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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Mukherjee, S., Ghosh, R.N., and Maxfield, F.R. (1997) Endocytosis. Physiol Rev. 77, 759–803.
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.
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.
Murray, J.W. and Wolkoff, A.W. (2005) Assay of Rab4-dependent trafficking on microtubules. Methods Enzymol. 403, 92–107.
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.
Inoue, S. (1986) Video Microscopy. Plenum Press, New York.
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.
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.
Waterman-Storer, C.M. (1998) In: Current Protocols in Cell Biology, pp. 13.1.6–13.1.7. John Wiley, New York.
Lee, G.E., et al. (2006) Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro. J. Virol. 80, 4264–4275.
Neufeld, D.S. (1997) Isolation of rat liver hepatocytes. Methods Mol. Biol. 75, 145–151.
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.
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.
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.
Wang, Z. and Sheetz, M.P. (2000) The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity. Biophys. J. 78, 1955–1964.
Higuchi, H. and Endow, S.A. (2002) Directionality and processivity of molecular motors. Curr. Opin. Cell Biol. 14, 50–57.
Murray, J.W., Bananis, E., and Wolkoff, A.W. (2002) Immunofluorescence microchamber technique for characterizing isolated organelles. Anal. Biochem. 305, 55–67.
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.
Gyoeva, F.K. and Gelfand, V.I. (1991) Coalignment of vimentin intermediate filaments with microtubules depends on kinesin. Nature 353, 445–448.
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.
Brady, S.T. (1991) Molecular motors in the nervous system. Neuron 7, 521–533.
Harrison, R.E. and Huebner, E. (1997) Unipolar microtubule array is directly involved in nurse cell-oocyte transport. Cell Motil. Cytoskelet. 36, 355–362.
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.
Kishino, A. and Yanagida, T. (1988) Force measurements by micromanipulation of a single actin filament by glass needles. Nature 334, 74–76.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Murray, J.W., Wolkoff, A.W. (2007). In Vitro Motility System to Study the Role of Motor Proteins in Receptor-Ligand Sorting. In: Sperry, A.O. (eds) Molecular Motors. Methods in Molecular Biology™, vol 392. Humana Press. https://doi.org/10.1007/978-1-59745-490-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-490-2_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-665-8
Online ISBN: 978-1-59745-490-2
eBook Packages: Springer Protocols