Isolation of Melanosomes

Hidenori Watabe1, Tsuneto Kushimoto1, Julio C. Valencia1, Vincent J. Hearing1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.14
DOI:  10.1002/0471143030.cb0314s26
Online Posting Date:  April, 2005
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Abstract

The methods used to purify early and late melanosomes are detailed. These methods include the use of highly pigmented cells to maximize recovery and the use of various sucrose density gradients to separate melanosome fractions based on their density (which is determined in large part by the amount of dense melanin pigment that they contain). Early melanosomes lacking pigment must be further purified using free‐flow electrophoresis.

Keywords: melanosomes; free‐flow electrophoresis; subcellular fractionation; melanin; pigment

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Early and/or Late Melanosomes from Cultured Melanoma Cells by Density Gradient Centrifugation
  • Alternate Protocol 1: Preparation of Early Melanosomes from Cultured Melanoma Cells by Inverse Density Gradient Centrifugation
  • Alternate Protocol 2: Preparation of Early Melanosomes from Cultured Pigmented Melanoma Cells by Density Gradient Centrifugation and Free‐Flow Electrophoresis (FFE)
  • Basic Protocol 2: Preparation of Early and/or Late Melanosomes from Melanoma Tissues by Density Gradient Centrifugation
  • Support Protocol 1: Tyrosinase Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Early and/or Late Melanosomes from Cultured Melanoma Cells by Density Gradient Centrifugation

  Materials
  • MNT‐1 human melanoma cells
  • Dulbecco's modified Eagle medium (DMEM), supplemented (see recipe)
  • PBS ( appendix 2A)
  • Cell homogenization medium–1 (CHM‐1; see recipe)
  • Sucrose step gradient, 1.0 to 2.0 M (see recipe)
  • Melanosome wash buffer (MWB; see recipe)
  • 150‐cm2 culture dishes (Becton Dickinson Labware)
  • Tabletop centrifuge, 4°C
  • Dounce glass/glass homogenizer
  • 15‐ml centrifuge tubes
  • 36.5‐ml polyallomer centrifuge tubes (Seton)
  • Beckman SW‐28 swinging‐bucket rotor
  • 3‐in., 20‐G needle attached to a 12‐ml syringe
  • 50‐ml Oak Ridge centrifuge tubes (Nalge Nunc)
  • Sorvall SS‐34 centrifuge rotor
  • Additional reagents and equipment for mammalian cell culture (unit 1.1)

Alternate Protocol 1: Preparation of Early Melanosomes from Cultured Melanoma Cells by Inverse Density Gradient Centrifugation

  • MNT‐1 human melanoma cells or SK‐MEL‐28 amelanotic human melanoma cells (American Type Culture Collection)
  • DMEM, supplemented (for MNT‐1 cells; see recipe), or minimal essential medium, supplemented (for SK‐MEL‐28 cells; see recipe)
  • 2.0 M sucrose (see recipe)
  • Sucrose step gradient, 0.25 to 1.8 M (see recipe)
  • Sucrose step gradient, 0.25 to 1.4 M (see recipe)

Alternate Protocol 2: Preparation of Early Melanosomes from Cultured Pigmented Melanoma Cells by Density Gradient Centrifugation and Free‐Flow Electrophoresis (FFE)

  • 0.3% (w/v) trypsin
  • 0.6% (w/v) soybean trypsin inhibitor
  • FFE chamber buffer: 0.25 M sucrose in 1× TEA, pH 7.4 (see recipe for 100×)
  • Octopus‐PZE FFE apparatus (FFE Weber)

Basic Protocol 2: Preparation of Early and/or Late Melanosomes from Melanoma Tissues by Density Gradient Centrifugation

  Materials
  • B16 melanoma cells
  • Eagle's minimal essential medium (Sigma) containing 10% (v/v) heat‐inactivated FBS (unit 1.2; HyClone)
  • C57BL/6 mice
  • PBS ( appendix 2A)
  • Cell homogenization medium–2 (CHM‐2; see recipe)
  • Sucrose step gradient, 1.0 to 2.0 M (see recipe)
  • MWB (see recipe)
  • 150‐cm2 culture dishes (Bacton Dickinson Labware)
  • Potter‐Elvehjem glass homogenizer
  • 15‐ml centrifuge tubes
  • Tabletop centrifuge, 4°C
  • 36.5‐ml polyallomer centrifuge tubes (Seton)
  • Beckman SW‐28 swinging‐bucket rotor
  • 3‐in., 20‐G needle attached to a 12‐ml syringe
  • 50‐ml Oak Ridge centrifuge tubes (Nalge Nunc)
  • Sorvall SS‐34 centrifuge rotor
  • Additional reagents and equipment for mammalian cell culture (unit 1.1)
NOTE: All protocols using live animals must first be reviewed and approved by an institutional animal care and use committee (IACUC) and must conform to governmental regulations regarding the care and use of laboratory animals.

