Bonnie Howell

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 “dynamitin” protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

Spindle Checkpoint Protein Dynamics at Kinetochores in Living Cells

BACKGROUND To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.

Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.

Purification of a WD Repeat Protein, EMAP, That Promotes Microtubule Dynamics through an Inhibition of Rescue

The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 μm EMAP to 21 μm tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.