Self-organizing actin waves as planar phagocytic cup structures

Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as “frustrated” phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed.Key words: actin waves, Dictyostelium, membrane tension, pattern formation, phagocytosis, PI3-kinase, PI(3,4,5)P3, self-organization     

The Three-Dimensional Dynamics of Actin Waves, a Model of Cytoskeletal Self-Organization

Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization.     

Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of "comet tails" that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P(2) and PI(3,4,5)P(3) on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P(2) and PI(3,4,5)P(3) and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P(2) and PI(3,4,5)P(3) formation and signaling in endomembranes.     

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.     

Contractile proteins in phagocytosis: an example of cell surface-to-cytoplasm communication.

Phagocytosis is a prime example of a cellular event in which cell surface perturbation activates the assembly of a filamentous gel beneath the plasma membrane. This gel may be responsible for movement of the membrane around ingestible particles. The molecular mechanism of these events is being approached by the purification of actin, myosin and associated proteins from phagocytic cells and by the study of a human disease, neutrophil actin dysfunction. Novel contractile proteins discovered in mammalian phagocytes include a cofactor that regulates actin:myosin interaction and an actin-binding protein that promotes assembly and gelation of actin. There is evidence that phagocytosis alters the state of the actin-binding protein, and that this alteration may be an early event in the assembly of the actin gel. Cytochalasin B, which inhibits phagocytosis, acts by interfering with the interaction between actin-binding protein and actin. Actin polymerized poorly in the neutrophils of a human infant, and the affected neutrophils were deficient in phagocytosis. Actin assembly is important in phagocytosis and is amenable to biochemical analysis.     

Reconstitution of an Actin Cortex Inside a Liposome

The composite and versatile structure of the cytoskeleton confers complex mechanical properties on cells. Actin filaments sustain the cell membrane and their dynamics insure cell shape changes. For example, the lamellipodium moves by actin polymerization, a mechanism that has been studied using simplified experimental systems. Much less is known about the actin cortex, a shell-like structure underneath the membrane that contracts for cell movement. We have designed an experimental system that mimicks the cell cortex by allowing actin polymerization to nucleate and assemble at the inner membrane of a liposome. Actin shell growth can be triggered inside the liposome, which offers a useful system for a controlled study. The observed actin shell thickness and estimated mesh size of the actin structure are in good agreement with cellular data. Such a system paves the way for a thorough characterization of cortical dynamics and mechanics.     

Cytoskeletal mechanics during Shigella invasion and dissemination in epithelial cells

The actin cytoskeleton is key to the barrier function of epithelial cells, by permitting the establishment and maintenance of cell–cell junctions and cell adhesion to the basal matrix. Actin exists under monomeric and polymerized filamentous form and its polymerization following activation of nucleation promoting factors generates pushing forces, required to propel intracellular microorganisms in the host cell cytosol or for the formation of cell extensions that engulf bacteria. Actin filaments can associate with adhesion receptors at the plasma membrane via cytoskeletal linkers. Membrane anchored to actin filaments are then subjected to the retrograde flow that may pull membrane‐bound bacteria inside the cell. To induce its internalization by normally non‐phagocytic cells, bacteria need to establish adhesive contacts and trick the cell into apply pulling forces, and/or to generate protrusive forces that deform the membrane surrounding its contact site. In this review, we will focus on recent findings on actin cytoskeleton reorganization within epithelial cells during invasion and cell‐to‐cell spreading by the enteroinvasive pathogen Shigella, the causative agent of bacillary dysentery.     

Growth cone‐like waves transport actin and promote axonogenesis and neurite branching

Axonogenesis involves a shift from uniform delivery of materials to all neurites to preferential delivery to the putative axon, supporting its more rapid extension. Waves, growth cone‐like structures that propagate down the length of neurites, were shown previously to correlate with neurite growth in dissociated cultured hippocampal neurons. Waves are similar to growth cones in their structure, composition and dynamics. Here, we report that waves form in all undifferentiated neurites, but occur more frequently in the future axon during initial neuronal polarization. Moreover, wave frequency and their impact on neurite growth are altered in neurons treated with stimuli that enhance axonogenesis. Coincident with wave arrival, growth cones enlarge and undergo a marked increase in dynamics. Through their engorgement of filopodia along the neurite shaft, waves can induce de novo neurite branching. Actin in waves maintains much of its cohesiveness during transport whereas actin in nonwave regions of the neurite rapidly diffuses as measured by live cell imaging of photoactivated GFP‐actin and photoconversion of Dendra‐actin. Thus, waves represent an alternative axonal transport mechanism for actin. Waves also occur in neurons in organotypic hippocampal slices where they propagate along neurites in the dentate gyrus and the CA regions and induce branching. Taken together, our results indicate that waves are physiologically relevant and contribute to axon growth and branching via the transport of actin and by increasing growth cone dynamics. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

African swine fever virus induces filopodia-like projections at the plasma membrane

