Eukaryotic cell locomotion depends on the propagation of self-organized reaction-diffusion waves and oscillations of actin filament assembly

Actin filament (F-actin) assembly kinetics determines the locomotion and shape of crawling eukaryotic cells, but the nature of these kinetics and their determining reactions are unclear. Live BHK21 fibroblasts, mouse melanoma cells, and Dictyostelium amoebae, locomoting on glass and expressing Green Fluorescent Protein-actin fusion proteins, were examined by confocal microscopy. The cells demonstrated three-dimensional bands of F-actin, which propagated throughout the cytoplasm at rates usually ranging between 2 and 5 microm/min in each cell type and produced lamellipodia or pseudopodia at the cell boundary. F-actin's dynamic behavior and supramolecular spatial patterns resembled in detail self-organized chemical waves in dissipative, physico-chemical systems. On this basis, the present observations provide the first evidence of self-organized, and probably autocatalytic, chemical reaction-diffusion waves of reversible actin filament assembly in vertebrate cells and a comprehensive record of wave and locomotory dynamics in vegetative-stage Dictyostelium cells. The intensity and frequency of F-actin wavefronts determine locomotory cell projections and the rotating oscillatory waves, which structure the cell surface. F-actin assembly waves thus provide a fundamental, deterministic, and nonlinear mechanism of cell locomotion and shape, which complements mechanisms based exclusively on stochastic molecular reaction kinetics.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

Regulation of actin cytoskeleton by Rap1 binding to RacGEF1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.     

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.     

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.     

Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion

Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.     

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.     

Developmental cell death in dictyostelium does not require paracaspase

Apoptotic cell death often requires caspases. Caspases are part of a family of related molecules including also paracaspases and metacaspases. Are molecules of this family generally involved in cell death? More specifically, do non-apoptotic caspase-independent types of cell death require paracaspases or metacaspases? Dictyostelium discoideum lends itself well to answering these questions because 1) it undergoes non-apoptotic developmental cell death of a vacuolar autophagic type and 2) it bears neither caspase nor metacaspase genes and apparently only one paracaspase gene. This only paracaspase gene can be inactivated by homologous recombination. Paracaspase-null clones were thus obtained in each of four distinct Dictyostelium strains. These clones were tested in two systems, developmental stalk cell death in vivo and vacuolar autophagic cell death in a monolayer system mimicking developmental cell death. Compared with parent cells, all of the paracaspase-null cells showed unaltered cell death in both test systems. In addition, paracaspase inactivation led to no alteration in development or interaction with a range of bacteria. Thus, in Dictyostelium, vacuolar programmed cell death in development and in a monolayer model in vitro would seem not to require paracaspase. To our knowledge, this is the first instance of developmental programmed cell death shown to be independent of any caspase, paracaspase or metacaspase. These results have implications as to the relationship in evolution between cell death and the caspase family.     

血清反应因子参与RhoA诱导的人血管内皮细胞肌动蛋白骨架重构

为进一步阐明RhoA调控人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)肌动蛋白骨架重构的分子机制,用逆转录病毒感染并筛选出稳定表达持续活化型RhoA(Q63LRhoA)和主导抑制型RhoA(T19NRhoA)的HUVECs。应用免疫组化和Western blot方法分析去血清前后HUVECs血清反应因子(serum response factor,SRF)的表达及定位,Rhodamine-Phalloidine染色观察F-actin动态变化。结果显示,Q63LRhoA组细胞核中SRF表达增加,F-actin重排形成大量应力纤维;T19NRhoA组中SRF表达较弱,F-actin无明显改变,无应力纤维形成。去血清后,正常HUVECs(对照组)和感染细胞中SRF的表达均显著增加,但其亚细胞定位明显不同。对照组去血清培养3d,SRF主要定位在细胞核,去血清培养5d,SRF出核转位入细胞浆。Q63LRhoA组SRF发生核滞留,不随去血清培养时间延长发生出核转位现象。T19NRhoA组SRF的表达主要定位于细胞核周。对照组去血清培养3d,F-actin表达增加,同时形成大量应力纤维,去血清培养5d,细胞F-actin表达下调,应力纤维解聚。Q63LRhoA组F-actin重构持续发生并形成大量应力纤维,但不随去血清培养时间延长发生明显解聚。而T19NRhoA组F-actin表达不随去血清时间延长而增加。上述结果提示,RhoA介导HUVECs F-actin的重构与SRF的核转位现象密切相关。     

