Calmodulin Suppresses Synaptotagmin-2 Transcription In

Total Page:16

File Type:pdf, Size:1020Kb

Calmodulin Suppresses Synaptotagmin-2 Transcription In Supplemental Material can be found at: http://www.jbc.org/content/suppl/2010/09/08/M110.150151.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 44, pp. 33930–33939, October 29, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Calmodulin Suppresses Synaptotagmin-2 Transcription in Cortical Neurons*□S Received for publication, June 1, 2010, and in revised form, July 23, 2010 Published, JBC Papers in Press, August 20, 2010, DOI 10.1074/jbc.M110.150151 Zhiping P. Pang‡1,2, Wei Xu‡§1, Peng Cao§, and Thomas C. Su¨dhof‡§3 From the ‡Department of Molecular and Cellular Physiology and the §Howard Hughes Medical Institute, Stanford University, Palo Alto, California 94304-5543 ؉ Calmodulin (CaM) is a ubiquitous Ca2 sensor protein that modes of synaptic vesicle exocytosis are triggered by Ca2ϩ. The plays a pivotal role in regulating innumerable neuronal func- synchronous release mode exhibits an apparent Ca2ϩ cooper- tions, including synaptic transmission. In cortical neurons, ativity of ϳ5 (1–3), and the asynchronous release shows an ؉ most neurotransmitter release is triggered by Ca2 binding to apparent Ca2ϩ cooperativity of ϳ2 (3). The role of synaptotag- synaptotagmin-1; however, a second delayed phase of release, mins as primary Ca2ϩ sensors for synchronous neurotransmit- ؉ referred to as asynchronous release, is triggered by Ca2 binding ter release is well established (4–11). However, the molecular ؉ Downloaded from to an unidentified secondary Ca2 sensor. To test whether CaM identity of the Ca2ϩ sensor that mediates asynchronous release ؉ could be the enigmatic Ca2 sensor for asynchronous release, remains unknown. we now use in cultured neurons short hairpin RNAs that sup- Calmodulin (CaM)4 is a ubiquitous and essential Ca2ϩ-bind- press expression of ϳ70% of all neuronal CaM isoforms. Sur- ing protein that regulates a plethora of cellular processes, from prisingly, we found that in synaptotagmin-1 knock-out neurons, gene transcription to signal transduction to ion channels to the CaM knockdown caused a paradoxical rescue of synchro- membrane traffic (12–14). CaM is highly conserved in verte- www.jbc.org nous release, instead of a block of asynchronous release. Gene brates and is ubiquitously expressed. All CaM proteins are and protein expression studies revealed that both in wild-type composed of two lobes (i.e. the N- and C-lobes) that each con- and in synaptotagmin-1 knock-out neurons, the CaM knock- tain two E-F hand Ca2ϩ-binding motifs and are connected via a 2ϩ down altered expression of >200 genes, including that encoding flexible ␣-helix (15). Each E-F hand motif binds to one Ca at UMDNJ RW JOHNSON, on June 4, 2012 synaptotagmin-2. Synaptotagmin-2 expression was increased ion. Ca2ϩ binds to the N- and C-lobes in a cooperative manner, several-fold by the CaM knockdown, which accounted for the with the N-lobe binding Ca2ϩ with a lower affinity but faster paradoxical rescue of synchronous release in synaptotagmin-1 association and dissociation rates than the C-lobe (16, 17). The knock-out neurons by the CaM knockdown. Interestingly, the different Ca2ϩ binding characteristics probably confer onto CaM knockdown primarily activated genes that are preferen- CaM lobes specific target protein binding properties and func- tially expressed in caudal brain regions, whereas it repressed tions (18, 19). Apart from numerous cytoplasmic regulatory genes in rostral brain regions. Consistent with this correlation, functions, Ca2ϩ binding to CaM serves to activate transcription quantifications of protein levels in adult mice uncovered an