Thus, ARL2 phenocopies ARL2 and ARL2 siRNA in promoting Bekanamycin mitochondrial fragmentation and perinuclear clustering. When we compared the microtubule staining profiles of mock transfected cells with those expressing ARL2 or ARL2, and those Ribostamycin Sulfate depleted for ARL2, we found that microtubule profiles were indistinguishable at all times examined ; characterized by meshwork staining throughout the cytosol, extending to the cell periphery. In contrast, only cells expressing ARL2 or ARL2 displayed a clearly reduced density of microtubules. This effect on microtubules was seen 48 hours after transfection, which is later than when we observe mitochondrial defects. The strongest effect on loss of microtubules was seen with expression of the dominant active mutant, ARL2, which had no effects on mitochondrial fragmentation or clustering. Thus, the two point mutants whose expression cause very similar changes to mitochondrial morphology and clustering have very different effects on microtubules. Additionally, knockdown of ARL2 by siRNA did not alter microtubule density. Lastly, we showed that endosomal and lysosomal motility are not compromised at the same times that we observe mitochondrial motility loss. Taken together, these results strengthen our conclusions that ARL2 regulates multiple aspects of mitochondrial function and does so independently of its role in tubulin or microtubule dynamics. ARL3 is the closest paralog to ARL2 in humans, sharing 53% identity and 72% homology. With previous evidence of both shared and distinct functions in cells, we tested for effects of ARL3 siRNA or expression of dominant negative ARL3 on mitochondria. We reported previously the characterization of ARL3 siRNA reagents and their effects on Golgi, microtubules, and cytokinesis but did not specifically examine mitochondria in the earlier study. When ARL3 was depleted using an ARL3 SmartPool in HeLa cells, we observed no changes in mitochondrial morphology or localization after staining of fixed cells with HSP60 or cytochrome c 48 hours after transfection.