Support Protocol 1: Tyrosinase Assay

  Materials
  • Melanosome fractions to be assayed (see Basic Protocols protocol 11 and protocol 42 and Alternate Protocols protocol 21 and protocol 32)
  • Extraction buffer (see recipe)
  • Tyrosinase assay buffer (see recipe)
  • 25 µCi/ml l‐[U‐14C]tyrosine solution (100 mCi/mmol)
  • 0.1 N HCl
  • 1 g/liter unlabeled tyrosine in 0.1 N HCl
  • 95% ethanol
  • Acetone
  • Scintillation fluid
  • Vortex mixer
  • 96‐well microtiter plates
  • 2.5‐cm Whatman 3MM filter paper discs
  • 1‐liter Erlenmeyer flasks
  • Liquid scintillation counter
  • Scintillation vials
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Figures

Videos

Literature Cited

Literature Cited
   Araki, K., Horikawa, T., Chakraborty, A.K., Nakagawa, K., Itoh, H., Oka, M., Funasaka, Y., Pawelek, J.M., and Ichihashi, M. 2000. Small GTPase Rab3A is associated with melanosomes in melanoma cells. Pigment Cell Res. 13:332‐336.
   Basrur, V., Yang, F., Kushimoto, T., Higashimoto, Y., Yasumoto, K., Valencia, J., Muller, J., Vieira, W.D., Watabe, H., Shabanowitz, J., Hearing, V.J., Hunt, D.F., and Appella, E. 2003. Proteomic analysis of early melanosomes: Identification of novel melanosomal proteins. J. Protein Res. 2:69‐79.
   Dell'Angelica, E.C. 2003. Melanosome biogenesis: Shedding light on the origin of an obscure organelle. Trends Cell Biol. 13:503‐506.
   Dell'Angelica, E.C., Mullins, C., Caplan, S., and Bonifacino, J.S. 2000. Lysosome‐related organelles. FASEB J. 14:1265‐1278.
   Donovan, J. and Brown, P. 1995. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1‐1.6.10. John Wiley & Sons, Hoboken, N.J.
   Hearing, V.J. and Ekel, T.M. 1976. Mammalian tyrosinase: A comparison of tyrosine hydroxylation and melanin formation. Biochem. J. 157:549‐557.
   Kobayashi, T., Urabe, K., Orlow, S.J., Higashi, K., Imokawa, G., Kwon, B.S., Potterf, S.B., and Hearing, V.J. 1994. The pmel17/silver locus protein: Characterization and investigation of its role in mammalian melanogenesis. J. Biol. Chem. 269:29198‐29205.
   Kushimoto, T., Basrur, V., Valencia, J., Matsunaga, J., Vieira, W.D., Ferrans, V.J., Muller, J., Appella, E., and Hearing, V.J. 2001. A new model for melanosome biogenesis based on the purification and mapping of early melanosomes. Proc. Natl. Acad. Sci. U.S.A. 98:10698‐10703.
   Kushimoto, T., Valencia, J.C., Costin, G.E., Toyofuku, K., Watabe, H., Yasumoto, K., Rouzaud, F., Vieira, W.D., and Hearing, V.J. 2003. The Seiji Memorial Lecture—The melanosome: An ideal model to study cellular differentiation. Pigment Cell Res. 16:237‐244.
   Kwon, B.S., Chintamaneni, C.D., Kozak, C.A., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Barton, D.E., Francke, U., Kobayashi, Y., and Kim, K.K. 1991. A melanocyte‐specific gene, Pmel 17, maps near the silver coat color locus on mouse chromosome 10 and is a syntenic region on human chromosome 12. Proc. Natl. Acad. Sci. U.S.A. 88:9228‐9232.
   Marks, M.S. and Seabra, M.C. 2001. The melanosome: Membrane dynamics in black and white. Nat. Rev. Mol. Cell Biol. 2:738‐748.
   Raposo, G. and Marks, M.S. 2002. The dark side of lysosome‐related organelles: Specialization of the endocytic pathway for melanosome biogenesis. Traffic 3:237‐248.
   Seiji, M., Fitzpatrick, T.B., Simpson, R.T., and Birbeck, M.S. 1963a. Chemical composition and terminology of specialized organelles (melanosomes and melanin granules) in mammalian melanocytes. Nature 197:1082‐1084.
   Seiji, M., Shimao, K., Birbeck, M.S., and Fitzpatrick, T.B. 1963b. Subcellular localization of melanin biosynthesis. Ann. N.Y. Acad. Sci. 100:497‐533.
   Wakamatsu, K. and Ito, S. 2002. Advanced chemical methods in melanin determination. Pigment Cell Res. 15:174‐183.
   Watabe, H., Valencia, J.C., Yamaguchi, Y., and Hearing, V.J. 2003. Physiological regulation of skin and hair pigmentation. J. Clin. Dermatol. 32:S2‐S8.
   Watabe, H., Valencia, J.C., Yasumoto, K., Kushimoto, T., Ando, H., Muller, J., Vieira, W.D., Mizoguchi, M., Appella, E., and Hearing, V.J. 2004. Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity. J. Biol. Chem. 279:7971‐7981.
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