Tropical tree foliage supplementation in ruminants improves rumen fermentation and the bacterial profile and decreases methane production.

The main of this study was to evaluate the effect of supplementation of tropical tree foliage in ruminant diets on the in vitro fermentation, bacterial population, volatile fatty acids (VFAs), and enteric CH4 production. Seven experimental diets were evaluated: a control treatment of Pennisetum purpureum (T7) and six treatments of P. purpureum supplemented (30%) with the foliage of Neomillspaughia emargiata (T1), Tabernaemontana amygdalifolia (T2), Caesalpinia gaumeri (T3), Piscidia piscipula (T4), Leucaena leucocephala (T5), and Havardia albicans (T6). The T2, T7, and T5 treatments had the highest (p < 0.05) digestibility of dry matter. Overall, supplementation increased (p < 0.05) the concentrations of propionic and butyric acid and decreased acetic acid. Methanogenic bacteria decreased (p < 0.05) in T1, T2, T5, and T6. Ruminococcus albus decreased in T1, T2, T3, and T5 and Selenomonas ruminiantum increased in T3. Fibrobacter succinogenes increased, except in T5. Methane production decreased (p < 0.05) in T1, T4, T5, and T6. The supplementation with Leucaena leucocephala, Tabernaemontana amygdalifolia, Neomillspaughia emargiata, Piscidia piscipula, Havardia albicans, and Caesalpinia gaumeri is a potential alternative nutritional strategy for ruminants that results in positive changes in VFAs profile, a decrease on CH4 production and methanogenic bacteria, and changes on fibrolytic and non-fibrolytic bacteria composition.HIGHLIGHTSTropical tree foliage supplementation increased propionic and butyric acid and decreased acetic acid concentrations.Fibrolytic, non-fibrolytic, and Methanogenic bacteria were selectively modulated with the supplementation of tropical tree foliage.The enteric methane (CH4) production decreased with the supplementation of tree foliage.The supplementation of Tabernaemontana amygdalifolia and Leucaena leucocephala had the highest digestibility and is a potential alternative nutritional strategy for ruminants.

Pre-transplant shedding of BK virus in urine is unrelated to post-transplant viruria and viremia in kidney transplant recipients.

BK virus-(BKV) associated nephropathy (BKVN) is a major cause of allograft injury in kidney transplant recipients. In such patients, subclinical reactivation of latent BKV infection can occur in the pre-transplant period. The purpose of this study was to determine whether urinary BKV shedding in the immediate pre-transplant period is associated with a higher incidence of viruria and viremia during the first year after kidney transplantation. We examined urine samples from 34 kidney transplant recipients, using real-time quantitative polymerase chain reaction to detect BKV. Urine samples were obtained in the immediate pre-transplant period and during the first year after transplant on a monthly basis. If BKV viruria was detected, blood samples were collected and screened for BKV viremia. In the immediate pre-transplant period, we detected BKV viruria in 11 (32.3%) of the 34 recipients. During the first year after transplantation, we detected BKV viruria in all 34 patients and viremia in eight (23.5%). We found no correlation between pre-transplant viruria and post-transplant viruria or viremia (p = 0.2). Although reactivation of latent BKV infection in the pre-transplant period is fairly common among kidney transplant recipients, it is not a risk factor for post-transplant BKV viruria or viremia.

Evaluation of the sensitivity of IgG and IgM ELISA in detecting Schistosoma mansoni infections in a low endemicity setting.

Schistosomiasis is a major public health concern, with 200 million people infected worldwide. In Brazil, this disease has been reported in 19 states, and its prevalence in the city of Barra Mansa in Rio de Janeiro State is 1 %. The parasitological diagnostic methods currently available in these areas lack sensitivity; however, enzyme-linked immunosorbent assays (ELISAs) have been employed successfully for the diagnosis of schistosomiasis by using antibodies against antigens of Schistosoma mansoni adult worms and eggs, and for the detection of circulating antigens. The objective of this study was to determine systematically the prevalence of S. mansoni infection in the peripheral areas of Barra Mansa. A cross-sectional study was conducted from April to December 2011 by using probabilistic sampling that collected 610 fecal samples and 612 serum samples. ELISA-IgG with total extracts and ELISA-IgM with trichloroacetic acid-soluble fractions were employed to detect antibodies against S. mansoni and were compared with the Kato-Katz and Hoffman parasitological techniques. Among the individuals studied, anti-S. mansoni antibodies were detected in 11.16 % (n = 71) by ELISA-IgG and in 20.75 % (n = 132) by ELISA-IgM, while the parasitological techniques showed 0.82 % (n = 5) positivity. The agreement between the two ELISA tests was 85.38 % (n = 543), and 8.65 % (n = 55) of the serum samples showed positive results in both tests. The higher positivity of the ELISA-IgM test corroborates the results of previous reports and indicates that the test may be a useful tool in epidemiological studies, particularly in areas of low endemicity for S. mansoni.

