The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT Interactions between intracellular membranes and microtubules (MT) 1 determine the structure and positioning of subcellular organelles and direct their dynamic movements. MT can associate with the organelles either dynamically or in a stable fashion. Dynamic interactions are required for organelle traffic and involve microtubule motors such as kinesin and dynein (1, 2). Stable interactions are responsible for the positioning and structural maintenance of the endoplasmic reticulum (ER) (3), and mitochondria (4 -9). However, it is not known whether MT can influence the function of mitochondria and ER. Taxol (paclitaxel) and nocodazole, which correspondingly stabilize and disrupt MT, change the structure of the cytoskeleton. In proliferating cells, these drugs prevent normal mitotic spindle formation and cause the cells to halt mitosis and to initiate apoptosis. In comparison with MT-disrupting drugs, Taxol is less toxic and has recently been promoted for the treatment of ovarian, breast, lung, and prostate cancers (10), but it is often accompanied by serious peripheral neuropathies (11), the origin and mechanisms of which are yet unclear. Only one intracellular target has been established for Taxol thus far; it binds to a site located on the inner surface of the MT wall (12) and enhances lateral contacts between tubulin dimers (13). Recent evidence obtained in non-neuronal cells (14, 15) suggests that Taxol can directly target tubulin bound to mitochondria. We show here that Taxol and nocodazole depolarized mitochondria and released previously stored Ca 2ϩ in brain stem pre-Bötzinger complex neurons. Both effects were inhibited by cyclosporin A (CsA) and 2-aminoethoxydiphenyl borate (APB), commonly used blockers of mitochondrial permeability transition pore (mPTP). mPTP opening was validated by using the mPTP-specific calcein/Co 2ϩ imaging technique (16). The effects of Taxol were not mediated by enhanced Ca 2ϩ influx, leading to subsequent overload of mitochondria with Ca 2ϩ or overproduction of reactive oxygen species (ROS), the two main factors that can promote the opening of mPTP with subsequent initiation of apoptosis (17-19). Electron and optical microscopy revealed close interactions of MT with mitochondria in living neurons and isolated mitochondria. We therefore suggest that mPTP induction is triggered by modification of MT interactions with proteins of the outer mitochondrial membrane, e.g. the voltagedependent anion channel (VDAC) (9, 20 -23). In non-neuronal cells mitochondria and ER are closely opposed, and mitochondria can capture Ca 2ϩ released from the ER (21, 24). In neurons, the positions of mitochondria and ER a...