Microtubule/organelle motility assays.

This unit describes an in vitro assay that uses video-enhanced differential interference contrast (VE-DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme-nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE-DIC microscopy.