183 results on '"Heuser, John E."'
Search Results
152. High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits
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Goodenough, Ursula W., primary, Gebhart, Brian, additional, Mermall, Valerie, additional, Mitchell, David R., additional, and Heuser, John E., additional
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- 1987
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153. Arrest of pigment granule motion in erythrophores by quick-freezing
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Wallace, Ip, primary, Murphy, Douglas B., additional, and Heuser, John E., additional
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- 1984
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154. Procedure for freeze-drying molecules adsorbed to mica flakes
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Heuser, John E., primary
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- 1983
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155. REVIEW OF ELECTRON MICROSCOPIC EVIDENCE FAVOURING VESICLE EXOCYTOSIS AS THE STRUCTURAL BASIS FOR QUANTAL RELEASE DURING SYNAPTIC TRANSMISSION
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Heuser, John E., primary
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- 1989
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156. The origins and evolution of freeze-etch electron microscopy.
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Heuser, John E.
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FREEZE-etching , *ELECTRON microscope techniques , *CELL membranes , *BILAYER lipid membranes , *FREEZE fracturing , *TISSUE fixation (Histology) , *ULTRASTRUCTURE (Biology) , *FREEZE-drying - Abstract
The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future. [ABSTRACT FROM AUTHOR]
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- 2011
157. Membrane?cytoskeletal dynamics in a new dimension.
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Heuser, John E. and Donaldson, Julie G.
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CYTOSKELETON , *CELL membranes , *MICROTUBULES - Abstract
The recent Airlie House meeting on 'Cytoplasmic Organization and Membrane Traffic' (22-25 March 2001), sponsored by the Keith Porter Endowment, proved not to be the typical exchange of advances among specialists familiar with each other's work, but rather a series of interesting and diverse presentations that together illuminated the pace and pattern of membrane and cytoskeletal interactions in living cells. [ABSTRACT FROM AUTHOR]
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- 2001
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158. The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.
- Author
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Honda, Makoto, Minami, Itsunari, Tooi, Norie, Morone, Nobuhiro, Nishioka, Hisae, Uemura, Kengo, Kinoshita, Ayae, Heuser, John E., Nakatsuji, Norio, and Aiba, Kazuhiro
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GENETIC overexpression , *ALZHEIMER'S disease , *PRESENILINS , *GENETIC mutation , *HUMAN embryonic stem cells , *ELECTROPHYSIOLOGY - Abstract
Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery. [ABSTRACT FROM AUTHOR]
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- 2016
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159. Role of Disulfide Bonds in Acrp30/Adiponectin Structure and Signaling Specificity.
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Tsu-Shuen Tsao, Tomas, Eva, Murrey, Heather E., Hug, Christopher, Lee, David H., Ruderman, Neil B., Heuser, John E., and Lodish, Harvey F.
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SULFIDES , *CHEMICAL bonds , *BLOOD proteins , *LIPIDS , *GLUCOSE , *METABOLISM - Abstract
Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and-stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys[sup 22]-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys[sup 22] with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-κB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-α at Thr[sup 172] and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state. [ABSTRACT FROM AUTHOR]
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- 2003
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160. Endocytosis of Oxidized Low Density Lipoprotein through Scavenger Receptor CD36 Utilizes a Lipid Raft Pathway That Does Not Require Caveolin-1.
- Author
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Youchun Zeng, Nengbing Tao, K. Ravi, Koong-Nah Chung, Heuser, John E., and Lublin, Douglas M.
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ENDOCYTOSIS , *LIGANDS (Biochemistry) , *LOW density lipoproteins - Abstract
The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of OxLDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the giycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways. [ABSTRACT FROM AUTHOR]
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- 2003
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161. Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function.