Genetic approaches to molecular mechanisms of programmed cell death in Dictyostelium

Caspase-independent cell deaths have been observed in many species including the human. However, the molecular mechanisms which govern them are largely unknown. Our present work makes use of a model organism, the protist Dictyostelium discoideum, which displays a caspase-independent cell death during its development. In rich medium, Dictyostelium multiplies vegetatively as a unicellular organism, but in starvation conditions, Dictyostelium cells aggregate, differentiate and morphogenize into a multicellular structure, called sorocarp, containing a mass of spores supported by a stalk. Cells in the stalk are considered dead on the basis of non-regrowth in a rich medium and are vacuolized. This programmed cell death is therefore developmental and vacuolar, and in addition, caspase-independent since the Dictyostelium genome does not contain caspases genes. In order to study in detail this cell death without induction of development, an in vitro experimental protocol has been adopted, which enabled us to describe the cascade of morphological events during this cell death. An insertional mutagenesis approach, followed by appropriate selection or screening of mutants potentially resistant to death, attempted at establishing the cascade of molecular events leading to vacuolar death of Dictyostelium cells. A better understanding of alternative death pathways may allow to control different types of cell deaths in the cases of cancers or neurodegenerative diseases. In this short review, we will discuss briefly some generalities about the development of Dictyostelium in starvation conditions, and we will focus on the course of programmed cell death in Dictyostelium and on the genetic tools used to elucidate the corresponding molecular mechanisms.     

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.     

Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in Dictyostelium

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.     

A talin fragment as an actin trap visualizing actin flow in chemotaxis,endocytosis, and cytokinesis

A C-terminal 63-kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F-actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re-programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid-zone of a dividing cell.     

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle.

Vacuoles of Saccharomyces cerevisiae were visualized by phase-contrast microscopy. Visualization was enhanced by adding polyvinylpyrrolidone. Vacuolar segregation during the cell cycle was analysed in 42 individual cells of strain X2180 by time-lapse photomicrography. Within 15 min of bud emergence, more than 80% of the cells contained a vacuolar segregation structure in the form of either a tubule or an alignment of vesicles. The structure emerged from one point of the mother vacuole, then elongated and moved into the bud in a few minutes. The vacuolar segregation structure disappeared, usually within 20 min, before nuclear migration, leaving a separate vacuole in the bud. To test the generality of this observation several strains were grown in the presence of the vacuolar vital dye fluorescein isothiocyanate. The bud size was used to measure progress in the cell cycle. All strains formed vacuolar segregation structures in cells with small buds, although with variations in duration and timing in the cell cycle. In the presence of nocodazole vacuolar segregation occurred normally, thus, microtubules seem not to be essential in this process.     

Cytoskeletal alterations in Dictyostelium induced by expression of human cdc42

The rho family of small G proteins has been shown to be involved in controlling actin filament dynamics in cells. To evaluate the functional overlap between human and Dictyostelium G proteins, we conditionally expressed constitutively active human cdc42 (V12-cdc42) in Dictyostelium cells. Upon induction, cells adopted a unique morphology: a flattened shape with wrinkles running from the cell edge toward the center. The appearance of these wrinkles is highly dynamic so that the cells cycle between the wrinkled and relatively normal morphologies. Phalloidin staining indicates that the stellate wrinkles contain dense actin structures and also that numerous filopods project vertically from the center of these cells. Consistent with the hypothesis that cdc42 induces actin polymerization in vivo, cells expressing V12-cdc42 show an increase in the amount of F-actin associated with the cytoskeleton. This is accompanied by an increase in the association of the actin-binding proteins 34-kDa bundler, ABP-120 and alpha-actinin with the cytoskeleton. In conclusion, human cdc42 has various effects on the Dictyostelium actin cytoskeleton consistent with a conserved role of small GTPases in control of the cytoskeleton.     

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.