Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein–GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70–containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Eukaryotic cells use cytoskeletal motor proteins to transport many different intracellular cargos. Numerous kinesins and myosins have evolved to cope with the various transport needs that have arisen during eukaryotic evolution. Surprisingly, a single cytoplasmic dynein (a minus end-directed microtubule motor) carries out similarly diverse transport activities as the many different types of kinesin. How is dynein coupled to its wide range of cargos and how is it spatially and temporally regulated? The answer could lie in the several multifunctional adaptors, including dynactin, lissencephaly 1, nuclear distribution protein E (NUDE) and NUDE-like, Bicaudal d, Rod–ZW10–Zwilch and Spindly, that regulate dynein function and localization.

Dynactin, a large multisubunit complex, is required for intracellular transport by dynein; however, its cellular functions and mechanism of action are not clear. Prior studies suggested that dynactin increases dynein processivity by tethering the motor to the microtubule through its own microtubule binding domains. However, this hypothesis could not be tested without a recombinant source of dynactin. Here, we have produced recombinant dynactin and dynein in Saccharomyces cerevisiae, and examined the effect of dynactin on dynein in single-molecule motility assays. We show that dynactin increases the run length of single dynein motors, but does not alter the directionality of dynein movement. Enhancement of dynein processivity by dynactin does not require the microtubule (MT) binding domains of Nip100 (the yeast p150 Glued homolog). Dynactin lacking these MT binding domains also supports the proper localization and function of dynein during nuclear segregation in vivo. Instead, a segment of the coiled-coil of Nip100 is required for these activities. Our results directly demonstrate that dynactin increases the processivity of dynein through a mechanism independent of microtubule tethering.microtubule ͉ motor protein ͉ nuclear segregation ͉ p150 Glued ͉ single molecule D ynactin, a large (Ϸ1.2 MD) complex, was first identified as an activator of dynein-mediated, minus-end-directed vesicle transport (1, 2), and has subsequently been shown to be essential for nearly every cellular function of cytoplasmic dynein (3, 4). Several dynactin alleles have been linked to human neurological disease, which most likely results from a defect in intracellular trafficking (5, 6). Dynactin is composed of a filament of the actin-related protein Arp1, capped at each end by additional subunits. The barbed end subcomplex contains a dimer of the largest subunit, p150 Glued (Nip100 in yeast), which binds to dynein directly (4). The Nterminus of p150Glued contains a CAP-Gly domain and a basic region, both of which have been shown to bind microtubules (MTs) (7-9), followed by a coiled-coil that projects as a 24-nm stalk from the Arp1 filament (10, 11).Two general mechanisms have been proposed through which dynactin could aid the cellular function of dynein. First, many studies have provided evidence that dynactin is important for localizing cytoplasmic dynein to its proper intracellular cargo (4). Dynactin also might modulate dynein motor activity, an idea that has been explored through several in vitro motility assays. Dynactin has been proposed to increase the processivity of dynein, based on findings that dynactin increases the run length of dynein-coated beads in vitro (12). Increased processivity might be important in the cell for uninterrupted transport over long distance or for transport under high load. However, because dynactin and dynein were nonspecifically adsorbed onto beads in this study, it could not be determined whether dynein-dynactin complexes were observed, or if dynactin bound separately from dynein on the bead surface ...