by a number of distinct signaling pathways (14, 20). inverse relationship of CaM and synaptotagmin-2 levels in CaM regulates neurotransmitter release by multiple mecha- mouse forebrain, brain stem, and spinal cord. Finally, we nisms, including binding to Munc13, regulating Ca2ϩ channels, employed molecular replacement experiments using a knock- and activating Ca2ϩ/CaM-dependent kinase II (CaMKII) (12– ؉ down rescue approach to show that Ca2 binding to the C-lobe 14, 20–24). In addition, CaM was proposed to directly function but not the N-lobe of CaM is required for suppression of synap- asaCa2ϩ sensor for Ca2ϩ-triggered exocytosis (25, 26), totagmin-2 expression in cortical neurons. Our data describe a prompting us to test here whether CaM may act as the Ca2ϩ ؉ previously unknown, Ca2 /CaM-dependent regulatory path- sensor for asynchronous release. For this purpose, we cultured way that controls the expression of synaptic proteins in the ros- cortical neurons from synaptotagmin-1 (Syt1) knock-out (KO) tral-caudal neuraxis. mice in which synchronous release is abolished and only asyn- chronous release remains (5, 6). We then analyzed the effects of shRNA-mediated knockdown (KD) of all CaM isoforms on Neurotransmitter release is mediated by two separate, com- neurotransmitter release, using a previously established lenti- peting pathways: synchronous and asynchronous releases. Both viral system that suppresses ϳ70% of neuronal CaM expression (24). We found that although KD of CaM had no significant * This work was supported, in whole or in part, by a National Institutes of Mental effect on asynchronous release, it surprisingly rescued the loss HealthGrant(toT.C.S.).ThisworkwasalsosupportedbyawardsfromNational Alliance for Research on Schizophrenia and Depression (to Z. P. P.). of synchronous release in Syt1 KO neurons. An unbiased □S The on-line version of this article (available at http://www.jbc.org) contains genome-wide gene expression profiling experiment revealed supplemental Tables S1 and S2 and Fig. S1. that the CaM KD induced a dramatic up-regulation of expres- 1 Both authors contributed equally to this work. 2 To whom correspondence may be addressed: Dept. of Molecular and Cellu- lar Physiology, Stanford University, 1050 Arastradero Rd., Palo Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: [email protected]. 4 The abbreviations used are: CaM, calmodulin; CaMKII, CaM-dependent 3 To whom correspondence may be addressed: Dept. of Molecular and kinase II; Syt, synaptotagmin; KO, knock-out; Syb, synaptobrevin; DIV, Cellular Physiology, Stanford University, 1050 Arastradero Rd., Palo day(s) in vitro; mIPSC, miniature inhibitory postsynaptic currents; VCP, Alto, CA 94304-5543. Tel.: 650-721-1421; E-mail: [email protected]. vasolin-containing protein. 33930 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 44•OCTOBER 29, 2010 Calmodulin Suppresses Synaptotagmin-2 Expression sion of Syt2 and synaptobrevin-1 (Syb1), which are normally ing CaM 1–3; or viruses with shRNAs and expression of shRNA expressed in the forebrain at low levels but are abundant in silent WT CaM cDNA (supplemental Table S1). Only the gene caudal brain regions (27–29). In addition, the expression of expression levels changed after CaM KD and rescued with WT other caudal synaptic genes was increased, whereas expression CaM in both experiments are included in the list. Full gene of rostral synaptic genes was decreased. Moreover, using expression array data are deposited to the NCBI Gene Expres- molecular replacement experiments, we show that the regula- sion Omnibus. tion of Syt2 expression by CaM requires Ca2ϩ binding to only Quantification of mRNA Level by Quantitative Real Time the C-lobe but not the