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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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Related in: MedlinePlus

Computer simulation of pole focusing by minus end–directed motor proteins. (A) Five K fibers and one aster of dynamic microtubules were simulated to study the pole formation in the mitotic spindle by dynein (green), Ncd (red), and EB1 (yellow) proteins. The K fibers were 10 times stiffer than single astral microtubule, and arranged in a parallel manner with the plus end upward, from which they were held in place firmly, fixed both in position, and rotation (e.g., immobile chromosomes, large white discs). The interaction of K fibers and microtubules was mediated by complexes able to cross-link any two fibers together (see zoomed regions a–c). The time sequence shows the process of K fiber focusing during this simulation. (B) Definition of K fiber distance in the simulation. (C) Influence of dynein dwell-time after reaching the microtubule minus end. Dynein is simulated alone, varying its detachment rate after reaching the minus end, and the other characteristics of the motor (Table I). Because these different parameters are chosen independently, the random sampling can be used to assess the influence of the dwell-time on the focusing. The quality of the focusing is measured likewise experimentally, and varies between 5 μm for nonfocused K fibers to ∼2 μm for a good focusing (see Materials and methods). The points are derived from 2,135 individual simulations and collected in bins based upon different minus end dwell times. The average and standard deviation of the data from these simulations are shown with open symbols (in order to simplify the graph, at dwell-time below 50 ms, circles represent the mean of more than one dwell time). These simulations reveal that the dwell-time has a strong influence on focusing, with a smooth transition around 100 ms. Dynein with a dwell time of 80 ms or more were able to focus the ends very well in some runs. See also Video 9. (D and E) Effect of plus end–localized Ncd–EB1 complexes on K fiber distance (see Table I for each parameter). In the simulations presented in D, the motor domain of Ncd–EB1 complex binds to C-MTs and the nonmotor domain to the K fiber, whereas it is reversed in E (Ncd motor domain binds to K fibers). Each point represents results of K fiber focusing from two simulations: one for dynein alone and one in which dynein was augmented by Ncd–EB1 (various motor parameters used, but the same set of dynein parameter was compared between plus and minus Ncd–EB1). The K fiber focusing result obtained for dynein alone defines the x-axis coordinate, while the focusing obtained after the addition of Ncd–EB1 defines the y-axis coordinate. Points on the diagonal show that Ncd–EB1 did not affect the outcome of dynein-mediated K fiber focusing. Points below the diagonal reveal a positive contribution of Ncd–EB1 to the focusing, while points above reflect a detrimental effect. These simulation reveal that Ncd–EB1, in a configuration where the Ncd motor domain interacts with C-MTs and the nonmotor domain interacts with K fibers, contributes positively to dynein-mediated K fiber focusing (points tend to be below the diagonal), and that this effect is particularly dramatic for simulations in which dynein was less effective for focusing on its own (larger values on the x-axis). In contrast, Ncd–EB1 complex has a strong negative effect on K fiber focusing in the configuration where the motor domain binds to K fibers. See also Video 10.
