Comparison of the intracellular distribution of cytoplasmic dynein and kinesin in cultured cells: motor protein location does not reliably predict function.

While immunolocalization methods have been used as a reasonable means to judge where a given molecule may be active in the cellular milieu, the correlation between distribution and function for proteins involved in intracellular transport may not be clear cut. To address the question of specificity and reproducibility of immunolocalization of microtubule-based motor proteins, we have co-localized cytoplasmic dynein and kinesin by immunofluorescence microscopy using two specific antibodies for each motor molecule. The results indicate that cytoplasmic dynein and kinesin appear to co-localize on a small subset of vesicles, but largely reside or accumulate on morphologically distinct organelles. In addition, anti-kinesin antibodies differing in their epitope specificity label different cellular compartments. To address the question of whether the distribution of motor molecules is representative of organelles that are undergoing active transport, we have altered the activity of vesicle trafficking pathways in fibroblasts using several different methods, including cytoplasmic acidification and disruption of cellular compartments with brefeldin A, nocodazole and okadaic acid. Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.