Sites Of Actin Polymerization Research Articles

Diacylglycerol kinase $zeta; regulates phosphatidylinositol 4-phosphate 5-kinase I$alpha; by a novel mechanism

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays an important role during actin polymerization and is produced by the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI), which are activated by phosphatidic acid (PA). As diacylglycerol kinases (DGKs) generate PA by phosphorylating diacylglycerol (DAG), we investigated whether DGKs were involved in controlling PIP2 levels by regulating PIP5KI activity. Here we show that expression of DGKζ significantly enhances PIP5KIα activity in thrombin-stimulated HEK293 cells, and DGK activity is required for this stimulation. We also observed that DGKζ co-immunoprecipitated and co-localized with PIP5KIα, suggesting that they reside in a regulated signaling complex. To explore the role of DGKζ in actin polymerization, we examined the subcellular distribution of DGKζ, PIP5KIα and actin, and found that these proteins co-localized with actin in lamellipodial protrusions. Supporting that PIP5KIα regulation occurs at the sites of actin polymerization, we found that PIP2 also accumulated in the actin-rich regions of lamellipodia. Significantly, in wounding assays, DGKζ, PIP5KIα and PIP2 accumulated at the leading edge of migrating A172 cells, where massive actin polymerization is known to occur. Combined, these data support a novel function for DGKζ: by generating PA, it stimulates PIP5KIα activity to increase local PIP2, which regulates actin polymerization.

Diacylglycerol kinase ζ regulates phosphatidylinositol 4-phosphate 5-kinase Iα by a novel mechanism

Phosphatidylinositol 4,5-bisphosphate (PIP 2) plays an important role during actin polymerization and is produced by the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI), which are activated by phosphatidic acid (PA). As diacylglycerol kinases (DGKs) generate PA by phosphorylating diacylglycerol (DAG), we investigated whether DGKs were involved in controlling PIP 2 levels by regulating PIP5KI activity. Here we show that expression of DGKζ significantly enhances PIP5KIα activity in thrombin-stimulated HEK293 cells, and DGK activity is required for this stimulation. We also observed that DGKζ co-immunoprecipitated and co-localized with PIP5KIα, suggesting that they reside in a regulated signaling complex. To explore the role of DGKζ in actin polymerization, we examined the subcellular distribution of DGKζ, PIP5KIα and actin, and found that these proteins co-localized with actin in lamellipodial protrusions. Supporting that PIP5KIα regulation occurs at the sites of actin polymerization, we found that PIP 2 also accumulated in the actin-rich regions of lamellipodia. Significantly, in wounding assays, DGKζ, PIP5KIα and PIP 2 accumulated at the leading edge of migrating A172 cells, where massive actin polymerization is known to occur. Combined, these data support a novel function for DGKζ: by generating PA, it stimulates PIP5KIα activity to increase local PIP 2, which regulates actin polymerization.

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation.

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.

Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration.

Ena/VASP proteins are a conserved family of actin regulatory proteins made up of EVH1, EVH2 domains, and a proline-rich central region. They have been implicated in actin-based processes such as fibroblast migration, axon guidance, and T cell polarization and are important for the actin-based motility of the intracellular pathogen Listeria monocytogenes. Mechanistically, these proteins associate with barbed ends of actin filaments and antagonize filament capping by capping protein (CapZ). In addition, they reduce the density of Arp2/3-dependent actin filament branches and bind Profilin at sites of actin polymerization. Vertebrate Ena/VASP proteins are substrates for PKA/PKG serine/threonine kinases. Phosphorylation by these kinases appears to modulate Ena/VASP function within cells, although the mechanism underlying this regulation remains to be determined.

Design of N-substituted Peptomer Ligands for EVH1 Domains

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

Molecular evolutionary convergence of the flight muscle protein arthrin in Diptera and hemiptera.

Uniquely, the asynchronous flight muscle myofibrils of many insects contain arthrin, a stable 1:1 conjugate between actin and ubiquitin. The function of arthrin is still unknown. Here we survey for the presence of arthrin in 63 species of insect across nine orders using Western blotting. Analysis of the evolutionary distribution shows that arthrin has evolved a limited number of times but at least once in the Diptera and once in the Hemiptera. However, the presence of arthrin does not correlate with any observed common features of flight mechanism, natural history, or morphology. We also identify the site of the isopeptide bond in arthrin from Drosophila melanogaster (Diptera) and Lethocerus griseus (Hemiptera) using mass spectrometry. In both species, the isopeptide bond is formed between lysine 118 of the actin and the C-terminal glycine 76 of ubiquitin. Thus, not only the ubiquitination of actin but also the site of the isopeptide bond has evolved convergently in Diptera and Hemiptera. In terms of the actin monomer, lysine 118 is near neither the binding sites of the major actin-binding proteins, myosin, tropomyosin, or the troponins, nor the actin polymerization sites. However, molecular modeling supports the idea that ubiquitin bound to an actin in one F-actin strand might be able to interact with tropomyosin bound to the actin monomers of the other strand and thereby interfere with thin filament regulation.

Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells.

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2–4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.

Watching the Signals

How signaling molecules function is not only a matter of when but also a matter of where. Kraynov et al. demonstrate that activation of the small guanosine triphosphate-binding protein Rac1 can be visualized in real time in living cells. Activation of Rac1, known to induce actin-based morphological changes, was restricted to sites of actin polymerization, independent of overall intracellular Rac distribution. Thus, cells can produce distinct behaviors through specific distributions of activated Rac1. Kraynov, V.S., Chamberlain, C., Bokoch, G.M., Schwartz, M.A., Slabaugh, S., and Hahn, K.M. (2000) Localized Rac activation dynamics visualized in living cells. Science 290 : 333-337. [Full Text] [Abstract]

Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain-containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization.

Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.

Recruitment of the Arp2/3 complex and mena for the stimulation of actin polymerization in growth cones by nerve growth factor.

The growth of axons and dendrites during development and regeneration is regulated by cues in the environment. Many of these cues regulate the actin cytoskeleton of the protrusive structures (like filopodia) of the growth cone that are essential for detecting and responding to cues. Nerve growth factor, which promotes the formation of protrusive structures, stimulated actin polymerization in rat sympathetic growth cones, resulting within 1-2 min in accumulations of F-actin at the distal edge and in splotches of F-actin farther back. We examined the potential involvement of a protein machinery important in at least certain types of actin polymerization in non-neuronal cells. Members of the Arp2/3 complex, p34-Arc and p21-Arc, heavily concentrated in the early accumulations of F-actin, as did one member of the Ena/VASP family (Mena) but not another (VASP). Retention of Arc proteins at preferred sites of actin polymerization did not require polymerization itself. Growth cones of differentiated PC12 cells were similar to sympathetic growth cones in their response to NGF. Introduction into these cells of a peptide that should block the binding of Ena/VASP family proteins to the protein complex at sites of actin polymerization reduced the formation of splotches and filopodia in response to NGF. These results point to the early involvement of the Arp2/3 complex and the Ena/VASP family in growth factor-stimulated actin polymerization that gives rise to protrusive structures at the growth cone.

Spatial control of actin polymerization during neutrophil chemotaxis.

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.

2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein, 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner ear to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation.

The maize actin-depolymerizing factor, ZmADF3, redistributes to the growing tip of elongating root hairs and can be induced to translocate into the nucleus with actin.

The maize actin depolymerizing factor, ZmADF3, binds G- and F-actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.

Involvement of p21racA, phosphoinositide 3-kinase, and vacuolar ATPase in phagocytosis of bacteria and erythrocytes by Entamoeba histolytica: suggestive evidence for coincidental evolution of amebic invasiveness.

Trophozoites of Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, phagocytose bacteria in the colonic lumen and erythrocytes (RBC) in host tissues. Because tissue invasion is an evolutionary dead end, it is likely that amebic pathogenicity is coincidentally selected, i.e., the same methods used to kill bacteria in the colonic lumen are used by parasites to damage host cells and cause disease. In support of this idea, the amebic lectin and pore-forming peptide are involved in binding and killing, respectively, bacteria and host epithelial cells. Here amebic phagocytosis of bacteria, RBC, and mucin-coated beads was disrupted by overexpression of E. histolytica p21(racA-V12), a ras-family protein involved in selection of sites of actin polymerization, which had been mutated to eliminate its GTPase activity. p21(racA-V12) transformants were also defective in capping and cytokinesis, while pinocytosis of fluorescent dextrans was not affected. Wortmannin, a fungal inhibitor of phosphoinositide 3-kinase, markedly inhibited phagocytosis of bacteria, RBC, and mucin-coated beads by wild-type amebae. In contrast to p21(racA-V12) overexpression, wortmannin abolished amebic pinocytosis of dextrans but had no inhibitory effects on capping. Inhibition of amebic vacuolar acidification by bafilomycin also decreased bacterial and RBC uptake. These results, which demonstrate similarities between mechanisms of phagocytosis of bacteria and RBC by amebae and macrophages, support the idea of coincidental selection of amebic genes encoding proteins that mediate destruction of host cells.

An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides.

We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two- to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.