Repeated cycles of rapid actin assembly and disassembly on epithelial cell phagosomes

We have found that early in infection of the intracellular pathogen Listeria monocytogenes in Madin-Darby canine kidney epithelial cells expressing actin conjugated to green fluorescent protein, F-actin rapidly assembles (approximately 25 s) and disassembles (approximately 30 s) around the bacteria, a phenomenon we call flashing. L. monocytogenes strains unable to perform actin-based motility or unable to escape the phagosome were capable of flashing, suggesting that the actin assembly occurs on the phagosome membrane. Cycles of actin assembly and disassembly could occur repeatedly on the same phagosome. Indirect immunofluorescence showed that most bacteria were fully internalized when flashing occurred, suggesting that actin flashing does not represent phagocytosis. Escherichia coli expressing invA, a gene product from Yersinia pseudotuberculosis that mediates cellular invasion, also induced flashing. Furthermore, polystyrene beads coated with E-cadherin or transferrin also induced flashing after internalization. This suggests that flashing occurs downstream of several distinct molecular entry mechanisms and may be a general consequence of internalization of large objects by epithelial cells.     

Modular complexes that regulate actin assembly in budding yeast

The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts. For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell. Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles. The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.     

Dynamin2 and cortactin regulate actin assembly and filament organization

The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Microfilaments and microtubules regulate recycling from phagosomes

It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fcgamma-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism for membrane recycling. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the cytoskeletal requirements are largely unknown. To study this vesicle-mediated recycling transport, we used a biochemical assay and we developed a method to follow this process by confocal fluorescence microscopy. Interestingly, recycling from the phagosomal compartment was increased when the actin cortex was thinned by inhibitors of F-actin. In contrast, depolymerization of microtubules diminished both phagocytosis and recycling from phagosomes. Our results suggest that actin and microtubules are needed not only for phagosome biogenesis but also at other steps along the phagocytic pathway.     

A flexible activin explains the membrane-dependent cooperative assembly of TGF-beta family receptors

A new crystal structure of activin in complex with the extracellular domain of its type II receptor (ActRIIb-ECD) shows that the ligand exhibits an unexpected flexibility. The motion in the activin dimer disrupts its type I receptor interface, which may account for the disparity in its affinity for type I versus type II receptors. We have measured the affinities of activin and its antagonist inhibin for ActRIIb-ECD and found that the affinity of the 2-fold symmetric homodimer activin for ActRIIb-ECD depends on the availability of two spatially coupled ActRIIb-ECD molecules, whereas the affinity of the heterodimer inhibin does not. Our results indicate that activin's affinity for its two receptor types is greatly influenced by their membrane-restricted setting. We propose that activin affinity is modulated by the ligand flexibility and that cooperativity is achieved by binding to two ActRII chains that immobilize activin in a type I binding-competent orientation.     

Tec kinases regulate actin assembly and cytokine expression in LPS-stimulated human neutrophils via JNK activation

The acute inflammatory response involves neutrophils wherein recognition of bacterial products, such as lipopolysaccharide (LPS), activates intracellular signaling pathways. We have shown that the mitogen-activated protein kinase (MAPK) c-Jun NH₂ terminal kinase (JNK) is activated by LPS in neutrophils and plays a critical role in monocyte chemoattractant protein (MCP)-1 expression and actin assembly. As the Tec family kinases are expressed in neutrophils and regulate activation of the MAPKs in other cell systems, we hypothesized that the Tec kinases are an upstream component of the signaling pathway leading to LPS-induced MAPKs activation in neutrophils. Herein, we show that the Tec kinases are activated in LPS-stimulated human neutrophils and that inhibition of the Tec kinases, with leflunomide metabolite analog (LFM-A13), decreased LPS-induced JNK, but not p38, activity. Furthermore, LPS-induced actin polymerization as well as MCP-1, tumor necrosis factor-α, interleukin-6, and interleukin-1β expression are dependent on Tec kinase activity.     

Phosphoinositides and Rho proteins spatially regulate actin polymerization to initiate and maintain directed movement in a one-dimensional model of a motile cell

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive cell motility, with parameter values derived from in vitro and in vivo studies. In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that the PIs filter noise, and define the front versus back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore the importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. We also suggest a mechanism to explain the spatial exclusion of Cdc42 and PTEN and the inability of cells lacking active Cdc42 to properly detect chemoattractant gradients.     

Regulating cellular actin assembly

Cellular actin assembly is tightly regulated. The study of pathogen motility has led to the identification of several cellular factors that are critical for controlling this process. Pathogens such as Listeria require Ena/VASP and Arp2/3 proteins to translate actin polymerization into movement. Recent work has extended these observations and uncovered some similarities and surprising differences in the way cells and pathogens utilize the actin cytoskeleton.     

Extension of filopodia by motor-dependent actin assembly.