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Ungar, Daniel, Oka, Toshihiko, Brittle, Elizabeth E., Vasile, Eliza, Lupashin, Vladimir V., Chatterton, Jon E., Heuser, John E., Krieger, Monty, and Waters, M. Gerard
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GOLGI apparatus , *CYTOSOL - Abstract
Characterizes the mammalian golgi-localized protein complex. Composition of conserved oligomeric golgi complex; Determinants of golgi apparatus structure; Identification of the COG5-containing protein in bovine brain cytosol.
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- 2002
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162. Structure of Arp2/3 Complex in Its Activated State and in Actin Filament Branch Junctions.
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Volkmann, Niels, Amann, Kurt J., Stoilova-McPhie, Svetla, Egile, Coumaran, Winter, Dirk C., Hazelwood, Larnele, Heuser, John E., Li, Rong, Pollard, Thomas D., and Hanein, Dorit
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ACTIN , *CRYOMICROSCOPY , *CHEMICAL structure - Abstract
Examines the structure of Arp2/3 complex in activated state and actin filament branch junctions. Use of electron cryomicroscopy and single-particle analysis to obtain the structure of WA-bound Arp2/3 complex; Details on the formation of actin filament branches; Calculation of three-dimensional reconstructions.
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- 2001
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163. Epsin 1 is a Polyubiquitin-Selective Clathrin-Associated Sorting Protein.
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Hawryluk, Matthew J., Keyel, Peter A., Mishra, Sanjay K., Watkins, Simon C., Heuser, John E., and Traub, Linton M.
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UBIQUITIN - Abstract
A correction to the article published by the authors in the March issue of this periodical which cited a paper about monoubiquitinated cargo molecules is presented.
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- 2006
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164. Surface structure of the COPII-coated vesicle.
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Matsuoka, Ken, Schekman, Randy, Orci, Lelio, and Heuser, John E.
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COATED vesicles , *ORGANELLES , *ELECTRON microscopy , *CYTOLOGY , *PROTEINS , *PHYSIOLOGY - Abstract
Studies the surface structure of the COPII-coated vesicle. Analysis pertaining to the spatial arrangement of COPII; Use of electron microscopy to explore the COPII subunit structure.
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- 2001
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165. The Structural Basis of Long-Term Potentiation in Hippocampal Synapses, Revealed by Electron Microscopy Imaging of Lanthanum-Induced Synaptic Vesicle Recycling.
- Author
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Heuser JE
- Abstract
Hippocampal neurons in dissociated cell cultures were exposed to the trivalent cation lanthanum for short periods (15-30 min) and prepared for electron microscopy (EM), to evaluate the stimulatory effects of this cation on synaptic ultrastructure. Not only were characteristic ultrastructural changes of exaggerated synaptic vesicle turnover seen within the presynapses of these cultures-including synaptic vesicle depletion and proliferation of vesicle-recycling structures-but the overall architecture of a large proportion of the synapses in the cultures was dramatically altered, due to large postsynaptic "bulges" or herniations into the presynapses. Moreover, in most cases, these postsynaptic herniations or protrusions produced by lanthanum were seen by EM to distort or break or "perforate" the so-called postsynaptic densities (PSDs) that harbor receptors and recognition molecules essential for synaptic function. These dramatic EM observations lead us to postulate that such PSD breakages or "perforations" could very possibly create essential substrates or "tags" for synaptic growth, simply by creating fragmented free edges around the PSDs, into which new receptors and recognition molecules could be recruited more easily, and thus, they could represent the physical substrate for the important synaptic growth process known as "long-term potentiation" (LTP). All of this was created simply in hippocampal dissociated cell cultures, and simply by pushing synaptic vesicle recycling way beyond its normal limits with the trivalent cation lanthanum, but we argued in this report that such fundamental changes in synaptic architecture-given that they can occur at all-could also occur at the extremes of normal neuronal activity, which are presumed to lead to learning and memory., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Heuser.)
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- 2022
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166. Introducing a mammalian nerve-muscle preparation ideal for physiology and microscopy, the transverse auricular muscle in the ear of the mouse.
