Advertisement

In Vitro Motility System to Study the Role of Motor Proteins in Receptor-Ligand Sorting

  • John W. Murray
  • Allan W. Wolkoff
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)

Abstract

This chapter presents fluorescence microscope assays that can be used to study microtubule (MT)-based movement and receptor-ligand sorting in vitro. The strategy is to isolate endosomes in a concentrated active form and store them in frozen aliquots for single use. Glass microchambers are then constructed and coated with fluorescent MTs, and the endosomes are thawed and bound to the MTs. Proteins of interest are then detected and quantified by immunofluorescence. For motility experiments, time-lapse movies are captured using multichannel fluorescence microscopy, and motility is initiated by the addition of ATP. Movies are later categorized and quantified for MT-based motility and other associated events such as endocytic fission. These techniques were developed to assess the role of MTs and MT motor proteins in endocytic processing within liver cells, and we have streamlined a rapid procedure for isolating abundant, highly motile endosomes from rat liver. Cultured cells and other organelles can also be examined, and many important biological questions concerning intracellular traffic and organelle composition can be studied by creative adaptation of the protocols that are presented.

Key Words

Dynein endosomes immunofluorescence in vitro kinesin microscopy microtubules motor proteins 

References

  1. 1.
    Wolkoff, A.W., Klausner, R.D., Ashwell, G., and Harford, J. (1984) Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex. J. Cell Biol. 98, 375–381.CrossRefPubMedGoogle Scholar
  2. 2.
    Mukherjee, S., Ghosh, R.N., and Maxfield, F.R. (1997) Endocytosis. Physiol Rev. 77, 759–803.PubMedGoogle Scholar
  3. 3.
    Oka, J.A. and Weigel, P.H. (1983) Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes. Biochim. Biophys. Acta 763, 368–376.CrossRefPubMedGoogle Scholar
  4. 4.
    Gruenberg, J., Griffiths, G., and Howell, K.E. (1989) Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J. Cell Biol. 108, 1301–1316.CrossRefPubMedGoogle Scholar
  5. 5.
    Murray, J.W. and Wolkoff, A.W. (2005) Assay of Rab4-dependent trafficking on microtubules. Methods Enzymol. 403, 92–107.CrossRefPubMedGoogle Scholar
  6. 6.
    Bananis, E., Nath, S., Gordon, K., Satir, P., Stockert, R.J., Murray, J.W., and Wolkoff, A.W. (2004) Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for dynein and kinesin. Mol. Biol. Cell 15, 3688–3697.CrossRefPubMedGoogle Scholar
  7. 7.
    Inoue, S. (1986) Video Microscopy. Plenum Press, New York.Google Scholar
  8. 8.
    Howard, J. and Hyman, A.A. (1993) Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence microscopy. Methods Cell Biol. 39, 105–113.CrossRefPubMedGoogle Scholar
  9. 9.
    Pollock, N., de Hostos, E.L., Turck, C.W., and Vale, R.D. (1999) Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium. J. Cell Biol. 147, 493–506.CrossRefPubMedGoogle Scholar
  10. 10.
    Waterman-Storer, C.M. (1998) In: Current Protocols in Cell Biology, pp. 13.1.6–13.1.7. John Wiley, New York.Google Scholar
  11. 11.
    Lee, G.E., et al. (2006) Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro. J. Virol. 80, 4264–4275.CrossRefPubMedGoogle Scholar
  12. 12.
    Neufeld, D.S. (1997) Isolation of rat liver hepatocytes. Methods Mol. Biol. 75, 145–151.PubMedGoogle Scholar
  13. 13.
    Huang, T., Deng, H., Wolkoff, A.W., and Stockert, R.J. (2002) Phosphorylation-dependent interaction of the asialoglycoprotein receptor with molecular chaperones. J. Biol. Chem. 277, 37798–37803.CrossRefPubMedGoogle Scholar
  14. 14.
    Bananis, E., Murray, J.W., Stockert, R.J., Satir, P., and Wolkoff, A.W. (2000) Microtubule and motor-dependent endocytic vesicle sorting in vitro. J. Cell Biol. 151, 179–186.CrossRefPubMedGoogle Scholar
  15. 15.
    Sarkar, S., Bananis, E., Nath, S., Anwer, M.S., Wolkoff, A.W., and Murray, J.W. (2006) PKCZeta is required for microtubule-based motility of vesicles containing the ntcp transporter. Traffic 7, 1078–1091.CrossRefPubMedGoogle Scholar
  16. 16.
    Wang, Z. and Sheetz, M.P. (2000) The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity. Biophys. J. 78, 1955–1964.CrossRefPubMedGoogle Scholar
  17. 17.
    Higuchi, H. and Endow, S.A. (2002) Directionality and processivity of molecular motors. Curr. Opin. Cell Biol. 14, 50–57.CrossRefPubMedGoogle Scholar
  18. 18.
    Murray, J.W., Bananis, E., and Wolkoff, A.W. (2002) Immunofluorescence microchamber technique for characterizing isolated organelles. Anal. Biochem. 305, 55–67.CrossRefPubMedGoogle Scholar
  19. 19.
    McDonald, D., Vodicka, M.A., Lucero, G., Svitkina, T.M., Borisy, G.G., Emerman, M., and Hope, T.J. (2002) Visualization of the intracellular behavior of HIV in living cells. J. Cell Biol. 159, 441–452.CrossRefPubMedGoogle Scholar
  20. 20.
    Gyoeva, F.K. and Gelfand, V.I. (1991) Coalignment of vimentin intermediate filaments with microtubules depends on kinesin. Nature 353, 445–448.CrossRefPubMedGoogle Scholar
  21. 21.
    Bananis, E., Murray, J.W., Stockert, R.J., Satir, P., and Wolkoff, A.W. (2003) Regulation of early endocytic vesicle motility and fission in a reconstituted system. J. Cell Sci. 116, 2749–2761.CrossRefPubMedGoogle Scholar
  22. 22.
    Brady, S.T. (1991) Molecular motors in the nervous system. Neuron 7, 521–533.CrossRefPubMedGoogle Scholar
  23. 23.
    Harrison, R.E. and Huebner, E. (1997) Unipolar microtubule array is directly involved in nurse cell-oocyte transport. Cell Motil. Cytoskelet. 36, 355–362.CrossRefGoogle Scholar
  24. 24.
    Murray, J.W., Bananis, E., and Wolkoff, A.W. (2000) Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process. Mol. Biol. Cell 11, 419–433.PubMedGoogle Scholar
  25. 25.
    Kishino, A. and Yanagida, T. (1988) Force measurements by micromanipulation of a single actin filament by glass needles. Nature 334, 74–76.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • John W. Murray
    • 1
    • 2
  • Allan W. Wolkoff
    • 1
    • 2
  1. 1.Marion Bessin Liver Research CenterAlbert Einstein College of MedicineBronx
  2. 2.Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronx

Personalised recommendations