N-lobe of CaM. Thus, our data show that PCR—The cultured cortical neurons were lysed, and total RNA Ca2ϩ binding to CaM regulates neurotransmitter release not was extracted and purified with a RNAqueous micro kit only in the short term by binding to target proteins (12–14, (Ambion) following the manufacturer’s instructions. The 20–24) but also on a longer time frame by modulating the mRNA level of individual genes was then analyzed by one-step expression of presynaptic proteins such as Syt2, thereby influ- quantitative real time PCR system with pre-made TaqMan௡ encing the properties of neurotransmitter release at a synapse. gene expression assays (Applied Biosystems). Briefly, 30 ng of RNA sample in 1 ␮l of volume was mixed with 10 ␮l of TaqMan EXPERIMENTAL PROCEDURES fast universal PCR master mix (twice), 0.1 ␮l of reverse tran- Neuronal Culture—Mouse cortical culture was made as scriptase (50 units/␮l), 0.4 ␮l of RNase inhibitor (20 units/␮l), ␮ ␮ ௡ described elsewhere (6, 24). Briefly, the primary cortical neu- 7.5 lofH2O, and 7 l of TaqMan gene expression assay for rons were isolated from postnatal day 0 pups of Syt1 deficient or the target gene (including the forward and reverse primers and Downloaded from wild-type mice, dissociated by papain digestion, and plated on the TaqMan FAM-MGB probe). The reaction mixture was Matrigel-coated circle glass coverslips. The neurons were cul- loaded onto ABI7900 fast real time PCR machine for 30 min of tured in vitro for 13–16 days in minimal essential medium reverse transcription at 48 °C followed by 40 PCR amplification (Invitrogen) supplemented with B27 (Invitrogen), glucose, cycles consisting of denaturation at 95 °C for 1 s and annealing transferrin, fetal bovine serum, and Ara-C (Sigma). and extension at 60 °C for 20 s. The amplification curve was www.jbc.org ⌬⌬ Lentivirus Packaging and Infection of Neuronal Culture—The collected and analyzed with Ct methods for relative quanti- packaging of lentiviruses and the infection of neurons with len- fication of mRNAs. The amount of mRNA of target genes, nor- tiviruses were described previously (24). Briefly, the lentiviral malized to that of an endogenous control and relative to the Ϫ⌬⌬ at UMDNJ RW JOHNSON, on June 4, 2012 expression vector (control vector L309 or the shRNAs carrying calibrator sample, is calculated by 2 Ct. In the current study, vectors) and three helper plasmids, the pRSV-REV, pMDLg/ GAPDH was used as the endogenous control, and the RNA pRRE, and vesicular stomatitis virus G protein were co-trans- samples derived from neurons infected with control vector fected into HEK 293T cells (ATCC, Manassas, VA) at 6, 2, 2, (L309) were used as calibrators.
Recommended publications
  • Annexin A2 Flop-Out Mediates the Non-Vesicular Release of Damps/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions
    cells Article Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions Hayato Matsunaga 1,2,† , Sebok Kumar Halder 1,3,† and Hiroshi Ueda 1,4,* 1 Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan; [email protected] (H.M.); [email protected] (S.K.H.) 2 Department of Medical Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523, Japan 3 San Diego Biomedical Research Institute, San Diego, CA 92121, USA 4 Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan * Correspondence: [email protected]; Tel.: +81-75-753-4536 † These authors contributed equally to this work. Abstract: Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum- free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 Citation: Matsunaga, H.; Halder, through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 S.K.; Ueda, H. Annexin A2 Flop-Out antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1.
