The Rap1–Cofilin-1 Pathway Coordinates Actin Reorganization and MTOC Polarization at the B Cell Immune Synapse Jia C
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The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy
bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. The kinetics of nucleotide binding to isolated Chlamydomonas axonemes using UV-TIRF microscopy M. Feofilova, M. Mahamdeh, J. Howard 1 bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Key words: dynein, axoneme, cilia, flagella, mantATP, fluorescent nucleotide Abbreviations ADP – adenosine diphosphate ATP – adenosine triphosphate MantATP - methylanthraniloyl adenosine triphosphate TIRF – total-internal-reflection fluorescence 2 bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Cilia and flagella are long, slender organelles found in many eukaryotic cells where they have sensory, developmental and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. -
Supplementary Materials
Supplementary Materials COMPARATIVE ANALYSIS OF THE TRANSCRIPTOME, PROTEOME AND miRNA PROFILE OF KUPFFER CELLS AND MONOCYTES Andrey Elchaninov1,3*, Anastasiya Lokhonina1,3, Maria Nikitina2, Polina Vishnyakova1,3, Andrey Makarov1, Irina Arutyunyan1, Anastasiya Poltavets1, Evgeniya Kananykhina2, Sergey Kovalchuk4, Evgeny Karpulevich5,6, Galina Bolshakova2, Gennady Sukhikh1, Timur Fatkhudinov2,3 1 Laboratory of Regenerative Medicine, National Medical Research Center for Obstetrics, Gynecology and Perinatology Named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia 2 Laboratory of Growth and Development, Scientific Research Institute of Human Morphology, Moscow, Russia 3 Histology Department, Medical Institute, Peoples' Friendship University of Russia, Moscow, Russia 4 Laboratory of Bioinformatic methods for Combinatorial Chemistry and Biology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 5 Information Systems Department, Ivannikov Institute for System Programming of the Russian Academy of Sciences, Moscow, Russia 6 Genome Engineering Laboratory, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia Figure S1. Flow cytometry analysis of unsorted blood sample. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S2. Flow cytometry analysis of unsorted liver stromal cells. Representative forward, side scattering and histogram are shown. The proportions of negative cells were determined in relation to the isotype controls. The percentages of positive cells are indicated. The blue curve corresponds to the isotype control. Figure S3. MiRNAs expression analysis in monocytes and Kupffer cells. Full-length of heatmaps are presented. -
Rap1 Acts Via Multiple Mechanisms to Position Canoe/Afadin and Adherens Junctions and Mediate Apical-Basal Polarity Establishment
bioRxiv preprint doi: https://doi.org/10.1101/170977; this version posted July 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Rap1 acts via multiple mechanisms to position Canoe/Afadin and adherens junctions and mediate apical-basal polarity establishment Teresa T. Bonello1, Kia Z. Perez-Vale2, Kaelyn D. Sumigray3, and Mark Peifer1,2,3* 1 Department of Biology, University of North Carolina at Chapel Hill, CB#3280, Chapel Hill, NC 27599-3280, USA 2 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 3 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Running Title Active Rap1 positions Canoe and AJs 6950 words * To whom correspondence should be addressed Email: [email protected] Phone: (919) 962-2272 Abbreviations used: α-cat, alpha-catenin; β-cat, beta-catenin; AJ, adherens junction; Arm, Armadillo; Baz, BazooKa; CA, constitutively active; Cno, Canoe; DE-cad, Drosophila E-cadherin; Dzy, Dizzy; GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GTP, guanosine triphosphate; IF, immunofluorescence; MIP, maximum intensity projection; RA, Ras-associated; RFP, red fluorescent protein; SAJ, spot adherens junction; shRNA, short hairpin RNA; TCJ, tricellular junction; WT, wildtype 1 bioRxiv preprint doi: https://doi.org/10.1101/170977; this version posted July 31, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. -
Cargo Specific Regulation of Cytoplasmic Dynein by Effector Proteins