- Author
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Heuser JE and Tenkova TI
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- Animals, Mice, Muscle Proteins, Muscle, Skeletal, Neuromuscular Junction, Membrane Proteins, Microscopy
- Abstract
A new mammalian neuromuscular preparation is introduced for physiology and microscopy of all sorts: the intrinsic muscle of the mouse ear. The great utility of this preparation is demonstrated by illustrating how it has permitted us to develop a wholly new technique for staining muscle T-tubules, the critical conductive-elements in muscle. This involves sequential immersion in dilute solutions of osmium and ferrocyanide, then tannic acid, and then uranyl acetate, all of which totally blackens the T-tubules but leaves the muscle pale, thereby revealing that the T-tubules in mouse ear-muscles become severely distorted in several pathological conditions. These include certain mouse-models of muscular dystrophy (specifically, dysferlin-mutations), certain mutations of muscle cytoskeletal proteins (specifically, beta-tubulin mutations), and also in denervation-fibrillation, as observed in mouse ears maintained with in vitro tissue-culture conditions. These observations permit us to generate the hypothesis that T-tubules are the "Achilles' heel" in several adult-onset muscular dystrophies, due to their unique susceptibility to damage via muscle lattice-dislocations. These new observations strongly encourage further in-depth studies of ear-muscle architecture, in the many available mouse-models of various devastating human muscle-diseases. Finally, we demonstrate that the delicate and defined physical characteristics of this 'new' mammalian muscle are ideal for ultrastructural study, and thereby facilitate the imaging of synaptic vesicle membrane recycling in mammalian neuromuscular junctions, a topic that is critical to myasthenia gravis and related diseases, but which has, until now, completely eluded electron microscopic analysis. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries., (Copyright © 2019. Published by Elsevier Ltd.)
- Published
- 2020
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167. Hybrid Cellular Metabolism Coordinated by Zic3 and Esrrb Synergistically Enhances Induction of Naive Pluripotency.
- Author
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Sone M, Morone N, Nakamura T, Tanaka A, Okita K, Woltjen K, Nakagawa M, Heuser JE, Yamada Y, Yamanaka S, and Yamamoto T
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- Animals, Cell Line, Cells, Cultured, Fibroblasts cytology, Fibroblasts metabolism, Homeodomain Proteins genetics, Humans, Induced Pluripotent Stem Cells metabolism, Kruppel-Like Factor 4, Mice, Inbred C57BL, Mice, Inbred ICR, Receptors, Estrogen genetics, Transcription Factors genetics, Up-Regulation, Cellular Reprogramming, Glycolysis, Homeodomain Proteins metabolism, Induced Pluripotent Stem Cells cytology, Oxidative Phosphorylation, Receptors, Estrogen metabolism, Transcription Factors metabolism
- Abstract
Naive pluripotent stem cells (PSCs) utilize both glycolysis and oxidative phosphorylation (OXPHOS) to satisfy their metabolic demands. However, it is unclear how somatic cells acquire this hybrid energy metabolism during reprogramming toward naive pluripotency. Here, we show that when transduced with Oct4, Sox2, and Klf4 (OSK) into murine fibroblasts, Zic3 and Esrrb synergistically enhance the reprogramming efficiency by regulating cellular metabolic pathways. These two transcription factors (TFs) cooperatively activate glycolytic metabolism independently of hypoxia inducible factors (HIFs). In contrast, the regulatory modes of the TFs on OXPHOS are antagonistic: Zic3 represses OXPHOS, whereas Esrrb activates it. Therefore, when introduced with Zic3, Esrrb restores OXPHOS activity, which is essential for efficient reprogramming. In addition, Esrrb-mediated OXPHOS activation is critical for the conversion of primed PSCs into the naive state. Our study suggests that the combinatorial function of TFs achieves an appropriate balance of metabolic pathways to induce naive PSCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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168. Polymer-coated pH-responsive high-density lipoproteins.