    [Show full text]
  • Is Synaptotagmin the Calcium Sensor? Motojiro Yoshihara, Bill Adolfsen and J Troy Littleton
    315 Is synaptotagmin the calcium sensor? Motojiro Yoshihara, Bill Adolfsen and J Troy Littletonà After much debate, recent progress indicates that the synaptic synaptotagmins, which are transmembrane proteins con- vesicle protein synaptotagmin I probably functions as the taining tandem calcium-binding C2 domains (C2A and calcium sensor for synchronous neurotransmitter release. C2B) (Figure 1a). Synaptotagmin I is an abundant cal- Following calcium influx into presynaptic terminals, cium-binding synaptic vesicle protein [8,9] that has been synaptotagmin I rapidly triggers the fusion of synaptic vesicles demonstrated via genetic studies to be important for with the plasma membrane and underlies the fourth-order efficient synaptic transmission in vivo [10–13]. The C2 calcium cooperativity of release. Biochemical and genetic domains of synaptotagmin I bind negatively-charged studies suggest that lipid and SNARE interactions underlie phospholipids in a calcium-dependent manner [9,14,15, synaptotagmin’s ability to mediate the incredible speed of 16–18]. There is compelling evidence that phospholipid vesicle fusion that is the hallmark of fast synaptic transmission. binding is an effector interaction in vesicle fusion, as the calcium dependence of this process ( 74 mM) and its Addresses rapid kinetics (on a millisecond scale) (Figure 1b) fit Picower Center for Learning and Memory, Department of Biology and reasonably well with the predicted requirements of Department of Brain and Cognitive Sciences, Massachusetts synaptic transmission [15]. In addition to phospholipid Institute of Technology, Cambridge, MA 02139, USA Ãe-mail: [email protected] binding, the calcium-stimulated interaction between synaptotagmin and the t-SNAREs syntaxin and SNAP- 25 [15,19–23] provides a direct link between calcium and Current Opinion in Neurobiology 2003, 13:315–323 the fusion complex.
    [Show full text]
  • A FAMILY of NEURONAL Ca2+-BINDING PROTEINS WITH
    Neuroscience Vol. 112, No. 1, pp. 51^63, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII: S0306-4522(02)00063-5 0306-4522 / 02 $22.00+0.00 www.neuroscience-ibro.com NECABS: A FAMILY OF NEURONAL Ca2þ-BINDING PROTEINS WITH AN UNUSUAL DOMAIN STRUCTURE AND A RESTRICTED EXPRESSION PATTERN S. SUGITA,1 A. HO and T. C. SUº DHOFÃ Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235, USA AbstractöCa2þ-signalling plays a major role in regulating all aspects of neuronal function. Di¡erent types of neurons exhibit characteristic di¡erences in the responses to Ca2þ-signals. Correlating with di¡erences in Ca2þ-response are expression patterns of Ca2þ-binding proteins that often serve as markers for various types of neurons. For example, in the cerebral cortex the EF-hand Ca2þ-binding proteins parvalbumin and calbindin are primarily expressed in inhibitory interneurons where they in£uence Ca2þ-dependent responses. We have now identi¢ed a new family of proteins called NECABs (neuronal Ca2þ-binding proteins). NECABs contain an N-terminal EF-hand domain that binds Ca2þ, but di¡erent from many other neuronal EF-hand Ca2þ-binding proteins, only a single EF-hand domain is present. At the C-terminus, NECABs include a DUF176 motif, a bacterial domain of unknown function that was previously not observed in eukaryotes. In rat at least three closely related NECAB genes are expressed either primarily in brain (NECABs 1 and 2) or in brain and muscle (NECAB 3).
    [Show full text]
  • Observation of Ls Time-Scale Protein Dynamics in the Presence of Ln Ions
    J Biomol NMR (2007) 37:79–95 DOI 10.1007/s10858-006-9105-y ARTICLE Observation of ls time-scale protein dynamics in the presence of Ln3+ ions: application to the N-terminal domain of cardiac troponin C Christian Eichmu¨ ller Æ Nikolai R. Skrynnikov Received: 20 September 2006 / Accepted: 2 October 2006 / Published online: 19 December 2006 Ó Springer Science+Business Media B.V. 2006 Abstract The microsecond time-scale motions in the environment of the reporting spin does not change. This N-terminal domain of cardiac troponin C (NcTnC) happens, for example, when the motion involves a ‘rigid’ loaded with lanthanide ions have been investigated by structural unit such as individual a-helix. Even more means of a 1HN off-resonance spin-lock experiment. significantly, the dispersions based on pseudocontact The observed relaxation dispersion effects strongly shifts offer better chances for structural characterization increase along the series of NcTnC samples containing of the dynamic species. This method can be generalized La3+,Ce3+, and Pr3+ ions. This rise in dispersion effects for a large class of applications via the use of specially is due to modulation of long-range pseudocontact shifts designed lanthanide-binding tags. by ls time-scale dynamics. Specifically, the motion in the coordination sphere of the lanthanide ion (i.e. in the Keywords Cardiac troponin C Á Spin-lock relaxation NcTnC EF-hand motif) causes modulation of the dispersion experiment Á ls–ms protein dynamics Á paramagnetic susceptibility tensor which, in turn, causes Lanthanide Á Pseudocontact shift Á Calmodulin modulation of pseudocontact shifts. It is also probable that opening/closing dynamics, previously identified in Ca2+–NcTnC, contributes to some of the observed Introduction dispersions.
