non-myristoylated human Arf6 bound to GTPγS. Unlike their GDP-bound forms, the active forms of Arf6 and Arf1 are very similar. Thus, the switch regions are discriminatory elements between Arf isoforms in their inactive but not in their active forms, a property that may generalize to other families of small G proteins. This suggests that GTP-bound Arfs may establish specific interactions outside the switch regions and/or be recognized in their cellular context rather than as isolated proteins. The structure also allows further insight into the lack of spontaneous GTPase activity of Arf proteins.     

Gelsolin-independent podosome formation in dendritic cells

Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.     

The LEA proteins and trehalose loving couple: a step forward in anhydrobiotic engineering

Arf family GTP-binding proteins function in the regulation of membrane-trafficking events and in the maintenance of organelle structure. They act at membrane surfaces to modify lipid composition and to recruit coat proteins for the generation of transport vesicles. Arfs associate with membranes through insertion of an N-terminal myristoyl moiety in conjunction with an adjacent amphipathic alpha-helix, which embeds in the lipid bilayer when Arf1 is GTP-bound. In this issue of the Biochemical Journal, Lundmark et al. report that myristoylated Arfs in the presence of GTP bind to and cause tubulation of liposomes, and that GTP exchange on to Arfs is more efficient in the presence of liposomes of smaller diameter (increased curvature). These findings suggest that Arf protein activation and membrane interaction may initiate membrane curvature that will be enhanced further by coat proteins during vesicle formation.     

Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking

Coronins constitute an evolutionarily conserved family of WD-repeat actin-binding proteins, which can be clearly classified into two distinct groups based on their structural features. All coronins possess a conserved basic N-terminal motif and three to ten WD repeats clustered in one or two core domains. Dictyostelium and mammalian coronins are important regulators of the actin cytoskeleton, while the fly Dpod1 and the yeast coronin proteins crosslink both actin and microtubules. Apart from that, several coronins have been shown to be involved in vesicular transport. C. elegans POD-1 and Drosophila coro regulate the actin cytoskeleton, but also govern vesicular trafficking as indicated by mutant phenotypes. In both organisms, defects in cytoskeleton and trafficking lead to severe developmental defects ranging from abnormal cell division to aberrant formation of morphogen gradients. Finally, mammalian coronin 7 appears not to execute any cytoskeleton-related functions, but rather participates in regulating Golgi trafficking. Here, we review recent data providing more insight into molecular mechanisms underlying the regulation of F-actin structures, cytoskeletal rearrangements and intracellular membrane transport by coronin proteins and the way that they might link cytoskeleton with trafficking in development and disease.     

Actin dynamics at intracellular membranes: the Spir/formin nucleator complex

The assembly of actin monomers into filaments is a highly regulated basic cellular function. The structural organization of a cell, morphological changes or cell motility is dependent on actin filament dynamics. While within the last decade substantial knowledge has been acquired about actin dynamics at the cell membrane, today only little is known about the actin cytoskeleton and its functions at intracellular endosomal and organelle membranes. The Spir actin nucleators are specifically targeted towards endosomal membranes by a FYVE zinc finger membrane localization domain, and provide an important link to study the role of actin dynamics in the regulation of intracellular membrane transport. Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. Although Spir proteins can nucleate actin polymerization in vitro by themselves, they form a regulatory complex with the distinct actin nucleators of the formin subgroup (Fmn) of formins. A cooperative mechanism in actin nucleation has been proposed. Ongoing studies on the function and regulation of the Spir proteins in vesicle transport processes will reveal important insights into actin dynamics at intracellular membranes and how this regulates the highly directed and controlled routes of intracellular membrane trafficking.     

Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts

Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.     

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

WASP-interacting protein (WIP): working in polymerisation and much more

The migration of cells and the movement of some intracellular pathogens, such as Shigella and Vaccinia, are dependent on the actin-based cytoskeleton. Many proteins are involved in regulating the dynamics of the actin-based microfilaments within cells and, among them, WASP and N-WASP have a significant role in the regulation of actin polymerisation. The activity and stability of WASP is regulated by its cellular partner WASP-interacting protein (WIP) during the formation of actin-rich structures, including the immune synapse, filopodia, lamellipodia, stress fibres and podosomes. Here, we review the role of WIP in regulating WASP function by stabilising WASP and shuttling WASP to areas of actin assembly in addition to reviewing the WASP-independent functions of WIP.     

Regulating the actin cytoskeleton during vesicular transport

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.     

Rab and Arf Proteins in Genetic Diseases

Rab and ADP‐ribosylation factor (Arf) family proteins are master regulators of membrane trafficking and are involved in all steps of vesicular transport. These families of small guanine‐nucleotide‐binding (G) proteins are well suited to regulate membrane trafficking processes since their nucleotide state determines their conformation and the capacity to bind to a multitude of effectors, which mediate their functions. In recent years, several inherited diseases have been associated with mutations in genes encoding proteins belonging to these two families or in proteins that regulate their GTP‐binding cycle. The genetic diseases that are caused by defects in Rabs, Arfs or their regulatory proteins are heterogeneous and display diverse symptoms. However, these diseases mainly affect two types of subcellular compartments, namely lysosome‐related organelles and cilia. Also, several of these diseases affect the nervous system. Thus, the study of these diseases represents an opportunity to understand their etiology and the molecular mechanisms involved, as well as to develop novel therapeutic strategies .     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.