Dynein binds to beta-catenin and may tether microtubules at adherens junctions.

Interactions between microtubule and actin networks are thought to be crucial for mechanical and signalling events at the cell cortex. Cytoplasmic dynein has been proposed to mediate many of these interactions. Here, we report that dynein is localized to the cortex at adherens junctions in cultured epithelial cells and that this localization is sensitive to drugs that disrupt the actin cytoskeleton. Dynein is recruited to developing contacts between cells, where it localizes with the junctional proteins beta-catenin and E-cadherin. Microtubules project towards these early contacts and we hypothesize that dynein captures and tethers microtubules at these sites. Dynein immunoprecipitates with beta-catenin, and biochemical analysis shows that dynein binds directly to beta-catenin. Overexpression of beta-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing beta-catenin, the centrosome becomes disorganized and microtubules no longer appear to be anchored at the cortex. These results identify a novel role for cytoplasmic dynein in capturing and tethering microtubules at adherens junctions, thus mediating cross-talk between actin and microtubule networks at the cell cortex.

beta III spectrin binds to the Arp1 subunit of dynactin.

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.

Mitochondria generate ATP and are involved in the regulation of cytoplasmic calcium levels. It is thought that local demand for mitochondria differs between axons and dendrites. Moreover, it has been suggested that the distribution of both energy need and calcium flux in dendrites changes with patterns of synaptic activation, whereas the distribution of these demands in axons is stable. The present study sought to determine whether there are differences in mitochondrial movements between axons and dendrites that may relate to differences in local mitochondrial demand. We labeled the mitochondria in cultured hippocampal neurons with a fluorescent dye and used time-lapse microscopy to examine their movements. In both axons and dendrites, approximately one-third of the mitochondria were in motion at any one time. In both domains, approximately 70% of the mitochondria moved in the anterograde direction, whereas the remainder moved in the retrograde direction. The velocity of the movements in each direction in each domain ranged from 0.1 microm/sec to approximately 2 microm/sec, and the means and distributions of the velocities were similar. Only one difference in the behavior of mitochondria between axons and dendrites emerged from this analysis. Mitochondria in axons were more likely to move with a consistently rapid velocity than were those in dendrites. As a result, mitochondria in axons tended to travel farther than mitochondria in dendrites. These results suggest that the transport of mitochondria in axons and dendrites is similar despite any differences in mitochondrial demand between the two domains.

Role of microtubules and actin filaments in the movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.

The mitochondria in the axons and dendrites of neurons are highly motile, but the mechanism of these movements is not well understood. It has been thought that the transport of membrane-bounded organelles in axons, and perhaps also in dendrites, depends on molecular motors of the kinesin and dynein families. However, recent evidence has suggested that some organelle transport, including that of mitochondria, may proceed along actin filaments as well. The present study sought to determine the extent to which mitochondrial movements in neurons depend on microtubule-based and actin-based transport systems. The mitochondria in cultured hippocampal neurons were labeled with a fluorescent dye and the cells were treated with either nocodazole, a drug that disrupts the microtubule network or cytochalasin D or latrunculin B, drugs which disrupt the actin network. The movement of the mitochondria in the axons and dendrites of neurons after each of these drug treatments was then examined with time-lapse microscopy. Treatment with nocodazole, which depolymerizes microtubules, stopped most mitochondrial movements in both axons and dendrites. Treatment with cytochalasin D, which aggregates actin filaments, also inhibited most movements of mitochondria, but latrunculin B, which depolymerizes actin filaments, had virtually no effect. Together, these data suggest that most of the mitochondrial movements in both axons and dendrites are microtubule-based, but in each domain there may also be some movement along actin filaments.