    [Show full text]
  • Therapeutics of Pediatric Urinary Tract Infections
    iMedPub Journals ARCHIVES OF MEDICINE 2015 http://wwwimedpub.com Special Issue Synaptotagmin Functions as a Larry H Bernstein Calcium Sensor: How Calcium New York Methodist Hospital, Brooklyn, Ions Regulate the Fusion of New York, USA Vesicles with Cell Membranes during Neurotransmission Corresponding Author: Larry H Bernstein New York Methodist Hospital, Brooklyn, New York, USA Short Communication This article is part of a series of articles discussed the mechanism [email protected] of the signaling of smooth muscle cells by the interacting parasympathetic neural innervation that occurs by calcium Tel: 2032618671 triggering neuro-transmitter release by initiating synaptic vesicle fusion. It involves the interaction of soluble N-acetylmaleimide- sensitive factor (SNARE) and SM proteins, and in addition, the the tip, calcium enters the cell. In response, the neuron liberates discovery of a calcium-dependsent Syt1 (C) domain of protein- chemical messengers—neurotransmitters—which travel to the kinase C isoenzyme, which binds to phospholipids. It is reasonable next neuron and thus pass the baton. to consider that it differs from motor neuron activation of skeletal He further stipulates that synaptic vesicle exocytosis operates muscles, mainly because the innervation is in the involuntary by a general mechanism of membrane fusion that revealed domain. The cranial nerve rooted innervation has evolved itself to be a model for all membrane fusion, but that is uniquely comes from the spinal ganglia at the corresponding level of the regulated by a calcium-sensor protein called synaptotagmin. spinal cord. It is in this specific neural function that we find a Neurotransmission is thus a combination of electrical signal and mechanistic interaction with adrenergic hormonal function, a chemical transport.
    [Show full text]
  • Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine
    proteomes Article Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle Britta Eggers 1,2,* , Karin Schork 1,2, Michael Turewicz 1,2 , Katalin Barkovits 1,2 , Martin Eisenacher 1,2, Rolf Schröder 3, Christoph S. Clemen 4,5 and Katrin Marcus 1,2,* 1 Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum, 44801 Bochum, Germany; [email protected] (K.S.); [email protected] (M.T.); [email protected] (K.B.); [email protected] (M.E.) 2 Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, 44801 Bochum, Germany 3 Institute of Neuropathology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, 91054 Erlangen, Germany; [email protected] 4 German Aerospace Center, Institute of Aerospace Medicine, 51147 Cologne, Germany; [email protected] 5 Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, Medical Faculty, University of Cologne, 50931 Cologne, Germany * Correspondence: [email protected] (B.E.); [email protected] (K.M.) Abstract: Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser Citation: Eggers, B.; Schork, K.; microdissected and separately investigated by mass spectrometry to develop advanced proteomic Turewicz, M.; Barkovits, K.; profiles of all murine skeletal muscle fiber types.
