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Full-Text Articles in Cell Biology
Dictyostelium Discoideum Plasma Membranes Contain An Actin-Nucleating Activity That Requires Ponticulin, An Integral Membrane Glycoprotein, A. Shariff, Elizabeth J. Luna
Dictyostelium Discoideum Plasma Membranes Contain An Actin-Nucleating Activity That Requires Ponticulin, An Integral Membrane Glycoprotein, A. Shariff, Elizabeth J. Luna
Elizabeth J. Luna
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 …
Ponticulin Plays A Role In The Positional Stabilization Of Pseudopods, D. C. Shutt, D. Wessels, K. Wagenknecht, A. Chandrasekhar, Anne L. Hitt, Elizabeth J. Luna, D. R. Soll
Ponticulin Plays A Role In The Positional Stabilization Of Pseudopods, D. C. Shutt, D. Wessels, K. Wagenknecht, A. Chandrasekhar, Anne L. Hitt, Elizabeth J. Luna, D. R. Soll
Elizabeth J. Luna
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 …
Mutant Rac1b Expression In Dictyostelium: Effects On Morphology, Growth, Endocytosis, Development, And The Actin Cytoskeleton, S. Palmieri, Thomas Nebl, Robert Pope, D. Seastone, E. Lee, E. Hinchcliffe, Greenfield Sluder, D. Knecht, J. Cardelli, Elizabeth Luna
Mutant Rac1b Expression In Dictyostelium: Effects On Morphology, Growth, Endocytosis, Development, And The Actin Cytoskeleton, S. Palmieri, Thomas Nebl, Robert Pope, D. Seastone, E. Lee, E. Hinchcliffe, Greenfield Sluder, D. Knecht, J. Cardelli, Elizabeth Luna
Elizabeth J. Luna
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 …
Supervillin (P205): A Novel Membrane-Associated, F-Actin-Binding Protein In The Villin/Gelsolin Superfamily, Kersi N. Pestonjamasp, Robert K. Pope, J. D. Wulfkuhle, Elizabeth J. Luna
Supervillin (P205): A Novel Membrane-Associated, F-Actin-Binding Protein In The Villin/Gelsolin Superfamily, Kersi N. Pestonjamasp, Robert K. Pope, J. D. Wulfkuhle, Elizabeth J. Luna
Elizabeth J. Luna
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 …
Merlin Differs From Moesin In Binding To F-Actin And In Its Intra- And Intermolecular Interactions, L. Huang, E. Ichimaru, Kersi Pestonjamasp, X. Cui, H. Nakamura, G. Lo, F. Lin, Elizabeth Luna, H. Furthmayr
Merlin Differs From Moesin In Binding To F-Actin And In Its Intra- And Intermolecular Interactions, L. Huang, E. Ichimaru, Kersi Pestonjamasp, X. Cui, H. Nakamura, G. Lo, F. Lin, Elizabeth Luna, H. Furthmayr
Elizabeth J. Luna
The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 …
A Stable, High Capacity, F-Actin Affinity Column, Elizabeth Luna, Y. Wang, E. Voss, D. Branton, D. Taylor
A Stable, High Capacity, F-Actin Affinity Column, Elizabeth Luna, Y. Wang, E. Voss, D. Branton, D. Taylor
Elizabeth J. Luna
A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds 125I-heavy meromyosin and 125I-tropomyosin. The associations between the F-actin affinity matrix and the iodinated F-actin binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.
