Assay of Rab4-dependent trafficking on microtubules

We present an in vitro method to measure how Rab4 and other regulatory proteins affect microtubule-based organelle motility. The protocols utilize small-volume, disposable "microchambers" designed for epifluorescence, confocal, or other microscope platforms and into which microtubules, organelles, and primary and fluorescent secondary antibodies are added. Our work has focused on the isolation and use of endocytic vesicles from rat liver, and we present these protocols. However, the techniques can be adapted for other organelles or cell types. Multiple fluorescent probes, rapid image capture, and immunofluorescence under non-fixation conditions allow for measurements of the location and intensity changes of endogenous proteins upon addition of ATP or upon addition of other proteins or regulatory factors. We review measurements of microtubule-based motility as well as measurements for protein localization and protein segregation in vitro.