How an actin network might cause fountain streaming and nuclear migration in the syncytial Drosophila embryo [published erratum appears in J Cell Biol 1995 Sep;130(5):1231-4]

We show here using time-lapse video tapes that cytoplasmic streaming causes nuclear migration along the anterior-posterior axis (axial expansion) in the early syncytial embryo of Drosophila melanogaster. Using confocal microscopy and labeled phalloidin we explore the distribution of F-actin during axial expansion. We find that a network of F-actin fibers fills the cytoplasm in the embryo. This actin network partially disassembles around the nuclei during axial expansion. Our observations of normal development, fixed embryos, and drug injection experiments indicate that disassembly of the actin network generates cytoplasmic movements. We suggest that the cell cycle regulates disassembly of the actin network, and that this process may be mediated directly or indirectly by the microtubules. The cytoplasmic movements we observe during axial expansion are very similar to fountain streaming in the pseudopod of amoebae, and by analogy with the pseudopod we propose a working hypothesis for axial expansion based on solation-contraction coupling within the actin network.     

Colocalization of F-actin and 34-kilodalton actin bundling protein in Dictyostelium amoebae and cultured fibroblasts

The Ca2+-sensitive actin-binding protein isolated from Dictyostelium discoideum, 30,000-D protein (Fechheimer and Taylor: J. Biol. Chem. 259:4514-4520, 1984;) has recently been localized in filipodia of substrate-adhered amoebae (Fechheimer: J. Cell Biol. 104:1539-1551, 1987). We have determined that this protein has a Mr of 34,000 daltons and is strictly colocalized with actin filaments in both substrate-attached Dictyostelium amoebae and cultured fibroblasts. 3T3 fibroblasts, as well as normal and virally transformed rat kidney fibroblasts (NRK) contain a 34-kilodalton (kD) protein that cross-reacts specifically with antibody to the Dictyostelium bundling protein. Mammalian 34-kD protein is colocalized with F-actin in stress fibers and the cortical cytoskeleton in substrate-adhered fibroblasts. In substrate-adhered vegetative Dictyostelium, F-actin and 34-kD protein are concentrated in regions of the cell cortex exhibiting filipodia and membrane ridges. Multiple filipodia formed after exposure to the chemoattractant folic acid stain intensely for 34-kD protein, implying participation in the assembly of actin bundles during filipod formation. The cortex of pseudopodia also contained high concentrations of bundling protein, but pseudopod interiors did not. In contrast to vegetative Dictyostelium, F-actin and 34-kD protein were not colocalized in cells that had progressed through the developmental cycle. In fruiting bodies, 34-kD protein was detected by immunofluorescence microscopy only in prespore cells, while F-actin appeared in stalk cells and spores.     

The LRRK2-related Roco kinase Roco2 is regulated by Rab1A and controls the actin cytoskeleton

We identify a new pathway that is required for proper pseudopod formation. We show that Roco2, a leucine-rich repeat kinase 2 (LRRK2)-related Roco kinase, is activated in response to chemoattractant stimulation and helps mediate cell polarization and chemotaxis by regulating cortical F-actin polymerization and pseudopod extension in a pathway that requires Rab1A. We found that Roco2 binds the small GTPase Rab1A as well as the F-actin cross-linking protein filamin (actin-binding protein 120, abp120) in vivo. We show that active Rab1A (Rab1A-GTP) is required for and regulates Roco2 kinase activity in vivo and that filamin lies downstream from Roco2 and controls pseudopod extension during chemotaxis and random cell motility. Therefore our study uncovered a new signaling pathway that involves Rab1A and controls the actin cytoskeleton and pseudopod extension, and thereby, cell polarity and motility. These findings also may have implications in the regulation of other Roco kinases, including possibly LRRK2, in metazoans.     

Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion model

In this work we evaluate the cortical expansion model for amoeboid chemotaxis with regard to new information about molecular events in the cytoskeleton following chemotactic stimulation of Dictyostelium amoebae. A rapid upshift in the concentration of chemoattractant can be used to synchronize the motile behavior of a large population of cells. This synchrony presents an opportunity to study the biochemical basis of morphological changes such as pseudopod extension that are required for amoeboid chemotaxis. Changes in the composition and activity of the cytoskeleton following stimulation can be measured with precision and correlated with important morphological changes. Such studies demonstrate that activation of actin nucleation is one of the first and most crucial events in the actin cytoskeleton following stimulation. This activation is followed by incorporation of specific actin cross-linking proteins into the cytoskeleton, which are implicated in the extension of pseudopods and filopods. These results, as well as those from studies with mutants deficient in myosin, indicate that cortical expansion, driven by focal actin polymerization, cross-linking and gel osmotic swelling, is an important force for pseudopod extension. It is concluded that whereas three forces, frontal sliding, tail contraction, and cortical expansion may cooperate to produce amoeboid movement, the cortical expansion model offers the simplest explanation of how focal stimulation with a chemoattractant causes polarized pseudopod extension.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction

