Dynein antagonizes eg5 by crosslinking and sliding antiparallel microtubules - PubMed (original) (raw)

Dynein antagonizes eg5 by crosslinking and sliding antiparallel microtubules

Nick P Ferenz et al. Curr Biol. 2009.

Abstract

Mitotic spindle assembly requires the combined activity of various molecular motor proteins, including Eg5 and dynein. Together, these motors generate antagonistic forces during mammalian bipolar spindle assembly; 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), 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 by less than or greater than 5.5 microm, 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.

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Figures

Figure 1

Figure 1. Spindle Assembly Following Nocodazole Washout

(A–C) Selected images from time-lapse sequences of cells treated with and released from nocodazole under the three centrosomal configurations. A, B and C correspond to Movies S2, S3 and S4, respectively. In each case, a bipolar spindle assembles following nocodazole washout. In the first image of each sequence, centrosomes appear as white dots. Arrows subsequently mark the position of in focus centrosomes when three or more foci are present. The last image of each sequence is a maximum intensity projection. All times are relative to the final nocodazole washout (0:00) and are displayed as min: sec. (D) Percentage of fixed LLC-Pk1α cells at the indicated mitotic stages, present 60min post-4x washout. Bar = 10μm.

Figure 2

Figure 2. Spindle Bistability in the Absence of Eg5 Activity

(A–C) Selected images from time-lapse sequences of cells treated with nocodazole and monastrol, then released into monastrol-containing medium. A and B correspond to Movies S5 and S6, respectively. Nocodazole washout leads to bipolar spindle formation, except when proximal centrosomes are close to one another. Set up is as defined in Figure 1. Additionally, asterisks mark the position of out of focus centrosomes. In C, two mitotic cells have fused together; the top spindle is acentrosomal. Bar = 10μm.

Figure 3

Figure 3. Dynein is Required for Monopolar Spindle Formation

(A–C) Selected images from time-lapse sequences of cells treated with nocodazole and monastrol, injected with p150-CC1, then released into monastrol-containing medium. A corresponds to Movie S7. In each case, a bipolar spindle assembles after nocodazole washout. Set up is as defined in Figure 2. Bar = 10μm.

Figure 4

Figure 4. Mathematical Modeling

(A) Schematic of the mathematical model. The total force, F, acting on centrosomes is a function of Forces A, B and C (see text for descriptions). (B) Force versus intercentrosomal distance given by the model with a realistic chromosomal distribution at the spindle midplane (see Figure S6B) for L = 2; A = 1; C = 0.03 for uninhibited, co-inhibited (Eq. 4 in Supplemental Text, solid curve, B = 0) and Eg5-inhibited (Eq. 5 in Supplemental Text, dashed curve, B = 2) cells.

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