Isolation of Clathrin‐Coated Vesicles by Differential and Density Gradient Centrifugation

Martine Girard1, Patrick D. Allaire1, Francois Blondeau1, Peter S. McPherson1

1 Montreal Neurological Institute, McGill University, Montreal, Quebec
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.13
DOI:  10.1002/0471143030.cb0313s26
Online Posting Date:  April, 2005
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Abstract

Clathrin‐coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans‐Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D2O‐sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described. Furthermore, we describe a protocol in which differential centrifugation and a series of discontinuous sucrose gradients are used to isolate CCVs from rat liver. An approach to analyzing CCV purity by electron microscopy is also described.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of CCVs from Adult Rat Brain using Differential and Density Gradient Centrifugation
  • Alternate Protocol 1: Isolation of CCVs from Developing Rat Brain using Differential and Density Gradient Centrifugation
  • Alternate Protocol 2: Isolation of CCVs from Cell Lines
  • Basic Protocol 2: Isolation of CCVs from Adult Rat Liver using Differential and Density Gradient Centrifugation
  • Support Protocol 1: Analysis of CCV Purity by Electron Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of CCVs from Adult Rat Brain using Differential and Density Gradient Centrifugation

  Materials
  • 10 Sprague‐Dawley rats (150 to 200 g each)
  • 1× buffer A (see recipe) containing protease inhibitors (see recipes)
  • Ficoll‐sucrose solution (see recipe)
  • Deuterium oxide (D 2O; heavy water)–sucrose solution (see recipe)
  • 20% and 50% sucrose solutions in 1× buffer A (see recipe)
  • Glass‐Teflon homogenizers (assorted sizes; Wheaton) fitted to a power head
  • Sorvall high‐speed centrifuge equipped with SS‐34 fixed‐angle rotor (or equivalent)
  • Battery‐operated pipet filler
  • Ultracentrifuge with fixed‐angle (Sorvall T‐865, Beckman 45Ti, or equivalent) and swinging‐bucket (Sorvall AH‐629, Beckman SW‐28, or equivalent) rotors
  • 25‐G, ⅝‐in. needle
  • 13‐ml thin‐walled centrifuge tubes
  • 2‐mm‐diameter glass capillary tubes
  • Two‐chamber gradient maker
  • Peristaltic pump
  • Additional reagents and equipment for standard protein assays ( appendix 3H)
NOTE: Protease inhibitors are added (to a concentration of 1×) to 1× buffer A within 30 min of buffer use, except for PMSF, which should be added immediately after the buffer comes into contact with a protein sample.

Alternate Protocol 1: Isolation of CCVs from Developing Rat Brain using Differential and Density Gradient Centrifugation

  • 15 litters of P5 rat pups
  • P5 homogenization buffer (see recipe) containing protease inhibitors (see recipes)
  • Control buffer (see recipe) containing protease inhibitors (see recipes)
  • 10× buffer A (see recipe)
NOTE: Protease inhibitors are added (to a concentration of 1×) to P5 homogenization buffer, control buffer, and 1× buffer A within 30 min of buffer use, except for PMSF, which should be added to a given solution immediately after the solution comes into contact with protein samples.

Alternate Protocol 2: Isolation of CCVs from Cell Lines

  • HEK‐293, COS‐7, or HeLa cells
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Rubber stopper cut in half lengthwise (to yield straight edges along top and bottom)
  • Additional reagents and equipment for mammalian cell culture (unit 1.1)
NOTE: Protease inhibitors are added (to a concentration of 1×) to 1× buffer A within 30 min of buffer use, except for PMSF, which is added immediately after the buffer comes into contact with a protein sample.

Basic Protocol 2: Isolation of CCVs from Adult Rat Liver using Differential and Density Gradient Centrifugation

  Materials
  • Ten Sprague‐Dawley rats (150 to 200 g)
  • Liver homogenization buffer (see recipe) containing protease inhibitors (see recipes)
  • 10× and 1× MES buffers (see recipes) containing protease inhibitors (see recipes)
  • 5%, 10%, 20%, 30%, 40%, 45%, 50%, and 55%, and 60% (w/v) sucrose solutions in 1× MES buffer (see recipe)
  • Glass‐Teflon homogenizers (assorted sizes; Wheaton) fitted with power heads
  • Sorvall high‐speed centrifuge equipped with SS‐34 fixed‐angle rotor (or equivalent)
  • Battery‐operated pipet filler
  • Ultracentrifuge with fixed‐angle (Sorvall T‐865, Beckman 45Ti, or equivalent) and swinging‐bucket (Sorvall AH‐629, Beckman SW‐28, or equivalent) rotors
  • 16‐ and 36‐ml polypropylene tubes for Sorvall AH‐629 (or equivalent) swinging‐bucket rotor
  • 2‐mm‐diameter glass capillary tubes
NOTE: Protease inhibitors are added to liver homogenization buffer and 1× MES buffer within 30 min of buffer use, except for PMSF, which is added to a given solution immediately after the solution comes into contact with protein samples.