    [Show full text]
  • Sensors in Hormone Secretion from Excitable Endocrine Cells
    R1 RAPID COMMUNICATION C Involvement of calpain and synaptotagmin Ca2 sensors in hormone secretion from excitable endocrine cells Ebun Aganna, Jacky M Burrin1, Graham A Hitman and Mark D Turner Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Barts and The London, Queen Mary’s School of Medicine and Dentistry, University of London, Whitechapel, London E1 2AT, UK and 1Centre for Molecular Endocrinology, William Harvey Research Institute, Barts and The London, Queen Mary’s School of Medicine and Dentistry, University of London, Charterhouse Square, London EC1M 6BQ, UK (Requests for offprints should be addressed to M D Turner; Email: [email protected]) Abstract C The requirement for Ca2 to regulate hormone secretion from secretagog-stimulated secretion from both INS-1 and GH3 cells endocrine cells is long established, but the precise function of was completely abolished following pre-incubation with C Ca2 sensors in stimulus–secretion coupling remains unclear. the cysteine protease inhibitor E64, whereas stimulated In the current study, we examined the expression of calpain and secretion from AtT20 cells was modest and completely synaptotagmin in INS-1 pancreatic and GH3 and AtT20 insensitive to E64 inhibition. These results are in stark contrast pituitary cells, and investigated the sensitivity of hormone to synaptotagmin data. Synaptotagmin expression in AtT20 cells secretion from these cells to inhibition of the calpain family of is abundant, whereas INS-1 cells express extremely low C cysteine proteases. Little difference in expression of m-calpain levels of this Ca2 sensor, relative to the pituitary cells. We was observed between the different endocrine cells.
    [Show full text]
  • Synaptotagmin 7 Functions As a Ca2+-Sensor for Synaptic Vesicle Replenishment
    IN PRESS Synaptotagmin 7 Functions as a Ca2+-sensor for Synaptic Vesicle Replenishment Huisheng Liu (University of Wisconsin), Hua Bai (University of Wisconsin), Enfu Hui (University of Wisconsin), Lu Yang (University of Wisconsin), Chantell Evans (University of Wisconsin), Zhao Wang (University of Wisconsin), Sung Kwon (University of Wisconsin), and Edwin Chapman (University of Wisconsin) Abstract: Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. http://dx.doi.org/10.7554/elife.01524 Please address questions to [email protected]. Details on how to cite eLife articles in news stories and our media policy are available at http://www.elifesciences.org/news/for-the- press. Articles published in eLife may be read on the journal site at http://elife.elifesciences.org.
    [Show full text]
  • Endoplasmic Reticulum Stress Is Important for the Manifestations Ofα
    3306 • The Journal of Neuroscience, March 7, 2012 • 32(10):3306–3320 Neurobiology of Disease Endoplasmic Reticulum Stress Is Important for the Manifestations of ␣-Synucleinopathy In Vivo Emanuela Colla,1 Philippe Coune,2 Ying Liu,1 Olga Pletnikova,1 Juan C Troncoso,1 Takeshi Iwatsubo,3 Bernard L. Schneider,2 and Michael K. Lee1,4,5 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, 2Brain Mind Institute, Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Switzerland, 3Department of Neuropathology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku Tokyo 113-0030, Japan, and 4Department of Neuroscience and 5Institute for Translational Neuroscience, University of Minnesota, Minneapolis, Minnesota 55102 Accumulation of misfolded ␣-synuclein (␣S) is mechanistically linked to neurodegeneration in Parkinson’s disease (PD) and other ␣-synucleinopathies. However, how ␣S causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in ␣-synucleinopathy. Using the A53T mutant human ␣S transgenic (A53T␣S Tg) mouse model of ␣-synucleinopathy, we show that disease onset in the ␣S Tg model is coincident with induction of ER chaperones in neurons exhibiting ␣S pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2␣, indicating that ␣-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated ␣S monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M ␣S than the control cases.
