Centractin (Arp 1) is an actin-related protein that shares 53% sequence identity with conventional actin and is predicted to have a similar core structure for nucleotide binding. Biochemical studies of centractin, named for its apparent localization to the centrosome, reveal that it is a member of a stable macromolecular complex, dynactin. The dynactin complex consists of at least 9 polypeptides and has been implicated in activating dynein mediated vesicle transport along microtubules. Ultra-structural analysis, by rotary shadowing electron microscopy with antibody decoration, reveals that within the context of the dynactin complex, centractin forms a 37 nm filament resembling the structure of an actin filament with capping protein and p62 localizing to opposite ends.We have used transient transfection assays to investigate the effects of overexpression of centractin in cells. Mammalian PtK2 cells were transiently transfected with centractin cloned in the pcDNA3 vector under the control of a CMV promoter. The cells were transfected for 24 hours using Ca2+/DNA co-precipitates. The transfection efficiency was as high as 41 % as determined by direct counting of immunostained cells. Cells were fixed at time points ranging from 18 to 24 hours post washing using 1% formaldehyde plus 1% Triton X-100 (5 min), 0.05% glutaraldehyde (5 min), 1 % formaldehyde (5 min), and 0.05% sodium borohydride to block free aldehyde groups (3×5 min). Immunofluorescence employed an affinity-purified, rabbit polyclonal antibody to centractin followed by a Texas Red-labeled secondary antibody. Epifluorescence microscopy revealed that all of the cells overexpressing centractin contained centractin structures with novel morphology. Two distinct patterns were observed consistently: filamentous centractin localized throughout the cytoplasm (Figure la) and perinuclear centractin accumulations (Figure lb). The perinuclear accumulations appear to be highly three dimensional and curved in structure (confirmed by confocal microscopy), and often have associated filamentous structures (Figure lb). The addition of 33 micromolar nocodozole (30 min. at 4°C) to the transfected cells results in thinner centractin filaments (Figure lc).