Wound contraction can substantially reduce the amount of new tissue needed to reestablish organ integrity after tissue loss. Fibroblasts, rich in F-actin bundles, generate the force of wound contraction. Fibronectin-containing microfibrils link fibroblasts to each other and to collagen bundles and thereby provide transduction cables across the wound for contraction. The temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction have not been determined. To establish these relationships, we used a cutaneous gaping wound model in outbred Yorkshire pigs. Granulation tissue filled approximately 80% of the wound space by day 5 after injury while wound contraction was first apparent at day 10. Neither actin bundles nor fibronectin receptors were observed in 5-d wound fibroblasts. Although fibronectin fibrils were assembled on the surfaces of 5-d fibroblasts, few fibrils coursed between cells. Day-7 fibroblasts stained strongly for nonmuscle-type F-actin bundles consistent with a contractile fibroblast phenotype. These cells expressed fibronectin receptors, were embedded in a fibronectin matrix that appeared to connect fibroblasts to the matrix and to each other, and were coaligned across the wound. Transmission EM confirmed the presence of microfilament bundles, cell-cell and cell-matrix linkages at day 7. Fibroblast coalignment, matrix interconnections, and actin bundles became more pronounced at days 10 and 14 coinciding with tissue contraction. These findings demonstrate that granulation tissue formation, F-actin bundle and fibronectin receptor expression in wound fibroblasts, and fibroblast-matrix linkage precede wound contraction.     

Cytoimmunofluorescent localization of severin in Dictyostelium amoebae

Severin is a 40-kDa Ca2+-activated protein from Dictyostelium that rapidly fragments and disassembles actin filaments in vitro (S.S. Brown, K. Yamamoto, and J.A. Spudich, J. Cell Biol. 93, 205-210, 1982; and K. Yamamoto, J.D. Pardee, J. Reidler, L. Stryer, and J.A. Spudich. J. Cell Biol. 95, 711-719, 1982). To determine if severin is colocalized with actin filaments in vivo, we have used the agar-overlay technique of S. Yumura, H. Mori, and Y. Fukui (J. Cell Biol. 99, 894-899, 1984) to examine the intracellular locations of severin and F-actin in vegetative Dictyostelium amoebae. In rounded cells taken from suspension culture severin colocalized with F-actin at cortical edges while maintaining an endoplasmic presence. Both severin and F-actin were present throughout nascent pseudopods of motile cells, while severin appeared concentrated at the leading edge of fully developed pseudopods. Amoebae feeding on a bacterial lawn formed large phagocytic vesicles that were surrounded by an extensive cell cortex rich in severin. Streaming cells entering aggregates during the Dictyostelium developmental cycle showed severin staining throughout the cytoplasm with F-actin at the cortex. The preferential localization of severin in cytoplasmic regions of vegetative cells undergoing extensive actin cytoskeletal rearrangement prompts consideration of a role for severin-mediated disruption of actin filament networks during pseudopod extension and phagocytosis.     

Experimental evidence for the limiting role of enzymatic reactions in chemoattractant-induced pseudopod extension in human neutrophils

Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.     

First Records and Microhabitat Assessment of Protostelids in the Aberdare Region, Central Kenya

ABSTRACT. A rapid assessment survey on the occurrence and distribution of protosteloid amoebae was carried out in central Kenya. Samples of dead plant materials were collected from 46 study sites (each 20 × 20 m) situated along an elevation gradient (1,785–3,396 m) that encompassed five major land use/cover types. Twenty-four species and subspecific taxa were recovered and included 23 protostelids and one minute myxomycete, often included in surveys for protostelids. All of these were the first records for Kenya, and six were new for Africa. Numbers of taxa were highest in ground litter and aerial litter microhabitats (20 taxa each) and lowest on aerial bark (10) and ground bark (7). Relative species abundance was greatest in aerial litter, moderate in ground litter, and low on aerial and ground bark microhabitats. The most frequently occurring species on ground litter were Schizoplasmodiopsis pseudoendospora, Schizoplasmodiopsis amoeboidea , and Protostelium mycophaga var. mycophaga , whereas the most common species on aerial litter were P. mycophaga var. mycophaga and Soliformovum irregularis . Species richness and abundance decreased with increasing elevation.     

Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.

Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.     

Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.     

Chemotaxis: signalling modules join hands at front and tail

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.     

Changes in the association of actin-binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictyostelium discoideum

Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.     

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.     

The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones

Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin. We report that these collapsing factors had different effects. ML-7 induced F-actin reorganization consistent with debundling whereas serotonin and TPA predominately depolymerized and possibly debundled F-actin. Additionally, these collapsing factors induced the formation of a dense actin-ring around the central domain, the thicker proximal area of growth cones [Zhou and Cohan, 2001: J. Cell Biol. 153:1071-1083]. The formation of the actin-ring occurred subsequent to the loss of actin bundles. The ML-7-induced actin-ring was found to inhibit microtubule extension into the P-domain. Thus, ML-7, serotonin, and TPA induce growth cone collapse associated with a decline in radially aligned F-actin bundles through at least two mechanisms involving debundling of actin filaments and/or actin depolymerization.     

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility.

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB-). DMIB- cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB- cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB- cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB- cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB- cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB- cells.     

WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.     

Actin dynamics in Amoeba proteus motility

Summary. We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid. Correspondence and reprints: Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, ulica Pasteura 3, 02-093 Warszawa, Poland.     

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.