Smooth Muscle Archvillin: A Novel Regulator Of Signaling And Contractility In Vascular Smooth Muscle, Samudra S. Gangopadhyay, Norio Takizawa, Cynthia Gallant, Amy L. Barber, Hyun-Dong Je, Tara C. Smith, Elizabeth J. Luna, Kathleen G. Morgan
Smooth Muscle Archvillin: A Novel Regulator Of Signaling And Contractility In Vascular Smooth Muscle, Samudra S. Gangopadhyay, Norio Takizawa, Cynthia Gallant, Amy L. Barber, Hyun-Dong Je, Tara C. Smith, Elizabeth J. Luna, Kathleen G. Morgan
Elizabeth J. Luna
The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear. Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2. We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein. The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells. In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after …
Supervillin Modulation Of Focal Adhesions Involving Trip6/Zrp-1, Norio Takizawa, Tara C. Smith, Thomas Nebl, Jessica Lynn Crowley, Stephen J. Palmieri, Lawrence M. Lifshitz, Anka G. Ehrhardt, Laura M. Hoffman, Mary C. Beckerle, Elizabeth J. Luna
Supervillin Modulation Of Focal Adhesions Involving Trip6/Zrp-1, Norio Takizawa, Tara C. Smith, Thomas Nebl, Jessica Lynn Crowley, Stephen J. Palmieri, Lawrence M. Lifshitz, Anka G. Ehrhardt, Laura M. Hoffman, Mary C. Beckerle, Elizabeth J. Luna
Elizabeth J. Luna
Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress …
F-Actin And Myosin Ii Binding Domains In Supervillin, Yu Chen, Norio Takizawa, Jessica Crowley, Sang Oh, Cheryl Gatto, Taketoshi Kambara, Osamu Sato, Xiang-Dong Li, Mitsuo Ikebe, Elizabeth Luna
F-Actin And Myosin Ii Binding Domains In Supervillin, Yu Chen, Norio Takizawa, Jessica Crowley, Sang Oh, Cheryl Gatto, Taketoshi Kambara, Osamu Sato, Xiang-Dong Li, Mitsuo Ikebe, Elizabeth Luna
Elizabeth J. Luna
Detergent-resistant membranes contain signaling and integral membrane proteins that organize cholesterol-rich domains called lipid rafts. A subset of these detergent-resistant membranes (DRM-H) exhibits a higher buoyant density ( approximately 1.16 g/ml) because of association with membrane skeleton proteins, including actin, myosin II, myosin 1G, fodrin, and an actin- and membrane-binding protein called supervillin (Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., and Luna, E. J. (2002) J. Biol. Chem. 277, 43399-43409). To characterize interactions among DRM-H cytoskeletal proteins, we investigated the binding partners of the novel supervillin N terminus, specifically amino acids 1-830. We …
Domain Analysis Of Supervillin, An F-Actin Bundling Plasma Membrane Protein With Functional Nuclear Localization Signals, J. D. Wulfkuhle, I. E. Donina, N. H. Stark, Robert K. Pope, Kersi N. Pestonjamasp, M. L. Niswonger, Elizabeth J. Luna
Domain Analysis Of Supervillin, An F-Actin Bundling Plasma Membrane Protein With Functional Nuclear Localization Signals, J. D. Wulfkuhle, I. E. Donina, N. H. Stark, Robert K. Pope, Kersi N. Pestonjamasp, M. L. Niswonger, Elizabeth J. Luna
Elizabeth J. Luna
A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino …
The Integral Membrane Protein, Ponticulin, Acts As A Monomer In Nucleating Actin Assembly, C. P. Chia, A. Shariff, S. A. Savage, Elizabeth J. Luna
The Integral Membrane Protein, Ponticulin, Acts As A Monomer In Nucleating Actin Assembly, C. P. Chia, A. Shariff, S. A. Savage, Elizabeth J. Luna
Elizabeth J. Luna
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. …
Archvillin, A Muscle-Specific Isoform Of Supervillin, Is An Early Expressed Component Of The Costameric Membrane Skeleton, Sang W. Oh, Robert K. Pope, Kelly P. Smith, Jessica Lynn Crowley, Thomas Nebl, Jeanne B. Lawrence, Elizabeth J. Luna
Archvillin, A Muscle-Specific Isoform Of Supervillin, Is An Early Expressed Component Of The Costameric Membrane Skeleton, Sang W. Oh, Robert K. Pope, Kelly P. Smith, Jessica Lynn Crowley, Thomas Nebl, Jeanne B. Lawrence, Elizabeth J. Luna
Elizabeth J. Luna
The membrane skeleton protein supervillin binds tightly to both F-actin and membranes and can potentiate androgen receptor activity in non-muscle cells. We report that muscle, which constitutes the principal tissue source for supervillin sequences, contains a approximately 250 kDa isoform of supervillin that localizes within nuclei and with dystrophin at costameres, regions of F-actin membrane attachment in skeletal muscle. The gene encoding this protein, 'archvillin' (Latin, archi; Greek, archos; 'principal' or 'chief'), contains an evolutionarily conserved, muscle-specific 5' leader sequence. Archvillin cDNAs also contain four exons that encode approximately 47 kDa of additional muscle-specific protein sequence in the form of …
Membrane Cytoskeleton: Pip(2) Pulls The Strings, Thomas Nebl, Sang Oh, Elizabeth Luna
Membrane Cytoskeleton: Pip(2) Pulls The Strings, Thomas Nebl, Sang Oh, Elizabeth Luna
Elizabeth J. Luna
A recent application of optical tweezers has shown that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels control adhesion of the membrane bilayer to the underlying cytoskeleton, by regulated direct binding of PIP(2) to cytoskeletal proteins and/or indirect effects on cytoskeleton structure.