Carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin

CEA cell adhesion molecule 1 (CEACAM1), a type 1 transmembrane and homotypic cell adhesion protein belonging to the carcinoembryonic antigen (CEA) gene family and expressed on epithelial cells, is alternatively spliced to produce four major isoforms with three or four Ig-like ectodomains and either long (CEACAM1-L) or short (CEACAM1-S) cytoplasmic domains. When murine MC38 (methylcholanthrene-induced adenocarcinoma 38) cells were transfected with human CEACAM1-L and stimulated with sodium pervanadate, actin was found to co-localize with CEACAM1-L at cell-cell boundaries but not in untreated cells. When CEACAM1-L was immunoprecipitated from pervanadate-treated MC38/CEACAM1-L cells and the associated proteins were analyzed by two-dimensional gel analysis and mass spectrometry, actin and tropomyosin, among other proteins, were identified. Whereas a glutathione S-transferase (GST) fusion protein containing the l-isoform (GST-Cyto-L) bound poorly to F-actin in a co-sedimentation assay, the S-isoform fusion protein (GST-Cyto-S) co-sedimented with F-actin, especially when incubated with G-actin during polymerization (K(D) = 7.0 microm). Both GST-Cyto-S and GST-Cyto-L fusion proteins bind G-actin and tropomyosin by surface plasmon resonance studies with binding constants of 0.7 x 10(-8) and 1.0 x 10(-7) m for GST-Cyto-L to G-actin and tropomyosin, respectively, and 3.1 x 10(-8) and 1.3 x 10(-7) m for GST-Cyto-S to G-actin and tropomyosin, respectively. Calmodulin or EDTA inhibited binding of the GST-Cyto-L fusion protein to G-actin, whereas calmodulin and G-actin, but not EDTA, stimulated binding to tropomyosin. A biotinylated 14-amino acid peptide derived from the juxtamembrane portion of the cytoplasmic domain of CEACAM1-L associated with both G-actin and tropomyosin with K(D) values of 1.3 x 10(-5) and 1.8 x 10(-5) m, respectively. These studies demonstrate the direct interaction of CEACAM1 isoforms with G-actin and tropomyosin and the direct interaction of CEACAM1-S with F-actin.     

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.     

Measurements of spatiotemporal changes in G-actin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension

To understand the intracellular role of G-actin concentration in stimulus-induced actin assembly and lamellipodium extension during cell migration, we developed a novel technique for quantifying spatiotemporal changes in G-actin concentration in live cells, consisting of sequential measurements of fluorescent decay after photoactivation (FDAP) of Dronpa-labeled actin. Cytoplasmic G-actin concentrations decreased by ～40% immediately after cell stimulation and thereafter the cell area extended. The extent of stimulus-induced G-actin loss and cell extension correlated linearly with G-actin concentration in unstimulated cells, even at concentrations much higher than the critical concentration of actin filaments, indicating that cytoplasmic G-actin concentration is a critical parameter for determining the extent of stimulus-induced G-actin assembly and cell extension. Multipoint FDAP analysis revealed that G-actin concentration in lamellipodia was comparable to that in the cell body. We also assessed the cellular concentrations of free G-actin, profilin- and thymosin-β4-bound G-actin, and free barbed and pointed ends of actin filaments by model fitting of jasplakinolide-induced temporal changes in G-actin concentration.     

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.     

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.     

