Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends.
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.
Vaughan
K T
KT
University of Massachusetts Medical School, Worcester, MA 01605, USA.
Tynan
S H
SH
Faulkner
N E
NE
Echeverri
C J
CJ
Vallee
R B
RB
D000818:Animals; D019556:COS Cells; D002453:Cell Cycle; D003902:Detergents / Q000494:pharmacology; D000072159:Dynactin Complex; D004398:Dyneins / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins; D015739:Nocodazole / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D013696:Temperature
D003902:Detergents; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; C076744:cytoplasmic linker protein 170; D017830:Octoxynol; D004398:Dyneins; D015739:Nocodazole