Lee A. Ligon

Dynein binds to β-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 β-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 β-catenin, and biochemical analysis shows that dynein binds directly to β-catenin. Overexpression of β-catenin disrupts the cellular localization of dynein and also dramatically perturbs the organization of the cellular microtubule array. In cells overexpressing β-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.

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. J. Comp. Neurol. 427:351–361, 2000.

Recruitment of dynein to the Jurkat immunological synapse

Binding of T cells to antigen-presenting cells leads to the formation of the immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and focused secretion of effector molecules. Here, we show that upon activation of Jurkat cells microtubules project from the MTOC to a ring of the scaffolding protein ADAP, localized at the synapse. Loss of ADAP, but not lymphocyte function-associated antigen 1, leads to a severe defect in MTOC polarization at the immunological synapse. The microtubule motor protein cytoplasmic dynein clusters into a ring at the synapse, colocalizing with the ADAP ring. ADAP coprecipitates with dynein from activated Jurkat cells, and loss of ADAP prevents MTOC translocation and the specific recruitment of dynein to the synapse. These results suggest a mechanism that links signaling through the T cell receptor to translocation of the MTOC, in which the minus end-directed motor cytoplasmic dynein, localized at the synapse through an interaction with ADAP, reels in the MTOC, allowing for directed secretion along the polarized microtubule cytoskeleton.

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

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 μm/sec to approximately 2 μm/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. J. Comp. Neurol. 427:340–350, 2000.

Mutant superoxide dismutase disrupts cytoplasmic dynein in motor neurons

Cytoplasmic dynein and dynactin drive retrograde axonal transport in neurons, and mutations in dynein/dynactin cause motor neuron degeneration. To test whether defects in dynein/dynactin function are involved in the neurodegenerative disease amyotrophic lateral sclerosis, we examined neurotracer transport from muscle to motor neuron in a transgenic mouse model of amyotrophic lateral sclerosis. Significant inhibition was observed, which was temporally correlated with declines in muscle strength. No decrease in dynein/dynactin expression was observed, but immunohistochemistry suggests that dynein associates with aggregates of mutant Cu/Zn superoxide dismutase 1. Expression of mutant Cu/Zn superoxide dismutase 1 in primary motor neurons altered the cellular localization of dynein, suggesting an inhibition of dynein/dynactin function. Thus, inhibition of dynein/dynactin function may have a role in motor neuron degeneration in amyotrophic lateral sclerosis.

Microtubules Tethered at Epithelial Cell Junctions by Dynein Facilitate Efficient Junction Assembly

Efficient remodeling of cell–cell adhesions is critical during development and morphogenesis. Junctional components must be specifically and rapidly transported to sites of junction assembly. In this study, we show a mechanism by which this targeted trafficking may occur. Microtubules target epithelial adherens junctions, and the number of microtubules both projecting to and tethered at sites of contact is increased during junction assembly, consistent with an increased need for new material at the nascent junction. Cytoplasmic dynein is localized to sites of cell–cell contact, and microtubules project to dynein patches where they become tethered. Microinjection of anti‐dynein antibodies disrupts the tethering of microtubules, showing that the motor anchors them. Furthermore, disruption of dynein inhibits junction formation. Immunocytochemistry with antibodies to p120 catenin support the hypothesis that tethered microtubules serve as tracks for delivery of new components to forming junctions, suggesting a model in which material is targeted for delivery to sites of need through microtubules tethered by dynein.

Regulation of Dynactin through the Differential Expression of p150Glued Isoforms

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.

Microtubule binding proteins CLIP-170, EB1, and p150Glued form distinct plus-end complexes

Microtubule plus‐end proteins CLIP‐170 and EB1 dynamically track the tips of growing microtubules in vivo. Here we examine the association of these proteins with microtubules in vitro. CLIP‐170 binds tubulin dimers and co‐assembles into growing microtubules. EB1 binds tubulin dimers more weakly, so no co‐assembly is observed. However, EB1 binds to CLIP‐170, and forms a co‐complex with CLIP‐170 and tubulin that is recruited to growing microtubule plus ends. The interaction between CLIP‐170 and EB1 is competitively inhibited by the related CAP‐Gly protein p150Glued, which also localizes to microtubule plus ends in vivo. Based on these observations, we propose a model in which the formation of distinct plus‐end complexes may differentially affect microtubule dynamics in vivo.

The posttranslational modification of tubulin undergoes a switch from detyrosination to acetylation as epithelial cells become polarized

Polarity leads to a shift in tubulin modification and microtubule organization. In unpolarized epithelial cells, detyrosinated microtubules point to the spreading edge, but in polarized cells, acetylated microtubules point to the apical domain. In both cases the modified microtubules are oriented to support cargo transport to areas of high need.