In vitro effects of high glucose concentrations on membrane protein oxidation, G-actin and deformability of human erythrocytes

The object of this study was to examine the effect of elevated in vitro glucose concentrations on protein modification and functional changes in human erythrocytes. Groups were exposed to 5-45 mM glucose concentrations. The time effect of any changes was also evaluated. In erythrocyte ghosts, protein glycation and oxidation were evaluated using spectrophotometric methods. G-actin was measured by a DNase I inhibition assay in cell lysates. Erythrocyte deformability was assessed using a cell transit analyser. At 24 h, a significant protein oxidation (at 25 and 45 mM glucose; p < 0.05), and G-actin increase was observed for all concentrations (p < 0.05). At 48 h, a significant increase in glycation (25 and 45 mM glucose; p < 0.05), protein oxidation (p < 0.05), and G-actin (p < 0.05) was observed in all groups. A significant positive correlation was observed between glucose /protein oxidation, glucose/G-actin and protein oxidation/G-actin at 24 and 48 h. Our findings show that the oxidative effect of glucose on erythrocytes depends on concentration and incubation time. We also present the first evidence of increased G-actin in human erythrocytes exposed to high glucose concentrations as a diabetes model.     

Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to G-actin.     

Fluorescent staining of the actin cytoskeleton in human lymphocytes,monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system

Summary The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric G) remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.     

Group-specific component (vitamin D binding protein) prevents the interaction between G-actin and profilin

Profilin purified from human platelets formed a 1:1 molar ratio complex with rabbit skeletal muscle G-actin but was displaced by purified serum Gc (vitamin D binding protein) in a dose-dependent fashion as assessed by chromatography and ultrafiltration. This suggested that Gc and profilin competed for the same binding area on G-actin, with Gc-G-actin complexes being more stable than profilin-G-actin complexes in vitro. The binding domain for Gc on G-actin was localized to a 16,000-Da C-terminal fragment of G-actin generated by Staphylococcus aureus V8 protease, as judged by comigration on two-dimensional electrophoresis and also by overlaying electrophoresis gels with 125I-Gc. Previous studies have reported that residues 374 and 375 of G-actin are essential for binding of profilin. In this study, experiments involving tryptic removal of Cys-374 labeled with the fluorescent probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)-ethylenediamine showed that these C-terminal amino acids were not necessary for interaction with Gc.     

G-actin pool and actin messenger RNA during development of the apical processes of the retinal pigment epithelial cells of the chick.

It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15-E21) reaching lengths of 20 microns or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F-actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F-actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 +/- 0.5 micrograms/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 micrograms/ml (13 microM). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in beta actin mRNA and a four- to fivefold increase in message for gamma actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA.     

Regulation of cell structure and function by actin-binding proteins: villin's perspective

Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.     

Fluorescent staining of the actin cytoskeleton in human lymphocytes, monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system.

The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric "G") remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.     

Actin—membrane interactions: Association of G-actin with the red cell membrane

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G-actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G-actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside-out vesicles which have been stripped of endogenous actin and spectrin by low-ionic-strength incubation bind little G-actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G-actin can be increased up to 40-fold. Further, this crude spectrin extract can compete for and abolish G-actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G-actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G-actin is added to reconstituted inside-out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G-actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.     

Quantitative analysis of G-actin transport in motile cells

Cell migration is based on an actin treadmill, which in turn depends on recycling of G-actin across the cell, from the rear where F-actin disassembles, to the front, where F-actin polymerizes. To analyze the rates of the actin transport, we used the Virtual Cell software to solve the diffusion-drift-reaction equations for the G-actin concentration in a realistic three-dimensional geometry of the motile cell. Numerical solutions demonstrate that F-actin disassembly at the cell rear and assembly at the front, along with diffusion, establish a G-actin gradient that transports G-actin forward “globally” across the lamellipod. Alternatively, if the F-actin assembly and disassembly are distributed throughout the lamellipod, F-/G-actin turnover is local, and diffusion plays little role. Chemical reactions and/or convective flow of cytoplasm of plausible magnitude affect the transport very little. Spatial distribution of G-actin is smooth and not sensitive to F-actin density fluctuations. Finally, we conclude that the cell body volume slows characteristic diffusion-related relaxation time in motile cell from ∼10 to ∼100 s. We discuss biological implications of the local and global regimes of the G-actin transport.     

Stable interaction between G-actin and neurofilament light subunit in dopaminergic neurons

Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulated by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.     