Support Protocol 1: Analysis of CCV Purity by Electron Microscopy

  Materials
  • Fresh CCVs in 1× buffer A or 1× MES buffer (see protocol 1, protocol 4, protocol 2, or protocol 3)
  • 1× buffer A (see recipe), filtered
  • 1× MES buffer (see recipe), filtered (use only if CCVs were prepared using protocol 4)
  • 25% (w/v) glutaraldehyde solution
  • Nitrogen gas
  • Sodium cacodylate buffer (see recipe), 4°C
  • Osmium tetroxide solution (see recipe), 4°C
  • Tannic acid solution (see recipe), 4°C
  • Sodium sulfate solution (see recipe), 4°C
  • Uranyl acetate solution (see recipe), 4°C, filtered
  • Maleate buffer (see recipe), 4°C
  • 50%, 70%, 90%, 95%, and 100% (all v/v) ethanol
  • Propylene oxide
  • Epon solution (see recipe)
  • 3:1, 1:1, and 1:3 (all w/w) Epon solution (see recipe)/propylene oxide
  • Platform rocker
  • Filter unit (Baudhuin et al., ; also see annotation to step ), including 13‐mm‐diameter Millipore filter discs (pore size, 0.22 µm)
  • Glass vial
  • Small paraffin‐coated polypropylene containers (Peel‐A‐Way disposable embedding molds; Polysciences) with (see recipe) and without a coating of hardened Epon on the inside bottom surface
NOTE: Osmium tetroxide, glutaraldehyde, and propylene oxide must be handled and disposed of in accordance with protocols approved by Institutional Chemical Safety and Chemical Waste Committees. Uranyl acetate is radioactive and therefore must be handled and disposed of in accordance with protocols approved by an Institutional Radiation Safety Committee, as well as in accordance with protocols approved by Institutional Chemical Safety and Chemical Waste Committees.NOTE: All steps involving propylene oxide should be performed in a fume hood, as this compound is highly volatile.
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Figures

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Literature Cited

Literature Cited
   Baudhuin, P., Evrard, P., and Berthet, J. 1967. Electron microscopic examination of subcellular fractions: I: The preparation of representative samples from suspensions of particles. J. Cell Biol. 32:181‐191.
   Blitz, A.L., Fine, R.E., and Toselli, P.A. 1977. Evidence that coated vesicles isolated from brain are calcium‐sequestering organelles resembling sarcoplasmic reticulum. J. Cell Biol. 75:135‐147.
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   Daiss, J.L. and Roth, T.F. 1983. Isolation of coated vesicles: Comparative studies. Methods Enzymol. 98:337‐349.
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   Pearse, B.M. 1982. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc. Natl. Acad. Sci. U.S.A. 79:451‐455.
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   Rothman, J.E. and Fine, R.E. 1980. Coated vesicles transport newly synthesized membrane glycoproteins from endoplasmic reticulum to plasma membrane in two successive stages. Proc. Natl. Acad. Sci. U.S.A. 77:780‐784.
   Saito, S., Fujita, T., Komiya, Y., and Igarashi, M. 1992. Biochemical characterization of nerve growth cones isolated from both fetal and neonatal rat forebrains: The growth cone particle fraction mainly consists of axonal growth cones in both stages. Brain Res. Dev. Brain Res. 65:179‐184.
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   Wasiak, S., Legendre‐Guillemin, V., Puertollano, R., Blondeau, F., Girard, M., de Heuvel, E., Boismenu, D., Bell, A.W., Bonifacino, J.S., and McPherson, P.S. 2002. Enthoprotin: A novel clathrin‐associated protein identified through subcellular proteomics. J. Cell Biol. 158:855‐862.
   Woods, J.W., Woodward, M.P., and Roth, T.F. 1978. Common features of coated vesicles from dissimilar tissues: Composition and structure. J. Cell Sci. 30:87‐97.
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