Kinetochore Dynein Is Required for Chromosome Motion and Congression Independent of the Spindle Checkpoint

Abstract

During mitosis, the motor molecule cytoplasmic dynein plays key direct and indirect roles in organizing microtubules (MTs) into a functional spindle. At this time, dynein is also recruited to kinetochores, but its role or roles at these organelles remain vague, partly because inhibiting dynein globally disrupts spindle assembly [1-4]. However, dynein can be selectively depleted from kinetochores by disruption of ZW10 [5], and recent studies with this approach conclude that kinetochore-associated dynein (KD) functions to silence the spindle-assembly checkpoint (SAC) [6]. Here we use dynein-antibody microinjection and the RNAi of ZW10 to explore the role of KD in chromosome behavior during mitosis in mammals. We find that depleting or inhibiting KD prevents the rapid poleward motion of attaching kinetochores but not kinetochore fiber (K fiber) formation. However, after kinetochores attach to the spindle, KD is required for stabilizing kinetochore MTs, which it probably does by generating tension on the kinetochore, and in its absence, chromosome congression is defective. Finally, depleting KD reduces the velocity of anaphase chromosome motion by approximately 40%, without affecting the rate of poleward MT flux. Thus, in addition to its role in silencing the SAC, KD is important for forming and stabilizing K fibers and in powering chromosome motion.

Prime movers: the mechanochemistry of mitotic kinesins

“…The initial capture of kinetochores by the microtubule lattice and their subsequent transport to microtubule plus ends can be mediated by dynein 96 and the kinesin-7 family motor KIF10 [97][98][99] (see Fig. 1b, chromosome v; FIG.…”

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis

“…11 Thus, a number of different binding partners, together with a diverse distribution of specific subunits 12 and their phospho-regulation, 13 allow a single motor protein -Dyneinto be involved in numerous independent cellular functions, including chromosome congression. [14][15][16] Another kinetochore-localized motor protein involved in chromosome movements is the kinesin-7 CENP-E. Although it localizes at the kinetochore fibrous corona, 17,18 just like Dynein, 19 CENP-E works in opposite direction, having the ability to move chromosomes toward the plus-ends of microtubules.…”

The perpetual movements of anaphase

“…In a system working at 20% efficiency, the hydrolysis of 4 ATP units/sec/dynein molecule is a reasonable estimation, which means that, in theory, a single (flagellar) dynein motor produces a net power output that is 100x greater than the one required to move a chromosome. However, this argument looses weight by the fact that knocking down ~85% of ZW10, which should reduce but not fully deplete kinetochore dynein, causes a significant reduction of chromosome movement to the poles [104]. Taken together, these results indicate that, if directly involved, dynein motor activity at kinetochores plays a minor role in powering anaphase chromosome motion.…”

“…Subsequent work in Ptk1 cells overexpressing p50 dynamitin reported a 30% reduction in chromosome-to-pole velocity after abrogation of the SAC [86]. More recently, Yang and colleagues selectively decreased the kinetochore pool of dynein by knocking down ZW10 in human U2OS cells [104]. The authors reported a 40% reduction in chromosome-to-pole velocity during anaphase, suggesting that kinetochore dynein is important in powering chromosome motion.…”

“…It is somewhat surprising that the observed reduction corresponds only to about half the reported "pac-man" activity in U2OS cells [61], suggesting that other mechanisms or factors are involved. Moreover, it remains to be elucidated whether the observed reduction is a direct result of lack of dynein processive motion along K-fiber microtubules, or whether it is due to defective/unstable microtubule attachments, possibly associated with a defective SAC [103][104][105]. The recent discovery of spindly, a protein that targets dynein to kinetochores in Drosophila, C. elegans and humans, but which does not impair the recruitment of Mad2 to kinetochores [106][107][108], may prove to be an important tool for the clarification of the abovementioned issues.…”