University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins Mara Olenick University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, Biophysics Commons, and the Cell Biology Commons Recommended Citation Olenick, Mara, "Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins" (2018). Publicly Accessible Penn Dissertations. 3167. https://repository.upenn.edu/edissertations/3167 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3167 For more information, please contact [email protected]. Cargo Specific Regulation Of Cytoplasmic Dynein By Effector Proteins Abstract Axonal transport is vital for the development and survival of neurons. The transport of cargo and organelles from the axon to the cell body is driven almost completely by the molecular motor, cytoplasmic dynein. Yet, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Previous work has suggested that adaptor proteins provide a mechanism for cargo-specific egulationr of motors. During my thesis work, I have investigated the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. Using optogenetic and single molecule assays, I found that Hook proteins interact with both dynein and dynactin, to effectively activate dynein motility, inducing longer run lengths and higher velocities than the previously characterized dynein activator, BICD2. In addition, I found that complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of EB proteins yet cannot bind directly to microtubules. -
Rap1 Regulates Hepatic Stellate Cell Migration Through the Modulation of Rhoa Activity in Response to TGF‑Β1
INTERNATIONAL JOURNAL OF MOleCular meDICine 44: 491-502, 2019 Rap1 regulates hepatic stellate cell migration through the modulation of RhoA activity in response to TGF‑β1 MI-YOUNG MOON1, HEE-JUN KIM2, MO-JONG KIM2, SUNHO UHM1, JI‑WON PARK1, KI-TAE SUK3, JAE‑BONG PARK4, DONG-JUN KIM3 and SUNG-EUN KIM1 1Department of Internal Medicine, Hallym University Sacred Heart Hospital, College of Medicine, Hallym University, Anyang, Gyeonggi 14068; 2Ilsong Institute of Life Science, Hallym University, Anyang, Gyeonggi 14066; 3Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chuncheon, Gangwon 24253; 4Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Gangwon 24252, Republic of Korea Received November 1, 2018; Accepted May 28, 2019 DOI: 10.3892/ijmm.2019.4215 Abstract. Although the migration of hepatic stellate cells activation of RhoA in TGF‑β1-stimulated HSC‑T6 cells. These (HSCs) is important for hepatic fibrosis, the regulation of this findings suggest that TGF‑β1 regulates Rap1, resulting in the migration is poorly understood. Notably, transforming growth suppression of RhoA, activation of and formation of F‑actin factor (TGF)-β1 induces monocyte migration to sites of injury during the migration of HSCs. or inflammation during the early phase, but inhibits cell migra- tion during the late phase. In the present study, the role of Introduction transforming protein RhoA signaling in TGF-β1-induced HSC migration was investigated. TGF‑β1 was found to increase Hepatic fibrosis is characterized by the excessive deposition the protein and mRNA levels of smooth muscle actin and of extracellular matrix (ECM) mediated by activated hepatic collagen type I in HSC‑T6 cells. -
Intraflagellar Transport Dynein Is Autoinhibited by Trapping of Its Mechanical and Track-Binding Elements
Intraflagellar Transport Dynein is Autoinhibited by Trapping of its Mechanical and Track-Binding Elements Katerina Toropova1, Miroslav Mladenov1, Anthony J. Roberts1* 1Institute of Structural and Molecular Biology, Birkbeck, London, United Kingdom. *Correspondence should be addressed to A.J.R. ([email protected]). ABSTRACT Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT. 1 Introduction A major question in structural cell biology is how micrometer-scaled organelles are constructed and maintained. Cilia (also known as eukaryotic flagella) are multi-functional organelles that emanate from almost all cell types in the human body and many other eukaryotes 1. Non-motile cilia serve as “signaling antennae” in processes as diverse as morphogenesis, mechanosensation, and olfaction 2. -
Rap1a and Rap1b Ras-Family Proteins Are Prominently Expressed in the Nucleus of Squamous Carcinomas: Nuclear Translocation of GTP-Bound Active Form
