Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in real time, by transfecting them with different GDP- and GTP-locked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobility in the presence of wild-type and Rab mutants. Dominant-negative and dominant-positive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L, Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement, and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N, and Rab5 Q79L. Our data indicate that changes in the GDP-GTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton.
Copyright © 2012 Wiley Periodicals, Inc.