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Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
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. Here, we have 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 show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

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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, 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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fig6: 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, 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).

Mentions: Based upon our live cell imaging and computer simulation analyses, as well as data from others (Walczak et al., 1998; Maiato et al., 2004), we propose that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking, (2) “search and capture” of K fibers by the tip of growing C-MTs, and (3) K fiber transport on C-MTs (Fig. 6). Our RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus end–directed motor proteins in these processes. By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type, we find that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing outlined above.


Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins.

Goshima G, Nédélec F, Vale RD - J. Cell Biol. (2005)

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, 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171195&req=5

fig6: 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, 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).
Mentions: Based upon our live cell imaging and computer simulation analyses, as well as data from others (Walczak et al., 1998; Maiato et al., 2004), we propose that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking, (2) “search and capture” of K fibers by the tip of growing C-MTs, and (3) K fiber transport on C-MTs (Fig. 6). Our RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus end–directed motor proteins in these processes. By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type, we find that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing outlined above.

Bottom Line: Even though these two motors have overlapping functions, we show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes.Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs.From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

View Article: PubMed Central - PubMed

Affiliation: The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94107, USA.

ABSTRACT
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. Here, we have 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 show that Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. We also report a novel localization of Ncd to the growing tips of C-MTs, which we show is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, we propose a model on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis.

Show MeSH