Oncogene (2003) 22, 6243–6256 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form Raj S Mitra1,4, Zhaocheng Zhang1,4, Bradley S Henson1, David M Kurnit2, Thomas ECarey 1,3, Nisha J D’Silva*,1 1Department of Oral Medicine, Pathology and Oncology, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA; 2Departments of Pediatrics and Human Genetics, University of Michigan Medical School, Ann Arbor, MI 481091, USA; 3Department of Otolaryngology, Laboratory of Head and Neck Cancer Biology, The University of Michigan Medical School and the Comprehensive Cancer Center, Ann Arbor, MI 48109-0506, USA We recently showed that rap1 regulates growth and exclusively in eukaryotes (Takai et al., 2001). Those proliferation in normal keratinocytes, which provoked us SMGs that have been identified (currently 4100) have to investigate its expression and regulation in malignant homology to ras, and belong to one of the five cells. Rap1 is variably expressed in whole cell lysates of subfamilies namely ras, rho, rab, sar1/arf and ran squamous cell carcinoma (SCC) cell lines. Immunoblot (Takai et al., 2001). These ras-like proteins shuttle analysis of nuclear and cytosolic fractions and immuno- between inactive GDP- and active GTP-bound states. histochemistry revealed that in addition to cytoplasmic Ras subfamily proteins, including ras and rap1, are key expression, SCC cells also exhibit prominent punctate players in receptor-linked signaling pathways that rap1 expression in the nucleus. -
Distinct Actions of Rab3 and Rab27 Gtpases on Late Stages of Exocytosis of Insulin
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12182 Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin Victor A. Cazares1,2, Arasakumar Subramani1, Johnny J. Saldate1,2, Widmann Hoerauf1,2 and Edward L. Stuenkel1,2,∗ 1Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA 2Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA ∗Corresponding author: Edward L. Stuenkel, [email protected] Abstract Rab GTPases associated with insulin-containing secretory granules (SGs) readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 are key in targeting, docking and assembly of molecular complexes GTPases, but not of Rab27A, is essential for a kinetically rapid filling of governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A Rab27A are associated with insulin granules, yet elucidation of the GTPases demonstrate considerable functional overlap in building the distinct roles of these Rab families on exocytosis remains unclear. To readily releasable granule pool. Hence, while Rab3 and Rab27A coop- define specific actions of these Rab families we employ Rab3GAP erate to generate release-ready SGs in β-cells, they also direct unique and/or EPI64A GTPase-activating protein overexpression in β-cells from kinetic and functional properties of the exocytotic pathway. wild-type or Ashen mice to selectively transit the entire Rab3 fam- Keywords exocytosis, GTPase, insulin secretion, membrane fusion, Rab ily or Rab27A to a GDP-bound state. Ashen mice carry a sponta- proteins neous mutation that eliminates Rab27A expression. -
Discrete Dynamic Model of the Mammalian Sperm Acrosome Reaction
bioRxiv preprint doi: https://doi.org/10.1101/2021.03.07.434290; this version posted March 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Discrete dynamic model of the mammalian sperm 2 acrosome reaction: the influence of acrosomal pH 3 and biochemical heterogeneity 1;2∗ 3;4 2;5 4 Andr´esAldana , Jorge Carneiro , Gustavo Mart´ınez-Mekler and Alberto Darszon 1∗ 1 5 Departamento de Gen´eticay Fisiolog´ıaMolecular, Instituto de Biotecnolog´ıa, 6 Universidad Nacional Aut´onomade M´exico,Morelos, M´exico 2 7 Centro de Ciencias de la Complejidad, Universidad Nacional Aut´onomade M´exico, 8 Ciudad de M´exico,M´exico 3 9 Instituto Gulbenkian de Ci^encia,Oeiras, Oeiras, Portugal 4 10 Instituto de Tecnologia Qu´ımica e Biol´ogica Ant´onioXavier, Universidade Nova, 11 Oeiras, Portugal 5 12 Instituto de Ciencias F´ısicas,Universidad Nacional Aut´onomade M´exico,Morelos, 13 M´exico 14 Correspondence: 15 Alberto Darszon 16 [email protected] 17 Andr´esAldana 18 [email protected] 19 Abstract 20 The acrosome reaction (AR) is an exocytotic process essential for mammalian fertil- 21 ization. It involves diverse biochemical and physiological changes that culminate in the 22 release of the acrosomal content to the extracellular medium as well as a reorganization 23 of the plasma membrane (PM) that allows sperm to interact and fuse with the egg. -
Site-Specific Processing of Ras and Rap1 Switch I by a MARTX Toxin
