Microtubule binding by dynactin is required for microtubule organization but not cargo transport.
Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150(glued), binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150(glued), we replaced the wild-type p150(glued) in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150(glued) with DeltaN-p150(glued) has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150(glued) has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.
Kim
Hwajin
H
Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Ling
Shuo-Chien
SC
Rogers
Gregory C
GC
Kural
Comert
C
Selvin
Paul R
PR
Rogers
Stephen L
SL
Gelfand
Vladimir I
VI
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D004331:Drosophila melanogaster / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism / Q000502:physiology; D016547:Kinesins / Q000502:physiology; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D022101:Microtubule-Organizing Center / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure*; D020675:Peroxisomes / Q000502:physiology; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D017384:Sequence Deletion; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D004398:Dyneins; D016547:Kinesins