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Kim H, Okamoto H, Felber AE, Polomska A, Morone N, Heuser JE, Leroux JC, and Murakami T
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- Amino Acid Sequence, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents pharmacology, Cell Line, Tumor, Curcumin administration & dosage, Curcumin pharmacokinetics, Curcumin pharmacology, Doxorubicin administration & dosage, Doxorubicin pharmacokinetics, Doxorubicin pharmacology, Endosomes metabolism, Humans, Hydrogen-Ion Concentration, Neoplasms drug therapy, Neoplasms metabolism, Polyethylene Glycols chemistry, Recombinant Fusion Proteins chemistry, Antineoplastic Agents administration & dosage, Delayed-Action Preparations chemistry, Fatty Acids, Monounsaturated chemistry, Lipoproteins, HDL chemistry, Polymethacrylic Acids chemistry, Quaternary Ammonium Compounds chemistry
- Abstract
Intracellular drug delivery by nanoparticles is often hampered by their endosomal entrapment followed by their degradation in the lysosomal compartment and/or exocytosis. Here, we show that internalization and endosomal escape of cargoes in a cationized natural nanocarrier, high-density lipoprotein (HDL), can be controlled in a pH-dependent manner through stable complexation with a membranolytic anionic block polymer. A genetically and chemically cationized form of HDL (catHDL) is prepared for the first time by both genetic fusion with YGRKKRRQRRR peptide and incorporation of 1,2-dioleoyloxy-3-(trimethylammonium)propane. Upon addition of poly(ethylene glycol)-block-poly(propyl methacrylate-co-methacrylic acid) (PA), catHDL yields inhibition of internalization at neutral pH and its subsequent recovery at mildly acidic pH. catHDL forms a stable discoidal-shape complex with PA (catHDL/PA) (ca. 50 nm in diameter), even in the presence of serum. Significant enhancement of endosomal escape of a catHDL component is observed after a 1-h treatment of human cancer cells with catHDL/PA. Doxorubicin and curcumin, fluorescent anti-cancer drugs, encapsulated into catHDL/PA are also translocated outside of endosomes, compared with that into catHDL, and their cytotoxicities are enhanced inside the cells. These data suggest that catHDL/PA may have a potential benefit to improve the cellular delivery and endosomal escape of therapeutics under mildly acidic conditions such as in tumor tissues., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
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169. Allosteric activation of ADAMTS13 by von Willebrand factor.
- Author
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Muia J, Zhu J, Gupta G, Haberichter SL, Friedman KD, Feys HB, Deforche L, Vanhoorelbeke K, Westfield LA, Roth R, Tolia NH, Heuser JE, and Sadler JE
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- ADAM Proteins blood, ADAM Proteins genetics, ADAMTS13 Protein, Allosteric Regulation physiology, Enzyme Activation physiology, Humans, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, von Willebrand Factor genetics, von Willebrand Factor metabolism, ADAM Proteins chemistry, von Willebrand Factor chemistry
- Abstract
The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.
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- 2014
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170. Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E.
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Vitre B, Gudimchuk N, Borda R, Kim Y, Heuser JE, Cleveland DW, and Grishchuk EL
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- Anaphase, Animals, Cattle, Cell Line, Chromosomal Proteins, Non-Histone chemistry, Chromosomes, Human metabolism, Humans, Protein Binding, Protein Structure, Tertiary, Xenopus laevis, Chromosomal Proteins, Non-Histone physiology, Kinetochores metabolism, Metaphase, Microtubules metabolism
- Abstract
Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore-microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E-dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of "Bonsai" CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore-microtubule attachments during chromosome congression and segregation., (© 2014 Vitre, Gudimchuk, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2014
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171. Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets.