ARTICLE Received 8 Jan 2015 | Accepted 1 May 2015 | Published 8 Jun 2015 DOI: 10.1038/ncomms8396 OPEN Site-specific processing of Ras and Rap1 Switch I by a MARTX toxin effector domain Irena Antic1,*, Marco Biancucci1,*, Yueming Zhu2, David R. Gius2 & Karla J.F. Satchell1 Ras (Rat sarcoma) protein is a central regulator of cell growth and proliferation. Mutations in the RAS gene are known to occur in human cancers and have been shown to contribute to carcinogenesis. In this study, we show that the multifunctional-autoprocessing repeats-in- toxin (MARTX) toxin-effector domain DUF5Vv from Vibrio vulnificus to be a site-specific endopeptidase that cleaves within the Switch 1 region of Ras and Rap1. DUF5Vv processing of Ras, which occurs both biochemically and in mammalian cell culture, inactivates ERK1/2, thereby inhibiting cell proliferation. The ability to cleave Ras and Rap1 is shared by DUF5Vv homologues found in other bacteria. In addition, DUF5Vv can cleave all Ras isoforms and KRas with mutations commonly implicated in malignancies. Therefore, we speculate that this new family of Ras/Rap1-specific endopeptidases (RRSPs) has potential to inactivate both wild-type and mutant Ras proteins expressed in malignancies. 1 Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Ward 6-225, Chicago, Illinois 60611, USA. 2 Department of Radiation Oncology and Pharmacology, Feinberg School of Medicine, Northwestern University, 303 East Superior Avenue, Lurie 3-119, Chicago, Illinois 60611, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressedto K.J.F.S. -
The Dynein Family at a Glance Peter Höök and Richard B
Cell Science at a Glance 4369 The dynein family at a functions. Although at least 14 classes of accessory subunits bind; and a ~380 kDa kinesin and 17 classes of myosin have motor domain. The motor domain glance been identified, the dyneins fall into contains six discernible AAA ATPase Peter Höök* and Richard B. only two major classes, axonemal and units, identifying the dynein HC as a Vallee cytoplasmic dyneins, based on both divergent member of the AAA+ family functional and structural criteria. of ATPases (Neuwald et al., 1999). Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA. Axonemal dyneins are responsible for Members of the AAA+ family are *Author for correspondence (e-mail: ciliary and flagellar beating; cytoplasmic involved in a very wide range of [email protected]) dyneins are involved in intracellular functions but have a common feature: the Journal of Cell Science 119, 4369-4371 transport, mitosis, cell polarization and formation of ring-shaped oligomeric Published by The Company of Biologists 2006 directed cell movement complexes of the AAA ATPase module. doi:10.1242/jcs.03176 Within the AAA+ proteins, dynein All dynein forms that have occupies a divergent branch along with Three families of cytoskeletal motor been identified biochemically are midasin (Iyer et al., 2004). This branch protein – the myosins, kinesins and multisubunit proteins. Each has one to is characterized by the incorporation of dyneins – have evolved to mediate three heavy chains (HCs) of >500 kDa; all six AAA modules within a single transport of cells and of structures and these correspond to the number of giant polypeptide. -
Small Gtpases of the Ras and Rho Families Switch On/Off Signaling
International Journal of Molecular Sciences Review Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1, Patricia Gálvez-Martín 3,4 , Hadriano M Lacerda 5, Alejandro Lucia 6,7, Francisco Llavero 1,6,* and José Luis Zugaza 1,2,8,* 1 Achucarro Basque Center for Neuroscience, Science Park of the Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain; [email protected] (A.A.S.); [email protected] (M.L.M.) 2 Department of Genetics, Physical Anthropology, and Animal Physiology, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, 180041 Granada, Spain; [email protected] 4 R&D Human Health, Bioibérica S.A.U., 08950 Barcelona, Spain 5 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; [email protected] 6 Faculty of Sport Science, European University of Madrid, 28670 Madrid, Spain; [email protected] 7 Research Institute of the Hospital 12 de Octubre (i+12), 28041 Madrid, Spain 8 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: [email protected] (F.L.); [email protected] (J.L.Z.) Received: 25 July 2020; Accepted: 29 August 2020; Published: 31 August 2020 Abstract: Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs).