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)


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 


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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

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