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Kadota S, Minami I, Morone N, Heuser JE, Agladze K, and Nakatsuji N
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- Anti-Arrhythmia Agents pharmacology, Cell Culture Techniques methods, Cell Differentiation, Desmosomes ultrastructure, Electric Stimulation, Humans, Membrane Potentials drug effects, Myocardial Contraction drug effects, Sarcomeres ultrastructure, Sodium Channel Blockers pharmacology, Voltage-Sensitive Dye Imaging methods, Arrhythmias, Cardiac pathology, Models, Cardiovascular, Myocytes, Cardiac pathology, Pluripotent Stem Cells pathology, Tissue Engineering methods
- Abstract
Aims: Development of a human cell-derived reentrant arrhythmia model is needed for studying the mechanisms of disease and accurate drug response., Methods and Results: We differentiated human pluripotent stem cells (hPSCs) into cardiomyocytes, and then re-plated them into cell sheets that proved capable of forming electrically coupled assemblies. We monitored the function of these re-plated sheets optically with the Ca(2+) sensitive dye Fluo-4, and found that they generated characteristic waves of activity whose velocity and patterns of propagation depended upon the concentration of sodium channel blockers; lidocaine and tetrodotoxin, and also the time after re-plating, as well as the applied stimulation frequency. Importantly, reentrant spiral-wave propagation could be generated in these sheets by applying high-frequency stimulation, particularly when cell-density in the sheets was relatively low. This was because cardiac troponin T-positive cells were more non-homogeneously distributed at low cell densities. Especially in such sheets, we could terminate spiral waves by administering the anti-arrhythmic drugs; nifekalant, E-4031, sotalol, and quinidine. We also found that in these sheets, nifekalant showed a clear dose-dependent increase in the size of the unexcitable 'cores' of these induced spiral waves, an important parallel with the treatment for ventricular tachycardia in the clinical situation, which was not shown properly in cardiac-cell sheets derived from dissociated rodent hearts., Conclusions: We have succeeded in creating from hPSCs a valuable type of cardiomyocyte sheet that is capable of generating reentrant arrhythmias, and thus is demonstrably useful for screening and testing all sorts of drugs with anti-arrhythmic potential.
- Published
- 2013
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172. Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery.
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Tanaka T, Takahashi K, Yamane M, Tomida S, Nakamura S, Oshima K, Niwa A, Nishikomori R, Kambe N, Hara H, Mitsuyama M, Morone N, Heuser JE, Yamamoto T, Watanabe A, Sato-Otsubo A, Ogawa S, Asaka I, Heike T, Yamanaka S, Nakahata T, and Saito MK
- Subjects
- Animals, Carrier Proteins genetics, Carrier Proteins physiology, Cells, Cultured, Cryopyrin-Associated Periodic Syndromes drug therapy, Cryopyrin-Associated Periodic Syndromes genetics, Humans, Induced Pluripotent Stem Cells physiology, Infant, Mice, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Mutant Proteins genetics, Mutant Proteins physiology, NLR Family, Pyrin Domain-Containing 3 Protein, Cryopyrin-Associated Periodic Syndromes pathology, Drug Discovery methods, Induced Pluripotent Stem Cells pathology, Models, Theoretical, Mosaicism
- Abstract
Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1β secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1β secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.
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- 2012
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173. Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth.
- Author
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Soman NR, Baldwin SL, Hu G, Marsh JN, Lanza GM, Heuser JE, Arbeit JM, Wickline SA, and Schlesinger PH
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- Animals, Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacokinetics, Antineoplastic Agents therapeutic use, Breast Neoplasms drug therapy, Breast Neoplasms metabolism, Breast Neoplasms pathology, Cell Line, Tumor, Drug Carriers, Female, Humans, Liposomes, Melanoma, Experimental metabolism, Melanoma, Experimental pathology, Melitten pharmacokinetics, Melitten therapeutic use, Mice, Mice, Inbred C57BL, Mice, Nude, Microscopy, Electron, Transmission, Nanoparticles administration & dosage, Nanoparticles ultrastructure, Tissue Distribution, Melanoma, Experimental drug therapy, Melitten administration & dosage
- Abstract
The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.
- Published
- 2009
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174. Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress.
