Dynein Antagonizes Eg5 by Crosslinking and Sliding Antiparallel Microtubules

Abstract

Summary Mitotic spindle assembly requires the combined activity of various molecular motor proteins, including Eg5 [1] and dynein [2]. Together, these motors generate antagonistic forces during mammalian bipolar spindle assembly [3]; what remains unknown, however, is how these motors are functionally coordinated such that antagonism is possible. Given that Eg5 generates an outward force by crosslinking and sliding apart antiparallel microtubules (MTs) [4–6], we explored the possibility that dynein generates an inward force by likewise sliding antiparallel MTs. We reasoned that antiparallel overlap, and therefore the magnitude of a dynein-mediated force, would be inversely proportional to the initial distance between centrosomes. To capitalize on this relationship, we utilized a nocodazole washout assay to mimic spindle assembly. We found that Eg5 inhibition led to either monopolar or bipolar spindle formation, depending on whether centrosomes were initially separated less than or greater than 5.5μm, respectively. Mathematical modeling predicted this same spindle bistability in the absence of functional Eg5, and required dynein acting on antiparallel MTs to do so. Our results suggest that dynein functionally coordinates with Eg5 by crosslinking and sliding antiparallel MTs, a novel role for dynein within the framework of spindle assembly.

Modulated microtubule dynamics enable Hklp2/Kif15 to assemble bipolar spindles

“…If Eg5 is inhibited, minus end-directed antagonistic motors become dominant and prevent pole separation. Accordingly, attenuating antagonistic motors like HSET or dynein [53][54][55] or strengthening synergistic motors like Hklp2/Kif15 32,33 can compensate for reduced Eg5 activity and restore the equilibrium of forces. Reportedly, microtubule dynamics can indirectly influence this equilibrium through modulation of microtubule length and, thus, by varying the zone where cross-linking motors can act.…”

KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture

“…34 This has been suggested to generate an inward force that balances Eg5's outward push. KL1 may work in concert with dynein by increasing the number of microtubules available for dynein mediated force generation (Fig.…”

APC/C is an essential regulator of centrosome clustering

“…We hypothesized that Eg5 represents a strong candidate substrate of APC/C based on two reasons. First, Eg5 cross-links and slides microtubules 14 , thereby generating outward forces that are antagonized by dynein-dependent inward forces 15 . Depletion of dynein/dynactin subunits in our assays generated multipolar spindles in 4NCA cells (Fig.…”