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.

Binding of T cells to antigen-presenting cells leads to the formation of the immunological synapse, translocation of the microtubuleorganizing 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.I n T cells, engagement of the T cell receptor leads to formation of an immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and ultimately secretion of effector molecules (1-4). Translocation of the MTOC serves to focus secretion at the synapse and is required for effector function of both helper and cytotoxic T cells (5-8).Previous studies have reported that signaling through ZAP-70, LAT, SLP-76, elevation of intracellular calcium, and Cdc42 are required for MTOC translocation (9 -12). However, the downstream actions that control MTOC translocation remain to be determined. The microtubule motor cytoplasmic dynein is a good candidate to drive the translocation of the MTOC and the resulting polarization of the microtubule cytoskeleton observed in synapse formation (4). Here, we present data with Jurkat cells showing that a dynein complex is recruited to the synapse and that the recruitment of dynein depends on the protein ADAP. ADAP is a SLP-76-associated scaffold protein that links T cell receptor signaling to integrin clustering through its association with SKAP55 and may also be linked to actin dynamics by virtue of its Ena͞vasodilator-stimulated phosphoprotein binding domain (13)(14)(15)(16)(17)(18). Here, we show that ADAP is associated with dynein, and upon T cell activation it forms a ring at the synapse that colocalizes with dynein and microtubules. When ADAP expression is reduced by using antisense morpholino (MO) oligonucleotides, dynein fails to localize to the synapse and MTOC translocation is blocked. Together, these results show a direct connection between T cell signaling, recruitment of dynein to the synapse, and polarization of the microtubule cytoskeleton. ResultsPrevious studies showed that microtubules project toward a ring of lymphocyte function-associated antigen 1 (LFA-1) clustered at the immunological synapse (4). We used LFA-1-deficient Fig. 6, w...

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 ␤III spectrin isoform. We identify two Arp1-binding sites in ␤III spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to ␤III spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and ␤III spectrin co-purify on vesicles isolated from rat brain, and ␤III spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, ␤III spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, ␤III spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for ␤III spectrin. We hypothesize that the interaction between ␤III 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.The microtubule-based motor cytoplasmic dynein is involved in a wide range of cellular processes, including retrograde transport in neurons, trafficking of vesicles from the endoplasmic reticulum to the Golgi, mitotic spindle assembly, and potentially cytokinesis (reviewed in Ref. 1). These processes require the targeting of dynein to many different cellular cargoes. Whereas dynein clearly provides a motile force in all of these processes, the mechanisms linking dynein to these various cargoes have yet to be identified (reviewed in Ref. 2).Dynactin is a multi-subunit complex that binds both to microtubules (3) and to cytoplasmic dynein (4, 5). Disruption of the dynein-dynactin interaction blocks dynein-mediated transport both in vitro and in vivo (6 -9). The specific role of the interaction between dynein and dynactin is unknown. One possibility is that dynactin increases the processive nature of the movement of dynein along cellular microtubules (3, 10). A second but not mutually exclusive hypothesis is that dynactin links dynein to its cellular cargo (11).Although a dynactin-independent association between a dynein light chain and the rhodopsin receptor has been detected in rod cell vesicles (12), other studies suggest that dynactin is required to mediate the interaction of dynein with vesicular cargo. Antibodies that block the dynein-dynactin interaction deplete dynein from vesicles and inhibit microtubule-based vesicular transport (6, 7). Furthermore, disruption of the dynein-dynactin interaction by dynamitin overexpression inhibits dynein-mediated vesicle trafficking in cells (9). The nature of the interaction ...

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Glued has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics

Cytoplasmic dynein and kinesin I are both unidirectional intracellular motors. Dynein moves cargo toward the cell center, and kinesin moves cargo toward the cell periphery. There is growing evidence that bi-directional motility is regulated in the cell, potentially through direct interactions between oppositely oriented motors. We have identified a direct interaction between cytoplasmic dynein and kinesin I. Using the yeast two-hybrid assay and affinity chromatography, we demonstrate that the intermediate chain of dynein binds to kinesin light chains 1 and 2. The interaction is both direct and specific. Co-immunoprecipitation experiments demonstrate an interaction between endogenous proteins in rat brain cytosol. Double-label immunocytochemistry reveals a partial co-localization of vesicleassociated motor proteins. Together these observations suggest that soluble motors can interact, potentially allowing kinesin I to actively localize dynein to cellular sites of function. There is also a vesicle population with both dynein and kinesin I bound that may be capable of bi-directional motility along cellular microtubules.