- Author
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Huang J, Roth R, Heuser JE, and Sadler JE
- Subjects
- Antibodies pharmacology, Blood Platelets cytology, Blood Platelets metabolism, Cell Adhesion physiology, Endothelial Cells ultrastructure, Humans, Integrin alphaVbeta3 immunology, Microscopy, Immunoelectron, Oligopeptides metabolism, P-Selectin metabolism, Stress, Mechanical, U937 Cells, Umbilical Veins cytology, Endothelial Cells metabolism, Integrin alphaVbeta3 metabolism, Platelet Adhesiveness physiology, von Willebrand Factor metabolism
- Abstract
Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin alpha(v)beta(3) in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin alpha(v)beta(3) reduced the number of VWF strings formed. In addition, integrin alpha(v) decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the alpha(v) subunit reduced the expression of cell-surface integrin alpha(v)beta(3) and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca(2+) and Mg(2+) but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin alpha(v)beta(3) on human endothelial cells.
- Published
- 2009
- Full Text
- View/download PDF
175. The AP-2 adaptor beta2 appendage scaffolds alternate cargo endocytosis.
- Author
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Keyel PA, Thieman JR, Roth R, Erkan E, Everett ET, Watkins SC, Heuser JE, and Traub LM
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Animals, Arrestins genetics, Arrestins metabolism, Cell Line, Clathrin genetics, Clathrin metabolism, Gene Silencing, Humans, Mice, Mice, Knockout, Protein Subunits genetics, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Receptor, Angiotensin, Type 1 genetics, Receptor, Angiotensin, Type 1 metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transcription Factor AP-2 genetics, Transferrin genetics, Transferrin metabolism, beta-Arrestin 1, beta-Arrestins, Endocytosis physiology, Protein Subunits metabolism, Protein Transport physiology, Transcription Factor AP-2 metabolism
- Abstract
The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin bud sites.
- Published
- 2008
- Full Text
- View/download PDF
176. A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments.
- Author
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Sheridan SD, Yu X, Roth R, Heuser JE, Sehorn MG, Sung P, Egelman EH, and Bishop DK
- Subjects
- Base Pairing, Cell Cycle Proteins chemistry, DNA chemistry, DNA, Circular ultrastructure, DNA, Single-Stranded ultrastructure, DNA-Binding Proteins chemistry, Humans, Image Processing, Computer-Assisted, Microscopy, Electron, Transmission, Rad51 Recombinase chemistry, Saccharomyces cerevisiae Proteins chemistry, Cell Cycle Proteins ultrastructure, DNA ultrastructure, DNA-Binding Proteins ultrastructure, Rad51 Recombinase ultrastructure, Saccharomyces cerevisiae Proteins ultrastructure
- Abstract
The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.
- Published
- 2008
- Full Text
- View/download PDF
177. CENP-E combines a slow, processive motor and a flexible coiled coil to produce an essential motile kinetochore tether.
- Author
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Kim Y, Heuser JE, Waterman CM, and Cleveland DW
- Subjects
- Animals, Chromosomal Proteins, Non-Histone genetics, Chromosomal Proteins, Non-Histone metabolism, Chromosome Segregation, Kinetochores metabolism, Microtubules metabolism, Models, Biological, Molecular Motor Proteins genetics, Molecular Motor Proteins metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Spindle Apparatus metabolism, Xenopus Proteins chemistry, Xenopus Proteins genetics, Xenopus Proteins metabolism, Xenopus laevis, Chromosomal Proteins, Non-Histone chemistry, Kinetochores chemistry, Microtubules chemistry, Molecular Motor Proteins chemistry, Protein Structure, Secondary, Spindle Apparatus chemistry
- Abstract
The mitotic kinesin centromere protein E (CENP-E) is an essential kinetochore component that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although reduction in CENP-E leads to high rates of whole chromosome missegregation, neither its properties as a microtubule-dependent motor nor how it contributes to the dynamic linkage between kinetochores and microtubules is known. Using single-molecule assays, we demonstrate that CENP-E is a very slow, highly processive motor that maintains microtubule attachment for long periods. Direct visualization of full-length Xenopus laevis CENP-E reveals a highly flexible 230-nm coiled coil separating its kinetochore-binding and motor domains. We also show that full-length CENP-E is a slow plus end-directed motor whose activity is essential for metaphase chromosome alignment. We propose that the highly processive microtubule-dependent motor activity of CENP-E serves to power chromosome congression and provides a flexible, motile tether linking kinetochores to dynamic spindle microtubules.
