Two modes of microtubule sliding driven by cytoplasmic dynein.
Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin-streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.
Shima
Tomohiro
T
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
Kon
Takahide
T
Imamula
Kenji
K
Ohkura
Reiko
R
Sutoh
Kazuo
K
D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D001710:Biotin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004023:Dictyostelium / Q000737:chemistry; D004398:Dyneins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D004398:Dyneins