Microtubule/Organelle Motility Assays

Clare M. Waterman‐Storer1

1 University of North Carolina, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.1
DOI:  10.1002/0471143030.cb1301s00
Online Posting Date:  May, 2001
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Abstract

This unit describes an in vitro assay that uses video‐enhanced differential interference contrast (VE‐DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme‐nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE‐DIC microscopy.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: MT/Organelle Motility Assays
  • Support Protocol 1: Preparation of Simple Perfusion Chambers and Coverslips
  • Support Protocol 2: Preparation of Sea Urchin Sperm Axonemes
  • Support Protocol 3: Preparation of Porcine Brain Tubulin
  • Support Protocol 4: Preparation of Rat Liver Cell Cytosol
  • Support Protocol 5: Preparation of Rat Liver Organelle Fractions
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: MT/Organelle Motility Assays

  Materials
  • Axoneme fragments (see protocol 3)
  • Golgi or ER membranes (see protocol 6)
  • 45 µM purified brain tubulin (see protocol 4)
  • Rat liver cytosol (see protocol 5)
  • PM buffer (see recipe)
  • recipePM buffer containing 1 mM GTP
  • 20× energy regeneration system (see recipe)
  • 15 mM MgGTP, prepared by diluting 100 mM MgGTP stock (see recipe) in recipePM buffer
  • Valap (see recipe)
  • Simple perfusion chambers (see protocol 2)
  • Filter paper cut into 2‐cm squares
  • Humid chamber made of a 90‐mm glass petri dish containing moist paper towels
  • High‐resolution VE‐DIC microscope system (as described in Salmon and Tran, or equivalent)

Support Protocol 1: Preparation of Simple Perfusion Chambers and Coverslips

  Materials
  • Versa Clean dish detergent (Fisher)
  • 1 mM EDTA
  • 70% and 100% ethanol
  • 22 × 22–mm no. 1.5 coverslips (Corning)
  • Water‐bath sonicator
  • Double‐stick tape
  • Clay‐Adams precleaned Goldseal 3 × 1–in. microscope slides (Becton Dickinson)
  • Dumont no. 5 forceps

Support Protocol 2: Preparation of Sea Urchin Sperm Axonemes

  Materials
  • 4 male S. purpuratus sea urchins (Marinus)
  • 0.55 M KCl
  • Artificial sea water (mixes available from aquarium supply stores; prepare per manufacturer's instructions)
  • 20% (w/v) sucrose in distilled water
  • Isolation buffer (see recipe)
  • High‐salt buffer (see recipe)
  • Isolation buffer (see recipe) containing 50% glycerol
  • 60‐ml syringe with 18‐G needle
  • Tabletop clinical centrifuge
  • Refrigerated superspeed centrifuge (Sorvall RC‐5B) with Sorvall SS‐34 rotor (or equivalent)
  • 50‐ml polycarbonate centrifuge tubes (e.g., Sorvall)
  • 50‐ml Dounce glass homogenizer with type A and B pestles
NOTE: Because it is impossible to tell what sex a sea urchin is until it sheds gametes from the pores located on its dorsal surface, order at least twice as many animals as needed.

Support Protocol 3: Preparation of Porcine Brain Tubulin

  Materials
  • 100‐g P‐11 cellulose phosphate fibrous cation exchanger (Whatman)
  • 0.1 M HCl
  • 0.1 M and 10 M NaOH
  • 0.1 M MgSO 4
  • 10× and 1× column buffer (see recipe)
  • 3 fresh pig brains (use <3 hr after slaughter)
  • Homogenization buffer (see recipe; freshly prepared)
  • PM buffer (see recipe)
  • 100 mM MgATP (see recipe)
  • PMG buffer (see recipe)
  • 100 mM MgGTP (see recipe)
  • 1 M dithiothreitol (DTT; appendix 2A)
  • Glutamic acid, sodium salt
  • 2‐liter sintered‐glass filter funnel and 2‐liter sidearm Erlenmeyer flask
  • Waring blender
  • Temperature‐controlled ultracentrifuge (Beckman L7‐55) with Beckman 50.2Ti rotor (or equivalent)
  • 31.5‐ml thick‐walled polycarbonate ultracentrifuge tubes (e.g., Beckman) with screw caps
  • 30‐ml Dounce type A glass homogenizer
  • 44 × 250–mm adjustable volume column for low‐pressure liquid chromatography (e.g., Amicon model #95240 or equivalent)
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)

Support Protocol 4: Preparation of Rat Liver Cell Cytosol

  Materials
  • Fresh or flash‐frozen (Pel‐Freez) rat livers
  • PBS ( appendix 2A)
  • Homogenization buffer (see recipe)
  • recipeHomogenization buffer containing 0.5 mM MgGTP (from 100 mM MgGTP stock; see recipe)
  • PM buffer (see recipe) containing 0.25 M sucrose
  • Superspeed centrifuge (Sorvall RC‐5B) with Sorvall SS‐34 rotor (or equivalent)
  • Ultracentrifuge (Beckman L7‐55) with Beckman 50.2Ti and SW‐28 rotors (or equivalents)
  • 20‐ml glass homogenizer with Teflon pestle
  • Homogenizer or drill press
  • 50‐ml polycarbonate centrifuge tubes (e.g., Sorvall)
  • 31.5‐ml thick‐walled polycarbonate ultracentrifuge tubes (e.g., Beckman) with screw caps
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)

Support Protocol 5: Preparation of Rat Liver Organelle Fractions

  • Homogenization buffer (see recipe) containing 0.5 mM MgGTP (from 100 mM stock; see recipe) and 0.25 M sucrose
  • Homogenization buffer containing 0.5 mM MgGTP
  • 2.3 M sucrose
  • PM buffer (see recipe) containing 0.25 M sucrose and 0.5 mM GTP
  • 3 M KI stock (optional)
  • 0.5 M EDTA stock (optional; appendix 2A)
  • recipe0.5 M Na 2CO 3 stock, pH 11.5 (optional)
  • recipePM buffer containing 0.25 M sucrose (optional)
  • 25‐ml Ultraclear ultracentrifuge tubes (e.g., Beckman)
  • Beckman SW‐28 and 50.2Ti rotors or equivalents
  • 31.5‐ml thick‐walled polycarbonate ultracentrifuge tubes (e.g., Beckman) with screw caps
  • TLA tabletop ultracentrifuge (Beckman) with TLS‐55 swinging bucket rotor or equivalent and mini‐ultracentrifuge tubes (e.g., Beckman)
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   Coue et al., 1991 and Waterman‐Storer et al., 1995. See above.
  In these papers, in vitro assays show that organelles can be moved by microtubule polymerization and depolymerization in the absence of motor ATPase activity.
   Dabora and Sheetz, 1988 and Vale, and Hotani, 1988. See above.
  These papers were the first to use in vitro assays to reconstitute microtubule motor‐based organelle motility.
   Klausner et al., 1992. See above.
  This review clearly describes the roles and regulation of coat proteins in the morphology and movement of organelles through the secretory pathway.
  Salmon and Tran, 1998. See above.
  This paper provides an in‐depth description of the theory and practice of setting up the microscope system required to perform the MT/organelle motility assay.
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