A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150^Glued by live-cell imaging. Time-lapse analysis of p150^Glued revealed targeting to the plus ends of growing microtubules, requiring the NH₂-terminal cytoskeleton-associated protein–glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150^Glued phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150^Glued phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150^Glued phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150^Glued during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150^Glued–labeled microtubules just prior to transport, implicating microtubules and dynactin in a search–capture mechanism for minus-end–directed organelles.

The EB1 protein is a member of the exciting and enigmatic family of microtubule (MT) tip-tracking proteins. EB1 acts as an exquisite marker of dynamic MT plus ends in some cases, whereas in others EB1 is thought to directly dictate the behavior of the plus ends. How EB1 differentiates between these two roles remains unclear; however, a growing list of interactions between EB1 and other MT binding proteins suggests there may be a single mechanism. Adding another layer of complexity to these interactions, two studies published in this issue implicate EB1 in cross-talk between mitotic MTs and between MTs and actin filaments (Goshima et al., p. 229; Wu et al., p. 201). These results raise the possibility that EB1 is a central player in MT-based transport, and that the activity of MT-binding proteins depends on their ability or inability to interact with EB1.

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-γ mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.