Copyright (c) 2009 All rights reserved. http://works.bepress.com/lunae Recent documents in Elizabeth J. Luna en-us Sun, 31 May 2009 08:44:08 PDT 3600 http://works.bepress.com/lunae/36 http://works.bepress.com/lunae/36 Fri, 28 Mar 2008 15:50:18 PDT The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition. D. Vardar Actins Amino Acid Sequence Animals Carrier Proteins Chickens Chromatography, Gel Circular Dichroism Cytoskeleton Dose-Response Relationship, Drug Electrophoresis, Polyacrylamide Gel Kinetics Microfilament Proteins Models, Molecular Molecular Sequence Data Plasmids Protein Binding Protein Conformation Protein Folding Protein Structure, Tertiary Recombinant Proteins Sequence Homology, Amino Acid Software Temperature Ultraviolet Rays http://works.bepress.com/lunae/35 http://works.bepress.com/lunae/35 Fri, 28 Mar 2008 15:50:14 PDT 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. Shariff Actins Animals Carrier Proteins Cell Membrane Dictyostelium Electrophoresis, Polyacrylamide Gel Fungal Proteins Kinetics Macromolecular Substances Membrane Glycoproteins Microfilament Proteins Muscles Protein Binding Rabbits Spectrometry, Fluorescence http://works.bepress.com/lunae/34 http://works.bepress.com/lunae/34 Fri, 28 Mar 2008 15:50:10 PDT Activation of human platelets with thrombin transiently increases phosphorylation at (558)threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, approximately 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane. F. Nakamura Actins Amino Acid Sequence Biochemistry Blood Platelets Cytoskeleton Detergents Humans Microfilament Proteins Molecular Sequence Data Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphorylation Quaternary Ammonium Compounds Threonine http://works.bepress.com/lunae/33 http://works.bepress.com/lunae/33 Fri, 28 Mar 2008 15:50:08 PDT No abstract provided. Elizabeth J. Luna Actins Adenosine Triphosphate Animals Cell Line Indicators and Reagents Iodine Radioisotopes *Molecular Probe Techniques Phalloidine Protein Binding Proteins Succinimides http://works.bepress.com/lunae/32 http://works.bepress.com/lunae/32 Fri, 28 Mar 2008 15:50:04 PDT 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. D. C. Shutt Actins Animals Carrier Proteins Cell Movement Cell Size Chemotaxis Dictyostelium Microfilament Proteins Mutation Pseudopodia http://works.bepress.com/lunae/31 http://works.bepress.com/lunae/31 Fri, 28 Mar 2008 15:50:00 PDT Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations. S. J. Palmieri Actins Animals Cell Adhesion Cell Division Cell Membrane Cell Movement Cells, Cultured Chemotaxis Cytoskeleton Dictyostelium *Endocytosis Humans Mutagenesis, Site-Directed Neuropeptides Recombinant Fusion Proteins Sequence Homology Signal Transduction rac GTP-Binding Proteins rac1 GTP-Binding Protein http://works.bepress.com/lunae/30 http://works.bepress.com/lunae/30 Fri, 28 Mar 2008 15:49:57 PDT 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. Elizabeth J. Luna Actins Binding, Competitive Cell Membrane Chromatography, Affinity Cytoskeleton Dictyostelium Electrophoresis, Polyacrylamide Gel Membrane Proteins Microscopy, Electron Molecular Weight http://works.bepress.com/lunae/29 http://works.bepress.com/lunae/29 Fri, 28 Mar 2008 15:49:54 PDT Regions of plasma membrane involved in Dictyostelium discoideum intercellular adhesion resist solubilization with the nonionic detergent Triton X-100. Electron microscopy shows that these regions of the plasma membrane adhere to each other, forming many bi- and multilamellar structures. NaDodSO4/polyacrylamide gels of these regions contain major polypeptides at 225 kDa (residual myosin), 105 kDa, 88 kDa, 84 kDa, 47 kDa (residual actin), and 34 kDa. These membranes contain a subset of the total plasma membrane proteins, as analyzed by labeling of electrophoretically fractionated and blotted membrane proteins with radioiodinated Con A and by electrophoresis of membrane proteins from surface-labeled cells. Antibodies specific for gp80, a glycoprotein implicated in intercellular adhesion, intensely stain the 88-kDa and 84-kDa bands. Since these membrane regions resist Triton extraction, they appear to be stabilized by protein-protein interactions. Such stabilizing interactions may involve multivalent linkages with adjacent cells, or associations with intracellular actin and myosin, or both. Since these membranes appear to represent regions of intercellular contact, we call them "contact regions." H. M. Ingalls Animals Cell Adhesion *Cell Aggregation Cell Fractionation Cell Membrane Dictyostelium Membrane Proteins Microscopy, Electron Molecular Weight http://works.bepress.com/lunae/28 http://works.bepress.com/lunae/28 Fri, 28 Mar 2008 15:49:51 PDT 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. C. M. Goodloe-Holland Actins Adenosine Triphosphate Binding, Competitive Cell Membrane Cytoskeleton Dictyostelium Electrophoresis, Polyacrylamide Gel Kinetics Membrane Proteins Microscopy, Electron Myosin Subfragments Myosins Peptide Fragments http://works.bepress.com/lunae/27 http://works.bepress.com/lunae/27 Fri, 28 Mar 2008 15:49:46 PDT 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). A. L. Hitt Amino Acid Sequence Animals Base Sequence Carrier Proteins Cytoplasm Dictyostelium Fungal Proteins Genomic Library Glycosylphosphatidylinositols Membrane Glycoproteins Microfilament Proteins Molecular Sequence Data Polymerase Chain Reaction Protein Structure, Secondary