CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions

In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca²⁺-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1α. Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca²⁺, but the interaction of CBP1 with EF-1α was increased substantially by Ca²⁺. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca²⁺-dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation.     

Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes.

A protein purified from cytoskeletal fractions of Dictyostelium discoideum proved to be a member of the fimbrin/plastin family of actin-bundling proteins. Like other family members, this Ca(2+)-inhibited 67-kDa protein contains two EF hands followed by two actin-binding sites of the alpha-actinin/beta-spectrin type. Dd plastin interacted selectively with actin isoforms: it bound to D. discoideum actin and to beta/gamma-actin from bovine spleen but not to alpha-actin from rabbit skeletal muscle. Immunofluorescence labeling of growth phase cells showed accumulation of Dd plastin in cortical structures associated with cell surface extensions. In the elongated, streaming cells of the early aggregation stage, Dd plastin was enriched in the front regions. To examine how the bundled actin filaments behave in myosin II-driven motility, complexes of F-actin and Dd plastin were bound to immobilized heavy meromyosin, and motility was started by photoactivating caged ATP. Actin filaments were immediately propelled out of bundles or even larger aggregates and moved on the myosin as separate filaments. This result shows that myosin can disperse an actin network when it acts as a motor and sheds light on the dynamics of protein-protein interactions in the cortex of a motile cell where myosin II and Dd plastin are simultaneously present.     

The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.     

Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.     

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.     

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.     

Binding of plasma membrane glycoproteins to the cytoskeleton during patching and capping is consistent with an entropy-enhancement model

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.     

Fluorescence-mediated visualization of actin and myosin filaments in the contractile membrane-cytoskeleton complex of Dictyostelium discoideum

An intact complex that consisted of the cell membrane and cytoskeleton was prepared from Dictyostelium amoebae by an improved version of the method previously used by CLARKE et al. (1975). Proc. Natl. Acad. Sci. USA., 72: 1758-1762. After cells had attached tightly to a polylysine-coated coverslip in the presence of a divalent cation, the upper portions of the cells were removed with a jet of microfilament-stabilizing solution squirted from a syringe. The cell membranes left on the coverslip were immediately stained with tetramethylrhodamine-conjugated phalloidin for staining of actin filaments, and with antibody against myosin from Dictyostelium and a fluorescein-conjugated second antibody for staining of myosin. Networks of actin filaments and numerous rod-like structures of myosin (myosin filaments) aligned along them were observed on the exposed cytoplasmic surfaces of the cell membranes. These networks were similar to those observed in the cortex of fixed whole cells. Addition of ATP to these intact complexes of cell membrane and cytoskeleton caused the aggregation of both actin and myosin into several dot-like structures of actin on the cell membrane. Similar dot-like structures were also seen in the cortex of fixed whole cells, and their changes in distribution correlated with the motile activity of the cells. Transmission electron microscopy showed that these dot-like structures were composed of an electron-dense structure at the center, from which numerous actin filaments radiated outwards. These observations suggest that these novel dot-like structures are organizing centers for cortical actin filaments and may possibly be related to the adhesion of cells to the substratum.     

Actin-cytoskeleton dynamics in non-monotonic cell spreading

The spreading of motile cells on a substrate surface is accompanied by reorganization of their actin network. We show that spreading in the highly motile cells of Dictyostelium is non-monotonic, and thus differs from the passage of spreading cells through a regular series of stages. Quantification of the gain and loss of contact area revealed fluctuating forces of protrusion and retraction that dominate the interaction of Dictyostelium cells with a substrate. The molecular basis of these fluctuations is elucidated by dual-fluorescence labeling of filamentous actin together with proteins that highlight specific activities in the actin system. Front-to-tail polarity is established by the sorting out of myosin-II from regions where dense actin assemblies are accumulating. Myosin-IB identifies protruding front regions, and the Arp2/3 complex localizes to lamellipodia protruded from the fronts. Coronin is used as a sensitive indicator of actin disassembly to visualize the delicate balance of polymerization and depolymerization in spreading cells. Short-lived actin patches that co-localize with clathrin suggest that membrane internalization occurs even when the substrate-attached cell surface expands. We conclude that non-monotonic cell spreading is characterized by spatiotemporal patterns formed by motor proteins together with regulatory proteins that either promote or terminate actin polymerization on the scale of seconds.Key words: actin cytoskeleton, Arp 2/3 complex, cell adhesion, cell spreading, Coronin, Dictyostelium, myosin, self-organization, clathrin     

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.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

盘基网柄菌(Dictyostelium discoideum)吞噬作用中肌动蛋白多聚化及其调节

肌动蛋白是盘基网柄菌(Dictyostelium discoideum)细胞吞噬过程中的关键组分,通过其细胞内的定位和多聚化形式在确定的时间和地点连接特定的分子,使吞噬过程得以完成。profilin是肌动蛋白多聚化的重要调节分子,在磷脂酰肌醇信号转导与细胞骨架相交处起关键作用。许多小分子G蛋白参与细胞骨架调节,CAP蛋白是两者间重要连接分子。所以,吞噬作用是细胞内诸分子协同作用的结果。     

F-actin assembly in Dictyostelium cell locomotion and shape oscillations propagates as a self-organized reaction-diffusion wave.

The crawling locomotion and shape of eukaryotic cells have been associated with the stochastic molecular dynamics of actin and its protein regulators, chiefly Arp2/3 and Rho family GTPases, in making a cytoskeleton meshwork within cell extensions. However, the cell's actin-dependent oscillatory shape and extension dynamics may also yield insights into locomotory mechanisms. Confocal observations of live Dictyostelium cells, expressing a green fluorescent protein-actin fusion protein, demonstrate oscillating supramolecular patterns of filamentous actin throughout the cell, which generate pseudopodia at the cell edge. The distinctively dissipative spatio-temporal behavior of these structures provides strong evidence that reversible actin filament assembly propagates as a self-organized, chemical reaction-diffusion wave.     

EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.     

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.     

Uncovering a role for the tail of the Dictyostelium discoideum SadA protein in cell-substrate adhesion

Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesion deficiency, and overexpression of the SadA tail domain reduced adhesion in wild-type cells. We also show that SadA is closely associated with the actin cytoskeleton. Mutagenesis studies suggested that four serine residues in the tail, S924/S925 and S940/S941, may regulate association of SadA with the actin cytoskeleton. Glutathione S-transferase pull-down assays identified at least one likely interaction partner of the SadA tail, cortexillin I, a known actin bundling protein. Thus, our data demonstrate an important role for the carboxy-terminal cytoplasmic tail in SadA function and strongly suggest that a phosphorylation event in this tail regulates an interaction with cortexillin I. Based on our data, we propose a model for the function of SadA.     

Orientation of cells during slug formation inDictyostelium

Summary Scanning electron microscopic observations ofDictyostelium discoideum cell masses during slug formation revealed two populations around the anterior tip; one group of cells resembled elongated aggregation stream cells and their orientation suggested that they move to the tip, whereas the other group of cells were isodiametric and showed no obvious orientation. In seeking further evidence for a role of differential cAMP chemotaxis in the orientation and movement of slug cells the anterior prestalk cells were compared to the posterior prespore cells in two chemotaxis tests. When a cell mass is placed on cAMP agar the prestalk cells exhibited better movement to cAMP sources but when the gradient was generated in a diffusion chamber the prestalk cells did not. This evidence suggested that the cells which are better able to generate a cAMP gradient might form part of the anterior zone of the slug.