- Published
- 2008
- Full Text
- View/download PDF
178. Plasma membrane deformation by circular arrays of ESCRT-III protein filaments.
- Author
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Hanson PI, Roth R, Lin Y, and Heuser JE
- Subjects
- Animals, COS Cells, Cell Line, Cell Membrane chemistry, Cell Membrane metabolism, Chlorocebus aethiops, Endosomal Sorting Complexes Required for Transport, Endosomes metabolism, Humans, Microscopy, Electron, Mutation, Polymers metabolism, Transfection, Carrier Proteins metabolism, Cell Membrane ultrastructure, Vesicular Transport Proteins metabolism
- Abstract
Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used "deep-etch" electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis-deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.
- Published
- 2008
- Full Text
- View/download PDF
179. Assembly of Weibel-Palade body-like tubules from N-terminal domains of von Willebrand factor.
- Author
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Huang RH, Wang Y, Roth R, Yu X, Purvis AR, Heuser JE, Egelman EH, and Sadler JE
- Subjects
- Dimerization, Disulfides chemistry, Humans, Hydrogen-Ion Concentration, Image Processing, Computer-Assisted, Imaging, Three-Dimensional, Ions, Lasers, Light, Microscopy, Electron, Peptides chemistry, Protein Conformation, Protein Structure, Tertiary, Scattering, Radiation, Weibel-Palade Bodies physiology, Weibel-Palade Bodies chemistry, von Willebrand Factor chemistry
- Abstract
Endothelial cells assemble von Willebrand factor (VWF) multimers into ordered tubules within storage organelles called Weibel-Palade bodies, and tubular packing is necessary for the secretion of VWF filaments that can bind connective tissue and recruit platelets to sites of vascular injury. We now have recreated VWF tubule assembly in vitro, starting with only pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D'D3 domains. Assembly requires low pH and calcium ions and is reversed at neutral pH. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D'D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging.
- Published
- 2008
- Full Text
- View/download PDF
180. Molecular switches involving the AP-2 beta2 appendage regulate endocytic cargo selection and clathrin coat assembly.
- Author
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Edeling MA, Mishra SK, Keyel PA, Steinhauser AL, Collins BM, Roth R, Heuser JE, Owen DJ, and Traub LM
- Subjects
- Adaptor Proteins, Vesicular Transport genetics, Amino Acid Sequence, Arrestins chemistry, Arrestins genetics, Arrestins metabolism, Binding Sites, Crystallography, X-Ray, HeLa Cells, Humans, Models, Molecular, Molecular Sequence Data, Peptides genetics, Peptides metabolism, Protein Binding, Protein Structure, Secondary, Receptors, G-Protein-Coupled metabolism, Receptors, LDL metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Transcription Factor AP-2 genetics, Transcription Factor AP-2 metabolism, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, beta-Arrestins, Adaptor Proteins, Vesicular Transport metabolism, Clathrin metabolism, Clathrin-Coated Vesicles metabolism, Endocytosis physiology, Protein Conformation, Protein Structure, Tertiary, Transcription Factor AP-2 chemistry
- Abstract
Clathrin-associated sorting proteins (CLASPs) expand the repertoire of endocytic cargo sorted into clathrin-coated vesicles beyond the transmembrane proteins that bind physically to the AP-2 adaptor. LDL and GPCRs are internalized by ARH and beta-arrestin, respectively. We show that these two CLASPs bind selectively to the AP-2 beta2 appendage platform via an alpha-helical [DE](n)X(1-2)FXX[FL]XXXR motif, and that this motif also occurs and is functional in the epsins. In beta-arrestin, this motif maintains the endocytosis-incompetent state by binding back on the folded core of the protein in a beta strand conformation. Triggered via a beta-arrestin/GPCR interaction, the motif must be displaced and must undergo a strand to helix transition to enable the beta2 appendage binding that drives GPCR-beta-arrestin complexes into clathrin coats. Another interaction surface on the beta2 appendage sandwich is identified for proteins such as eps15 and clathrin, suggesting a mechanism by which clathrin displaces eps15 to lattice edges during assembly.
