First, through a specific interaction of dynactin with growing microtubule plus ends, dynactin appears to stabilize a stochastic interaction between membranes and microtubules

First, through a specific interaction of dynactin with growing microtubule plus ends, dynactin appears to stabilize a stochastic interaction between membranes and microtubules. rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150subunit of dynactin as a specific binding partner for the intermediate chains (ICs)* of cytoplasmic dynein, implicating dynactin as an adaptor or receptor for cytoplasmic dynein on cargo (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). To evaluate a role Amidopyrine for dynactin as a cytoplasmic dynein receptor, we determined the distribution of dynactin in cultured cells and identified a novel population of dynactin which localized to the plus ends of microtubules and colocalized with cytoplasmic dynein (Vaughan et al., 1999; Habermann et al., 2001). Interest in this novel microtubule plus-endCassociated population of dynactin was fueled further by the detection of additional proteins at these sites, including: cytoplasmic linker protein (CLIP)-170 (Pierre et al., 1992; Dujardin et al., 1998; Perez et al., 1999; Valetti et al., 1999; Vaughan et al., 1999); adenomatous polyposis coli (APC) (Nathke et al., 1996; Mimori-Kiyosue et al., 2000a); EB1 (Morrison et al., 1998; Faulkner et al., 2000; Mimori-Kiyosue et al., 2000b); CLIP-115 (Hoogenraad et al., 2000); CLASPs (Akhmanova et al., 2001); and LIS-1 (Faulkner et al., 2000; Coquelle et al., 2002). CLIP-170 has been proposed to attract dynactin to plus ends (Dujardin et al., 1998; Valetti et al., 1999; Coquelle et al., 2002). EB1 can associate with dynactin in vitro (Berrueta et al., 1999), colocalizes with dynactin at microtubule ends (Faulkner et al., 2000), and might also be involved in dynactin targeting. Cytoplasmic dynein also interacts with LIS-1 (Tai et al., 2002), and LIS-1 overexpression displaces dynactin but not EB1 from microtubule ends (Faulkner et al., 2000). Additional evidence for a functional link comes from analysis in genetic organisms such as (cytoplasmic dynein) targets to growing microtubules tips in a dynactin-dependent manner (Xiang et al., 2000). This complexity of proteins at Amidopyrine microtubule plus ends suggests an important function. To determine the molecular basis of dynactin targeting, we have expressed GFP-p150fusions in cultured cells. Live-cell imaging of GFP-p150reveals a dynamic and regulated interaction of dynactin with growing microtubule plus ends. We have mapped the site of p150phosphorylation and tested the impact of phosphorylation on microtubule binding. Finally, imaging of p150with minus-endCdirected organelles suggests that this dynamic association with microtubules is an important factor in the initial stages of membrane transport. Results Targeting of p150have not revealed a Amidopyrine specific association with plus ends (Paschal et al., 1993; Waterman-Storer et al., 1995), and p150(Waterman-Storer et al., 1995), Arp1 (Holleran et al., 1996), and p62 (Garces et al., 1999) were not specific for plus ends when overexpressed. To minimize limitations of fixation and staining, we generated a GFP fusion to the NH2 terminus of full-length p150and performed live-cell imaging on transfectants. We observed a wide range of expression levels which were binned into four groups based on phenotype and levels of GFP intensity (Fig. 1 E). Images collected from cells expressing GFP-p150at medium and high levels were consistent with previous studies in which the transfected p150decorated microtubules along their lengths and appeared to induce microtubule bundling (Waterman-Storer et al., 1995). Using sensitive cameras, we detected another population of cells expressing GFP-p150at lower levels in which the p150was detected at the distal ends of microtubules (Fig. 1 A). These labeled microtubule ends were similar to the Rabbit polyclonal to A1BG comet tail patterns observed previously for endogenous dynactin (Valetti et al., 1999; Vaughan et al., 1999) with intense labeling at the microtubule tips diminishing as a function of distance from the tips (Fig. 1 A, inset). Open in a separate window Figure 1. p150 (A, C, and D) or truncated p150interacted exclusively with microtubule plus ends undergoing elongation (Fig. 1 A; Video.