Isolation of a domain of villin retaining calcium-dependent interaction with G-actin, but devoid of F-actin fragmenting activity

Villin is an F-actin binding protein located in the microfilament bundle of intestinal epithelial cell microvilli. Extensive in vitro proteolysis with Staphylococcus aureus V8 protease results in the production of a stable domain (apparent Mr 44000) which can be isolated due to its Ca2+-dependent interaction with G-actin bound to immobilized DNase-I, the standard procedure for the purification of villin. This 44-kDa fragment retains a single Ca2+ binding site with an apparent Kd = 2 X 10(-6) M, binds to G-actin, and inhibits the rate of actin polymerization. However, the 44-kDa domain does not shown any Ca2+-activated severing activity nor does it compete with villin for F-actin binding. These results suggest that villin contains three domains: headpiece containing an F-actin binding site, 44-kDa fragment containing a G-actin binding site, and an amino-terminal fragment responsible for the Ca2+-dependent severing activity.     

Thymosin beta 4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes

Thymosin beta 4 (T beta 4), a 5-kD peptide which binds G-actin and inhibits its polymerization (Safer, D., M. Elzinga, and V. T. Nachmias. 1991. J. Biol. Chem. 266:4029-4032), appears to be the major G-actin sequestering protein in human PMNs. In support of a previous study by Hannappel, E., and M. Van Kampen (1987. J. Chromatography. 397:279-285), we find that T beta 4 is an abundant peptide in these cells. By reverse phase HPLC of perchloric acid supernatants, human PMNs contain approximately 169 fg/cell +/- 90 fg/cell (SD), corresponding to a cytoplasmic concentration of approximately 149 +/- 80.5 microM. On non-denaturing polyacrylamide gels, a large fraction of G-actin in supernatants prepared from resting PMNs has a mobility similar to the G-actin/T beta 4 complex. Chemoattractant stimulation of PMNs results in a decrease in this G-actin/T beta 4 complex. To determine whether chemoattractant induced actin polymerization results from an inactivation of T beta 4, the G-actin sequestering activity of supernatants prepared from resting and chemoattractant stimulated cells was measured by comparing the rates of pyrenyl-actin polymerization from filament pointed ends. Pyrenyl actin polymerization was inhibited to a greater extent in supernatants from stimulated cells and these results are qualitatively consistent with T beta 4 being released as G-actin polymerizes, with no chemoattractant-induced change in its affinity for G-actin. The kinetics of bovine spleen T beta 4 binding to muscle pyrenyl G-actin are sufficiently rapid to accommodate the rapid changes in actin polymerization and depolymerization observed in vivo in response to chemoattractant addition and removal.     

Mechanism of actin polymerization in cellular ATP depletion

Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.     

Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells

Addition of actin monomer (G-actin) to growing actin filaments (F-actin) at the leading edge generates force for cell locomotion. The polymerization reaction and its regulation have been studied in depth. However, the mechanism responsible for transport of G-actin substrate to the cell front is largely unknown; random diffusion, facilitated transport via myosin II contraction, local synthesis as a result of messenger ribonucleic acid localization, or F-actin turnover all might contribute. By tracking a photoactivatable, nonpolymerizable actin mutant, we show vectorial transport of G-actin in live migrating endothelial cells (ECs). Mass spectrometric analysis identified Myo1c, an unconventional F-actin-binding motor protein, as a major G-actin-interacting protein. The cargo-binding tail domain of Myo1c interacted with G-actin, and the motor domain was required for the transport. Local microinjection of Myo1c promoted G-actin accumulation and plasma membrane ruffling, and Myo1c knockdown confirmed its contribution to G-actin delivery to the leading edge and for cell motility. In addition, there is no obvious requirement for myosin II contractile-based transport of G-actin in ECs. Thus, Myo1c-facilitated G-actin transport might be a critical node for control of cell polarity and motility.     

Profilin isoforms in Dictyostelium discoideum

Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.