When exiting the cell vaccinia virus induces actin polymerization and formation of a characteristic actin tail on the cytosolic face of the plasma membrane, directly beneath the extracellular particle. The actin tail acts to propel the virus away from the cell surface to enhance its cell-to-cell spread. We now demonstrate that African swine fever virus (ASFV), a member of the Asfarviridae family, also stimulates the polymerization of actin at the cell surface. Intracellular ASFV particles project out at the tip of long filopodia-like protrusions, at an average rate of 1.8 microm min(-1). Actin was arranged in long unbranched parallel arrays inside these virus-tipped projections. In contrast to vaccinia, this outward movement did not involve recruitment of Grb2, Nck1 or N-WASP. Actin polymerization was not nucleated by virus particles in transit to the cell periphery, and projections were not produced when the secretory pathway was disrupted by brefeldin A treatment. Our results show that when ASFV particles reach the plasma membrane they induce a localized nucleation of actin, and that this process requires interaction with virus-encoded and/or host proteins at the plasma membrane. We suggest that ASFV represents a valuable new model for studying pathways that regulate the formation of filopodia.     

Mechanically induced actin-mediated rocketing of phagosomes

Actin polymerization can be induced in Dictyostelium by compressing the cells to bring phagosomes filled with large particles into contact with the plasma membrane. Asymmetric actin assembly results in rocketing movement of the phagosomes. We show that the compression-induced assembly of actin at the cytoplasmic face of the plasma membrane involves the Arp2/3 complex. We also identify two other proteins associated with the mechanically induced actin assembly. The class I myosin MyoB accumulates at the plasma membrane-phagosome interface early during the initiation of the response, and coronin is recruited as the actin filaments are disassembling. The forces generated by rocketing phagosomes are sufficient to push the entire microtubule apparatus forward and to dislocate the nucleus.     

Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells

BACKGROUND: In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS: Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS: The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.     

Effect of phosphatidyl-L-serine and vinculin on actin polymerization

The effects of phosphatidyl-L-serine (PS) and/or vinculin on actin polymerization are examined by spectrophotometry, viscometry and electrophoresis. Actin polymerization is inhibited by PS alone and stimulated by PS and vinculin. The results suggest that actin does not directly adhere to cell membrane and that vinculin is a protein which is involved in structures connecting actin microfilaments to cell membranes.     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Dynamic polymorphism of actin as activation mechanism for cell motility

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.     

Direct determination of actin polarity in the cell

Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton.     

Pathogenicity of bacteria and the actin cytoskeleton

Actin system of eukaryotic cells creates the driving force for alteration of the phagocytic cytoplasmatic membrane shape, which is needed for cell movement in the space and for microorganism capturing. Manipulation by actin cytoskeleton mediated through specialized bacterial products can promote proliferation of bacteria in the host. Published reports indicate that bacterial regulation of the actin system activity can be carried out by two modes: 1) by bacterial interactions with surface receptors regulating the cytoskeleton status and 2) by introduction of bacterial products targeted to the cytoskeleton components into the cells. Intracellular pathogens (Legionella) possess ligands which interact with eukaryotic receptors and type IV secretion system fit for translocation of heretofore unknown effector molecules into the cytoplasm. This can result in stimulation of actin polymerization activity and accelerated phagocytosis of the bacteria with rapid multiplication in tissues. By contrast, representatives of extracellular pathogens (Clostridium) produce substances penetrating inside the eukaryotic cells and destroying the actin network, thus making capturing and intracellular digestion of these microorganisms impossible.     

Cotton annexin proteins participate in the establishment of fiber cell elongation scaffold

Cotton plant is one of the most important economic crops in the world which supplies natural fiber for textile industry. The crucial traits of cotton fiber quality are fiber length and strength, which are mostly determined by the fiber elongation stage. Annexins are assumed to be involved in regulating fiber elongation, but direct evidences remain elusive. Recently, we have investigated the activities of fiber-specific expressed annexins AnGb5/6 and their interacted proteins in cotton. AnGb5 and 6 can interact reciprocally to generate a protein macro-raft in cell membrane. This macro-raft is probably a stabilized scaffold for Actin1 organization. The actin assembling direction and density are correlated with AnGb6 gene expression and fiber expanding rate among three fiber length genotypes. These results suggest that annexins may act as the adaptor that linked fiber cell membrane to actin assembling. Due to the strong Ca²⁺ and lipid binding ability of annexins, these results also indicate that annexins complex may function as an intermediate to receive Ca²⁺ or lipid signals during fiber elongation.     

Propagating cell-membrane waves driven by curved activators of actin polymerization

Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape.     

Listeria monocytogenes cell invasion: a new role for cofilin in co-ordinating actin dynamics and membrane lipids

Actin reorganization, mediated by the actin dynamizing protein cofilin, is essential for host cell invasion by the intracellular pathogenic bacterium Listeria monocytogenes. During invasion, the InlB bacterial surface ligands closely interact with host cell Met receptors to induce phagocytosis. In this issue of Molecular Microbiology, Han et al., 2011 clearly demonstrate that phospholipase D (PLD)-dependent production of membrane phosphatidic acid is required for invasion. They further show that the phosphorylated form of cofilin, which is inactive in actin binding, is necessary for the activation of the PLD1 isoform. Although cofilin-independent PLD2 can also mediate internalization, it is a phospho-cofilin-dependent balanced production of phosphatidic acid that is required for optimal Listeria internalization. Cofilin-dependent membrane lipid remodelling has important implications for cofilin function that go well beyond its direct effects on actin.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.