Use of Dynasore, the Small Molecule Inhibitor of Dynamin, in the Regulation of Endocytosis

https://doi.org/10.1016/S0076-6879(07)38006-3Get rights and content

Abstract

The large GTPase dynamin is essential for clathrin‐dependent coated‐vesicle formation. Dynasore is a cell‐permeable small molecule that inhibits the GTPase activity of dynamin1, dynamin2 and Drp1, the mitochondrial dynamin. Dynasore was discovered in a screen of ∼16,000 compounds for inhibitors of the dynamin2 GTPase. Dynasore is a noncompetitive inhibitor of dynamin GTPase activity and blocks dynamin‐dependent endocytosis in cells, including neurons. It is fast acting (seconds) and its inhibitory effect in cells can be reversed by washout. Here we present a detailed synthesis protocol for dynasore, and describe a series of experiments used to analyze the inhibitory effects of dynasore on dynamin in vitro and to study the effects of dynasore on endocytosis in cells.

Introduction

Dynamin functions in membrane tubulation and fission of budding vesiculo‐tubular structures. It is essential for clathrin‐dependent endocytosis from the plasma membrane, for the fission of plasma membrane caveolae to form free transport vesicles, and for vesicle formation at the trans‐Golgi network (Cao 2000, Corda 2002, Nichols 2003, Takei 2005). It also appears to participate in actin comet formation and transport of macropinosomes and in the function of podosomes, probably by interaction with actin‐binding proteins. A related role in membrane fission has also been assigned to homolog proteins of dynamin (Dnm1 in mammalian cells and Drp1 in yeast) in the biogenesis of mitochondria and peroxisomes (Koch 2005, Schrader 2006).

Section snippets

Dynamin

Dynamin (for recent reviews, see Kirchhausen 1999, Praefcke 2004, Thompson 2001, Wiejak 2002, Yang 1999) is a multidomain protein of ∼100 kDa containing a GTPase module, a lipid‐binding pleckstrin homology (PH) domain, a GTPase effector domain (GED), and a proline/arginine‐rich C‐terminal segment (PRD) containing amino‐acid sequences that bind to the SH3 domains of other proteins. Dynamin is unusual among GTPases because its affinity for GDP and GTP is rather low (10 to 25 μM) when compared to

Dynamin and the Actin Cytoskeleton

Dynamin, alone or in combination with amphiphysin, can form membrane tubes of dimensions similar to those on collars of deeply invaginated clathrin coated pits (Takei et al., 1999). This was the first indication that a coated pit might not be a required template for dynamin function. Dynamin colocalizes with actin in growth cones (Torre et al., 1994), and binds to a number of proteins involved in the regulation of actin cytoskeleton. They include profilin, cortactin, syndapin (a partner of

The “Chemical Genetics” Discovery Approach

In the last decade, a number of laboratories have engaged in medium‐ and high‐throughput phenotype‐based screens of libraries of chemical compounds in an approach dubbed “chemical genetics.” The stated goal is to identify small molecules that disrupt the function of proteins or protein complexes (Gura, 2000). The Institute of Chemistry and Cell Biology (ICCB) at Harvard Medical School, now the ICCB‐Longwood, is a major screening center for this approach. Using its facilities, we have identified

Why Do We Need Interfering Small Molecules?

Interfering small molecules allow researchers to freeze biological processes at interesting points. This is particularly useful in the investigation of transient phenomena, such as membrane traffic. Much of the recent progress in understanding protein trafficking pathways has been achieved using approaches based on genetic dissection and morphological and biochemical analysis. However, the dynamic nature of these events (Cole et al., 1996) makes it particularly difficult to use slow techniques

Synthesis of Dynasore

We identified dynasore in a screen of ∼16,000 compounds (part of the Diverset E, Chembridge Library) for inhibition of the GST‐Grb2‐stimulated GTPase activity of dynamin2 (Macia et al., 2006) (assay described below). Here we describe our synthesis of dynasore (Fig. 6.3). Our approach is based on the strategy of Ling et al. (2001) for the synthesis of benzoic acid arylidenehydrazides. Dynasore (C18H14N2O4, molecular weight 322.31 g/mol) (1) is easily synthesized on gram scale in two steps from

Storage Conditions for Dynasore

Dynasore is stored as a dry solid under argon in the dark at −20°. Dynasore can also be stored at −20° or −80° in the dark (no need to flash freeze) as a 200‐mM solution in DMSO under argon. Aliquots of 10 to 20 μl are stored in 0.5‐ml microcentrifuge tubes. After adding argon and closing the cap, the microcentrifuge tubes are sealed with parafilm. To avoid the capture of moisture, the DMSO aliquots of dynasore are warmed up to room temperature before opening. The aqueous solution of dynasore

Protein expression

We express human dynamin in SF9 insect cells (Spodoptera frugiperda, GIBCO‐BRL, Gaithersburg, MD) grown in SF‐900 II SFM (GIBCO‐BRL) essentially as described (Damke et al., 2001). Using the Bac‐to‐Bac baculovirus expression system (GIBCO‐BRL), a full‐length, cDNA encoding human dynamin1 containing a 6‐His‐tag at the N‐terminus is subcloned into the baculovirus vector pFastBac. A bacmid is generated after transposition in Escherichia coli, and several independent clones are selected to transfect

