Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends

Abstract

Cytoplasmic dynein is a minus end-directed microtubule motor responsible for centripetal organelle movement and several aspects of chromosome segregation. Our search for cytoplasmic dynein-interacting proteins has implicated the dynactin complex as the cytoplasmic dynein ‘receptor’ on organelles and kinetochores. Immunofluorescence microscopy using a total of six antibodies generated against the p150Glued, Arp1 and dynamitin subunits of dynactin revealed a novel fraction of dynactin-positive structures aligned in linear arrays along the distal segments of interphase microtubules. Dynactin staining revealed that these structures colocalized extensively with CLIP-170. Cytoplasmic dynein staining was undetectable, but extensive colocalization with dynactin became evident upon transfer to a lower temperature. Overexpression of the dynamitin subunit of dynactin removed Arp1 from microtubules but did not affect microtubule-associated p150Glued or CLIP-170 staining. Brief acetate treatment, which has been shown to affect lysosomal and endosomal traffic, also dispersed the Golgi apparatus and eliminated the microtubule-associated staining pattern. The effect on dynactin was rapidly reversible and, following acetate washout, punctate dynactin was detected at microtubule ends within 3 minutes. Together, these findings identify a region along the distal segments of microtubules where dynactin and CLIP-170 colocalize. Because CLIP-170 has been reported to mark growing microtubule ends, our results indicate a similar relationship for dynactin. The functional interaction between dynactin and cytoplasmic dynein further suggests that this these regions represent accumulations of cytoplasmic dynein cargo-loading sites involved in the early stages of minus end-directed organelle transport.

Auto‐reverse nuclear migration in bipolar mammalian cells on micropatterned surfaces

“…The role of Lis1 in nucleokinesis was directly demonstrated by altered migratory behavior of cerebellar granule cells explanted from a Lis1 knockout mouse (Hirotsune et al, 1998). A number of other proteins like dynactin and CLIP (Vaughan et al, 1999), which are known to be involved in nuclear positioning of mammalian cells, also specifically localize on MT plus ends. Nevertheless, the functional role of these proteins, as well as the operational integration of this multi-component process remains unclear.…”

Suppression of Microtubule Dynamic Instability by the +TIP Protein EB1 and Its Modulation by the CAP-Gly Domain of p150Glued

“…One of the important +TIP binding partners of EB1 is p150 Glued , a subunit of dynactin which is a major component of the dynein-dynactin motor complex (15). p150 Glued localizes at the plus ends of growing microtubules along with other +TIPs including EB1 and CLIP170 (16). p150 Glued has been shown to bind directly to EB1 and to microtubules (17,18) and in cells appears to be involved in dynein-mediated transport of cargo along microtubules and required for the maintenance of radial microtubule arrays (19).…”

“…Microtubule associated or accessory proteins (MAPs) are known to regulate dynamic instability behaviors, both in vitro and in cells ( , ). The plus end tracking proteins (+TIPs) are MAPs that localize to and track the plus ends of growing microtubules in cells ( − ). +TIPs, which include EB1 (end binding protein 1), the p150 Glued subunit of dynactin, CLIP170 (cytoplasmic linker protein 170) and APC (adenomatous polyposis coli protein), are strongly conserved and found in species from yeast to humans.…”

Role of Dynactin in the Intracellular Localization and Activation of Cytoplasmic Dynein

“…The MT plus ends inside a cell are bound by end-binding proteins such as EB1, which additionally recruits the endosome–MT linker protein CLIP-170 . Dynactin was found to localize to plus ends of MTs in conjunction with CLIP-170, and a fraction of these plus end-localized dynactin spots were associated with cytoplasmic dynein . In fact, the 74 kDa dynein intermediate chain IC 74 was observed to track the growing plus ends of MTs in live HeLa cells .…”