[Genetic factors associated with long COVID]. / Factores genéticos asociados a long COVID.

INTRODUCTION: The variability in expression and evolution of COVID is not completely explained by clinical factors. In fact, genetic factors play an important role. Moreover, it is unknown whether the genetic factor that contribute to susceptibility and severity are also involved in the onset and evolution of long-COVID. The objective of this review is to gather information from literature to understand which genetic factors are involved in the onset of persistent COVID. MATERIAL AND METHODS: Systematic review in PubMed and bioRxiv and medRxiv repositories based on MeSH-descriptors and MeSH-terms related to COVID and genetic factors. Using these terms 2715 articles were pooled. An initial screening performed by authors independently, selected 205 articles of interest. A final deeper screening a total of 85 articles were chosen for complete reading and summarized in this review. RESULTS: Although ACE2 and TMPSS6 are involved in COVID susceptibility, their involvement in long-COVID has not been found. On the other hand, the severity of the disease and the onset of long-COVID has been associated with different genes involved in the inflammatory and immune response. Particularly interesting has been the association found with the FOXP4 locus. CONCLUSIONS: Although studies on long-COVID are insufficient to fully comprehend the cause, it is clear that the current identified genetic factors do not fully explain the progression and onset of long-COVID. Other factors such as polygenic action, pleiotropic genes, the microbiota and epigenetic changes must be considered and studied.

Molecular links between the cytoskeleton and membranes.

This review covers recent advances in non-erythroid spectrin re-distributions during development, structural motifs recently discovered in ankyrin, band 4.2, band 4.1, ezrin, talin, and myosin I, and our present understanding of actin-membrane interactions at focal adhesions and in liver, platelet, and Dictyostelium discoideum plasma membranes.

Membrane interactions with the actin cytoskeleton.

Recent advances have been made in our understanding of the direct binding of actin to integral membrane proteins. New information has been obtained about indirect actin-membrane associations through spectrin superfamily members and through proteins at the cytoplasmic surfaces of focal contacts and adherens junctions.

Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

A membrane cytoskeleton from Dictyostelium discoideum. III. Plasma membrane fragments bind predominantly to the sides of actin filaments.

The binding between sonicated Dictyostelium discoideum plasma membrane fragments and F-actin on Sephacryl S-1000 beads was found to be competitively inhibited by myosin subfragment-1. This inhibition is MgATP-sensitive, exhibits a Ki of approximately 5 X 10(-8) M, and is reciprocal, since membranes inhibit the binding of 125I-heavy meromyosin to F-actin on beads. These experiments demonstrate that membrane binding and S-1 binding to F-actin on beads are mutually exclusive and, therefore, that the membrane fragments bind predominantly to the sides, rather than to the ends, of the actin filaments. This conclusion is supported by electron micrographs that show many lateral associations between membrane fragments and bead-associated actin filaments. Such lateral associations could play an important role in the organization and lateral movement of membrane proteins by the cytomusculature.

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum.

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross-linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell.

F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.

Binding and assembly of actin filaments by plasma membranes from Dictyostelium discoideum.

The binding of native, 125I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 micrograms/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 micrograms/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. In kinetic experiments, actin added as monomers bound to membranes at a rate of 0.6 microgram ml-1 min-1, while pre-polymerized actin bound at a rate of 3.0 micrograms ml-1 min-1. Even in the absence of phalloidin, actin bound to membranes at concentrations well below the normal critical concentration. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. We conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins.

A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads.

In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluorescein-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads). Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at approximately 0.6 microgram of membrane protein per microgram of bead-bound F-actin, (c) rapid with a t1/2 of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads. This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E.J., V. M. Fowler, J. Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88:396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes. Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions. Using F-actin beads as an affinity column for actin-binding proteins, we show that at least 12 integral polypeptides in D. discoideum plasma membranes bind to F-actin directly or indirectly. At least four of these polypeptides appear to span the membrane and are thus candidates for direct transmembrane links between the cytoskeleton and the cell surface.

Ponticulin is an atypical membrane protein.

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium.

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm.

A biochemical assay employing DNase-I affinity chromatography, two-dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two-dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity.

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

Ponticulin plays a role in the positional stabilization of pseudopods.

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.