    [Show full text]
  • Actin Filament Disassembly Is a Sufficient Final Trigger for Exocytosis in Nonexcitable Cells Shmuel Muallem, Katarzyna Kwiatkowska, Xin Xu, and Helen L
    Actin Filament Disassembly Is a Sufficient Final Trigger for Exocytosis in Nonexcitable Cells Shmuel Muallem, Katarzyna Kwiatkowska, Xin Xu, and Helen L. Yin Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040 Abstract. Although the actin cytoskeleton has been Direct visualization and quantitation of actin filaments implicated in vesicle trafficking, docking and fusion, showed that B-thymosin, like agonists, induced actin its site of action and relation to the Ca2÷-mediated ac- depolymerization at the apical membrane where exo- tivation of the docking and fusion machinery have cytosis occurs. Blocking actin depolymerization by not been elucidated. In this study, we examined the phalloidin or neutralizing fl-thymosin by complexing role of actin filaments in regulated exocytosis by in- with exogenous actin prevented exocytosis. These troducing highly specific actin monomer-binding findings show that the cortical actin network acts as a proteins, the ~-thymosins or a gelsolin fragment, into dominant negative clamp which blocks constitutive streptolysin O-permeabilized pancreatic acinar cells. exocytosis. In addition, actin filaments also have a These proteins had stimulatory and inhibitory ef- positive role. High concentrations of the actin depoly- fects. Low concentrations elicited rapid and robust merizing proteins inhibited all phases of exocytosis. exocytosis with a profile comparable to the initial The inhibition overrides stimulation by agonists and all phase of regulated exocytosis, but without raising downstream effectors tested, suggesting that exocytosis [Ca2+], and even when [Ca 2÷] was clamped at low lev- cannot occur without a minimal actin cytoskeletal els by EGTA. No additional cofactors were required. structure. hE final steps of regulated exocytosis involve vesicle In the present study, we used a different approach to exam- docking, triggering, and membrane fusion.
    [Show full text]
  • Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact
    ARTICLE https://doi.org/10.1038/s41467-021-21090-x OPEN Conserved arginine residues in synaptotagmin 1 regulate fusion pore expansion through membrane contact Sarah B. Nyenhuis1,4, Nakul Karandikar 1, Volker Kiessling 2,3, Alex J. B. Kreutzberger 2,3,5, Anusa Thapa1, ✉ Binyong Liang2,3, Lukas K. Tamm 2,3 & David S. Cafiso 1,2,3 2+ 1234567890():,; Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the fusion probability and kinetics but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore. 1 Department of Chemistry, University of Virginia, Charlottesville, VA, USA. 2 Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA. 3 Center for Membrane Biology, University of Virginia, Charlottesville, VA, USA. 4Present address: Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA.
    [Show full text]
  • The Crystal Structure of Calcium-Free Human M-Calpain Suggests an Electrostatic Switch Mechanism for Activation by Calcium
    The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium Stefan Strobl*†‡, Carlos Fernandez-Catalan*†, Marianne Braun†, Robert Huber†, Hajime Masumoto§, Kazuhiro Nakagawa§, Akihiro Irie§, Hiroyuki Sorimachi§¶, Gleb Bourenkowʈ, Hans Bartunikʈ, Koichi Suzuki§, and Wolfram Bode†** †Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, D 82 152 Planegg-Martinsried, Germany; §Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; and ʈArbeitsgruppe Proteindynamik Max-Planck-Gesellschaft Arbeitsgruppen für Strukfurelle Molekularbiologie, c͞o Deutsches Elektronen Synchrotron, D-22603 Hamburg, Germany Contributed by Robert Huber, November 16, 1999 Calpains (calcium-dependent cytoplasmic cysteine proteinases) are functioning of calpains, the structures of full-length calpain must implicated in processes such as cytoskeleton remodeling and signal to be known. We (10) and others (11) have communicated transduction. The 2.3-Å crystal structure of full-length het- crystals of full-length human and partially truncated rat m- -erodimeric [80-kDa (dI-dIV) ؉ 30-kDa (dV؉dVI)] human m-calpain calpain, respectively. In the following, we describe the funda crystallized in the absence of calcium reveals an oval disc-like mental properties of full-length human m-calpain and discuss the shape, with the papain-like catalytic domain dII and the two possible mechanisms of calcium activation. calmodulin-like domains dIV؉dVI occupying opposite poles, and the tumor necrosis factor ␣-like ␤-sandwich domain dIII and the Materials and Methods -N-terminal segments dI؉dV located between. Compared with Full-length human m-calpain containing an N-terminal GlyArg -papain, the two subdomains dIIa؉dIIb of the catalytic unit are ArgAspArgSer L-chain elongation was overexpressed in a bac rotated against one another by 50°, disrupting the active site and ulovirus expression system and was purified (12).
    [Show full text]