Immunolocalization of cytoplasmic dynein and dynactin subunits in culturedmacrophages: enrichment on early endocytic organelles

Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtub ules. In many cases, there is compelling evidence that dynactin functions i n part to attach dyneins to cargo organelles, but this may not always be th e case, We have localized three dynactin subunits (Arp1, p62 and p150(Glued )) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold lab eling of thawed cryosections. Using stereological techniques, we have quant ified the relative distributions of each of these subunits on specific memb rane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the su bunits tested exhibits the same distribution with respect to different memb rane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membr ane, the plasma membrane and vesicles in the Golgi region, An additional po ol of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling, Even when examined closely, membrane organelle s could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules, The similarities am ong the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit, In con trast, our results show that dynactin can associate with microtubule ends i n the absence of dynein, perhaps providing sites for subsequent organelle a nd dynein association to form a functional motility complex.