- Published
- 2006
- Full Text
- View/download PDF
181. Drosophila Spire is an actin nucleation factor.
- Author
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Quinlan ME, Heuser JE, Kerkhoff E, and Mullins RD
- Subjects
- Actins ultrastructure, Amino Acid Sequence, Animals, Binding Sites, Drosophila Proteins chemistry, Drosophila Proteins ultrastructure, Drosophila melanogaster cytology, Microfilament Proteins chemistry, Microfilament Proteins ultrastructure, Molecular Sequence Data, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Multiprotein Complexes ultrastructure, Protein Binding, Protein Structure, Quaternary, Protein Structure, Tertiary, Actin Cytoskeleton chemistry, Actin Cytoskeleton metabolism, Actins chemistry, Actins metabolism, Drosophila Proteins metabolism, Drosophila melanogaster metabolism, Microfilament Proteins metabolism
- Abstract
The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.
- Published
- 2005
- Full Text
- View/download PDF
182. Dual engagement regulation of protein interactions with the AP-2 adaptor alpha appendage.
- Author
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Mishra SK, Hawryluk MJ, Brett TJ, Keyel PA, Dupin AL, Jha A, Heuser JE, Fremont DH, and Traub LM
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Clathrin physiology, Endocytosis, Molecular Sequence Data, Phylogeny, Protein Subunits, Rats, Adaptor Protein Complex 2 chemistry
- Abstract
Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.
- Published
- 2004
- Full Text
- View/download PDF
183. TorsinA in the nuclear envelope.
- Author
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Naismith TV, Heuser JE, Breakefield XO, and Hanson PI
- Subjects
- Adenosine Triphosphatases genetics, Adenosine Triphosphate metabolism, Amino Acid Motifs, Animals, CHO Cells, COS Cells, Conserved Sequence, Cricetinae, Mutagens, Adenosine Triphosphatases metabolism, Dystonia Musculorum Deformans metabolism, Nuclear Envelope metabolism
- Abstract
Early-onset torsion dystonia, a CNS-based movement disorder, is usually associated with a single amino acid deletion (Delta E302/303) in the protein torsinA. TorsinA is an AAA+ ATPase in the endoplasmic reticulum, but what it does is unknown. Here, we use torsinA mutants with defects in ATP hydrolysis (E171Q, ATP-bound) and ATP binding (K108A, ATP-free) to probe torsinA's normal cellular function. Surprisingly, ATP-bound torsinA is recruited to the nuclear envelope (NE) of transfected cells, where it alters connections between inner and outer nuclear membranes. In contrast, ATP-free torsinA is diffusely distributed throughout the endoplasmic reticulum and has no effect on the NE. Among AAA+ ATPases, affinity for substrates is high in the ATP-bound and low in the ATP-free state, leading us to propose that component(s) of the NE may be substrates for torsinA. We also find that the disease-promoting Delta E302/303 mutant is in the NE, and that this relocalization, as well as the mutant's previously described ability to induce membranous inclusions, is eliminated by the K108A ATP-binding mutation. These results suggest that changes in interactions involving torsinA in the NE could be important for the pathogenesis of dystonia and point to torsinA and related proteins as a class of ATPases that may operate in the NE.
- Published
- 2004
- Full Text
- View/download PDF
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