A variety of mechanisms have been proposed to explain the forward extension of cytoplasm in advancing cells and axonal growth cones, including actin polymerization and osmotic swelling. Based on our observations of the filopodia of cultured neuronal growth cones, we propose a mechanism involving motor-induced extension and retraction. We observed that filopodia (actin-based protrusions 0.2-0.5 mu in diameter) extend and retract from growth cone lamellae at the same rate. Further, force is generated at the tips of filopodia which is sufficient to produce compressive buckling of the proximal portion of the filopodium. From our analysis of these movements we suggest that a motor protein powers both the extension and retraction of filopodia.     

Phalloidin enhances actin assembly by preventing monomer dissociation

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation with actin in the presence of equimolar phalloidin in 100 mM KCl, 1 mM MgCl2 and 0.5 mM ATP at pH 7.5 resulted in a slightly smaller association rate constant at the preferred end than in the absence of the drug (3.36 +/- 0.14 X 10(6) M-1 s-1 vs. 2.63 +/- 0.22 X 10(6) M-1 s- 1, control vs. experimental). In the presence of phalloidin, the dissociation rate constant at the preferred end was reduced from 0.317 +/- 0.097 s-1 to essentially zero. Consequently, the critical concentration at the preferred end dropped from 0.10 microM to zero in the presence of the drug. There was no detectable change in the rate constant of association at the nonpreferred end in the presence of phalloidin (0.256 +/- 0.015 X 10(6) M-1 s-1 vs. 0.256 +/- 0.043 X 10(6) M-1 s-1, control vs. experimental); however, the dissociation rate constant was reduced from 0.269 +/- 0.043 s-1 to essentially zero. Thus, the critical concentration at the nonpreferred end changed from 1.02 microM to zero in the presence of phalloidin. Dilution-induced depolymerization at both the preferred and nonpreferred ends was prevented in the presence of phalloidin. Thus, phalloidin enhances actin assembly by lowering the critical concentration at both ends of actin filaments, a consequence of reducing the dissociation rate constants at each end.     

Processive acceleration of actin barbed-end assembly by N-WASP

Neuronal Wiskott–Aldrich syndrome protein (N-WASP)–activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3, N-WASP binds actin-filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We use nanofibers coated with N-WASP WWCA domains as model cell surfaces and single-actin-filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed-end assembly. Individual barbed ends captured by WWCA domains grow at or below their diffusion-limited assembly rate. At high filament densities, however, overlapping filaments form buckles between their nanofiber tethers and myosin attachment points. These buckles grew ∼3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native polyproline (PP) region show similar rate enhancements in the absence of profilin, but profilin slows barbed-end acceleration from constructs containing the PP region. Increasing Mg²⁺ to enhance filament bundling increases the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed-end bundling. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion.     

Detection of actin assembly by fluorescence energy transfer

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor- labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor- labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.     

Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency.     

Microtubule tips redirect actin assembly

In fission yeast, a protein complex that rides on the plus end of growing microtubules regulates establishment of new sites of actin cable assembly, necessary for cell growth, from one to both ends of the rod-shaped cells. Martin et al. describe a direct molecular link between the microtubule tip complex and the formin for3p, the nucleator for assembly of actin cables, which is necessary for this regulatory switch.     

Actin assembly induced by polylysine beads or purified phagosomes: quantitation by a new flow cytometry assay

BACKGROUND: Actin assembly on biological membranes is a poorly understood process. We have previously shown that phagosomal membranes could induce actin assembly in the presence of thymosin beta4 (an actin sequestering protein that inhibits nonspecific nucleation), via the barbed ends of actin filaments. METHODS: Here, we have developed an in vitro system based on fluorescein-labeled G (monomeric) actin and flow cytometry analysis, which allowed us to quantify de novo actin assembly on the cytoplasmic side of purified phagosomes. To standardize the system, we also used latex beads covalently coupled with polylysine, which efficiently promote actin nucleation. RESULTS: Flow cytometry analysis showed that the percentage of polylysine beads positive for F-actin filaments increased in a time- and G-actin concentration-dependent manner. Incubation of phagosomes with reagents affecting actin dynamics allowed us to extend our previous data showing that the phagosomal membranes assemble actin filaments de novo. Finally, our results pin-point a potential role for gelsolin as a positive regulator of actin assembly on the phagosomal membrane. CONCLUSIONS: We propose that our system could facilitate the development of other in vitro assays for the analysis of actin assembly and its links to signaling in cells.     

Cellular motility driven by assembly and disassembly of actin filaments

Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.     

Direct dynamin-actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.     

The actin cytoskeleton in presynaptic assembly

Dramatic morphogenetic processes underpin nearly every step of nervous system development, from initial neuronal migration and axon guidance to synaptogenesis. Underlying this morphogenesis are dynamic rearrangements of cytoskeletal architecture. Here we discuss the roles of the actin cytoskeleton in the development of presynaptic terminals, from the elaboration of terminal arbors to the recruitment of presynaptic vesicles and active zone components. The studies discussed here underscore the importance of actin regulation at every step in neuronal circuit assembly.