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Mechanisms of spindle pole focusing

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. We characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, we showed that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes (Fig 3). We also reported a novel localization of Ncd to the growing tips of C-MTs (Movie 2), which we showed is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process, in collaboration with Dr. Francois Nedelec (EMBL), suggested that the plus end localization of Ncd could facilitate the capture and transport of K fibers along centrosomal microtubules (Movie 3).

Fig. 3 - Distinct effects of Ncd and cytoplasmic dynein on pole coalescence. Representative spindle morphology after RNAi of indicated genes. Green, tubulin; blue, DNA.

Movie 2 - Ncd-GFP dynamics in the S2 metaphase spindle.

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Movie 3 - Computer modeling reveals that the microtubule motors dynein (green) and EB1 (yellow)-localized Ncd (red) focus metaphase spindle microtubules to the poles.

We used these results and simulations to build a model to explain how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis (Fig 4).

Fig. 4 - A model for three-step pole focusing by minus end–directed motors. (A) During spindle assembly process in early mitosis, or even during metaphase when steady-state length of bipolar spindle is maintained, centrosomal MTs (C-MTs) and kinetochore microtubule bundles (K fibers) are often discontinuous. (B) Inter-K fiber cross-linking by minus end–directed motors (predominantly Ncd in S2 cells). (C) Search and capture of K fibers by Ncd, which is recruited to the microtubule plus end. Note that EB1 protein is required for plus end tracking of Ncd but is not described in this cartoon figure. Because Ncd has two microtubule binding domains, Ncd enables C-MTs to "search" for, then "capture," and generate force upon K fibers. Our computer simulation supports the herein described configuration of Ncd where the nonmotor domain of Ncd binds to K fibers. (D) Next, minus end–directed motors (primarily processive dynein motor) transport the K fibers along C-MTs to the pole (bottom). Our simulations suggest that the search and capture mechanism in C can potentially facilitate such motor-mediated transport. However, in the case where a C-MT locates spontaneously closely to a K fiber, dynein can cross-link and transport the K fiber without the help of plus end–tracking Ncd (top).

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updated 4/9/07

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