Buffers and Reagents

  • Acid wash buffer: glycine 0.1 M, pH 2.5, and 150 mM NaCl

  • Penicillin/streptomycin solution: 1× corresponds to 100 units/ml of penicillin and 100 mg/ml of streptomycin

  • HEPES column buffer (HCB): 20 mM HEPES, pH 7, 2 mM EGTA, 1 mM MgCl2, and 1 mM dithiothreitol (DTT)

  • HCB250: HCB supplemented with 250 mM NaCl

  • K‐PO4 hydroxyapatite buffer: 50 or 500 mM, pH 7.2, 1 mM DTT, and 1 μM calpain inhibitor

  • GTPase buffer: 10 mM Tris, pH 7.2, 2 mM MgCl2, and 20 μM GTPγP32 (2000 dpm/pmol)

  • Acid‐washed charcoal: 10%

Acknowledgment

We thank Matthew D. Shair for use of his laboratory facilities to carry out the synthesis of dynasore. We also thank members of the Kirchhausen lab who participated in the discovery and characterization of dynasore activity, including Chris Brunner, Marcelo Erlich, Ramiro Massol, Werner Boll, and Emmanuel Boucrot. We acknowledge support from the National Institutes of Health (grants GM GM62566, GM03548, and GM075252 to T. K.).

References (59)

  • M. Schrader

    Shared components of mitochondrial and peroxisomal division

    Biochim. Biophys. Acta

    (2006)
  • S.M. Sweitzer et al.

    Dynamin undergoes a GTP‐dependent conformational change causing vesiculation

    Cell

    (1998)
  • P.A. Takizawa et al.

    Complete vesiculation of Golgi membranes and inhibition of protein transport by a novel sea sponge metabolite, ilimaquinone

    Cell

    (1993)
  • H.M. Thompson et al.

    Dynamin: switch or pinchase?

    Curr. Biol.

    (2001)
  • E. Torre et al.

    Dynamin 1 antisense oligonucleotide treatment prevents neurite formation in cultured hippocampal neurons

    J. Biol. Chem.

    (1994)
  • W.N. Yang et al.

    Endocytosis: Is dynamin a ‘blue collar’ or ‘white collar’ worker?

    Curr. Biol.

    (1999)
  • H. Cao et al.

    Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin

    J. Cell Sci.

    (2000)
  • J. Cerny et al.

    The small chemical vacuolin‐1 inhibits Ca(2+)‐dependent lysosomal exocytosis but not cell resealing

    EMBO Rep.

    (2004)
  • N.B. Cole et al.

    Diffusional mobility of Golgi proteins in membranes of living cells

    Science

    (1996)
  • D. Corda et al.

    Molecular aspects of membrane fission in the secretory pathway

    Cell. Mol. Life Sci.

    (2002)
  • H. Damke et al.

    Clathrin‐independent pinocytosis is induced in cells overexpressing a temperature‐sensitive mutant of dynamin

    J. Cell Biol.

    (1995)
  • H. Damke et al.

    Expression, purification, and functional assays for self association of dynamin-1.

    Methods Enzymol

    (1995)
  • Y. Feng et al.

    Exo1: A new chemical inhibitor of the exocytic pathway

    Proc. Natl. Acad. Sci. USA

    (2003)
  • Y. Feng et al.

    Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans‐Golgi network but not the Golgi apparatus in Exo2‐treated cells

    EMBO Rep.

    (2004)
  • Q. Guo et al.

    Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon‐COP

    J. Cell Biol.

    (1994)
  • T. Gura

    A chemistry set for life

    Nature

    (2000)
  • T. Hill et al.

    Small molecule inhibitors of dynamin I GTPase activity: development of dimeric tyrphostins

    J. Med. Chem.

    (2005)
  • R.B. Kelly

    New twists for dynamin

    Nat. Cell Biol.

    (1999)
  • M.M. Kessels et al.

    Mammalian Abp1, a signal‐responsive F‐actin‐binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

    J. Cell Biol.

    (2001)

    • Cited by (350)

    • Aichivirus A1 replicates in human intestinal epithelium and bronchial tissue: Lung–gut axis?

      2024, Virus Research

    • Uptake Pathway of Styrene Maleic Acid Copolymer-Coated Lipid Emulsions Under Acidic Tumor Microenvironment

      2024, Journal of Pharmaceutical Sciences

    • Basal interaction of the orphan receptor GPR101 with arrestins leads to constitutive internalization

      2024, Biochemical Pharmacology

    • A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis

      2024, Methods in Enzymology

    • Pre-exposure to titanium or iron oxide nanoparticles suppresses the subsequent cellular uptake of gold nanoparticles

      2023, Science of the Total Environment

    • Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion

      2024, Cellular and Molecular Life Sciences
    View all citing articles on Scopus
    View full text
    Copyright © 2008 Elsevier Inc. All rights reserved.