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fig5: Computer simulation of pole focusing by minus end–directed motor proteins. (A) Five K fibers and one aster of dynamic microtubules were simulated to study the pole formation in the mitotic spindle by dynein (green), Ncd (red), and EB1 (yellow) proteins. The K fibers were 10 times stiffer than single astral microtubule, and arranged in a parallel manner with the plus end upward, from which they were held in place firmly, fixed both in position, and rotation (e.g., immobile chromosomes, large white discs). The interaction of K fibers and microtubules was mediated by complexes able to cross-link any two fibers together (see zoomed regions a–c). The time sequence shows the process of K fiber focusing during this simulation. (B) Definition of K fiber distance in the simulation. (C) Influence of dynein dwell-time after reaching the microtubule minus end. Dynein is simulated alone, varying its detachment rate after reaching the minus end, and the other characteristics of the motor (Table I). Because these different parameters are chosen independently, the random sampling can be used to assess the influence of the dwell-time on the focusing. The quality of the focusing is measured likewise experimentally, and varies between 5 μm for nonfocused K fibers to ∼2 μm for a good focusing (see Materials and methods). The points are derived from 2,135 individual simulations and collected in bins based upon different minus end dwell times. The average and standard deviation of the data from these simulations are shown with open symbols (in order to simplify the graph, at dwell-time below 50 ms, circles represent the mean of more than one dwell time). These simulations reveal that the dwell-time has a strong influence on focusing, with a smooth transition around 100 ms. Dynein with a dwell time of 80 ms or more were able to focus the ends very well in some runs. See also Video 9. (D and E) Effect of plus end–localized Ncd–EB1 complexes on K fiber distance (see Table I for each parameter). In the simulations presented in D, the motor domain of Ncd–EB1 complex binds to C-MTs and the nonmotor domain to the K fiber, whereas it is reversed in E (Ncd motor domain binds to K fibers). Each point represents results of K fiber focusing from two simulations: one for dynein alone and one in which dynein was augmented by Ncd–EB1 (various motor parameters used, but the same set of dynein parameter was compared between plus and minus Ncd–EB1). The K fiber focusing result obtained for dynein alone defines the x-axis coordinate, while the focusing obtained after the addition of Ncd–EB1 defines the y-axis coordinate. Points on the diagonal show that Ncd–EB1 did not affect the outcome of dynein-mediated K fiber focusing. Points below the diagonal reveal a positive contribution of Ncd–EB1 to the focusing, while points above reflect a detrimental effect. These simulation reveal that Ncd–EB1, in a configuration where the Ncd motor domain interacts with C-MTs and the nonmotor domain interacts with K fibers, contributes positively to dynein-mediated K fiber focusing (points tend to be below the diagonal), and that this effect is particularly dramatic for simulations in which dynein was less effective for focusing on its own (larger values on the x-axis). In contrast, Ncd–EB1 complex has a strong negative effect on K fiber focusing in the configuration where the motor domain binds to K fibers. See also Video 10.

Mentions: To explore how minus end–directed motors and EB1 may contribute to the focusing of K fibers at the spindle pole, we applied computer simulations based upon our previous study that simulates establishment of bipolarity of two asters by varying properties of motors (Nedelec, 2002). Specifically, we sought to examine a possible role of an Ncd–EB1 complex at the plus ends of C-MTs. For simplicity, we used a half spindle in a two-dimensional simulation with five K fibers of constant length (6 μm) that have a 10-fold greater stiffness (result of microtubule bundling) compared with single C-MT (Fig. 5 A). The chromosomes to which the K fibers connect at the plus ends were not explicitly represented in the model; instead the fibers were immobilized at their plus end throughout the simulation (Fig. 5 A, white discs). Onto this microtubule array, we added a complex of minus end–directed motors (Nedelec, 2002), which can move toward the minus ends of K fibers and also can cross-bridge a K fiber to a C fiber microtubule thereby exerting forces between the centrosome and the K fiber (Fig. 5 A, green). Although we do not know if such a multiple motor complex exists in the spindle, it could be produced by multifunctional cargos such as Asp or NuMA that could bind multiple motors simultaneously. For our modeling, we endowed this motor complex with characteristics of dynein (e.g., speeds of 1 μm/s). This is consistent with our RNAi results indicating that dynein is the dominant motor for K fiber transport; however, also note that Ncd may also execute this function, albeit less efficiently, in the absence of dynein (e.g., after dynein RNAi). In some simulations, we also added Ncd–EB1 complex, in which EB1 (Fig. 5 A, yellow) is localized on the ∼1 μm distal plus ends of C-MTs to mimic our finding of microtubule plus end tracking of Ncd (Fig. 5 A, red). Because we do not know whether motor or tail domain of plus end–tracking Ncd binds to K fiber, we investigated the effect of each configuration. To evaluate the outcomes of these simulations, the distance between leftmost and rightmost K fibers was used (Fig. 5 B; analogous to the K fiber focusing measurement in Fig. 2 C). The simulation is described in more detail in the Materials and methods section. Note that we did not take into account other factors that contribute to pole focusing, such as inter-K fiber cross-linking (driven by Ncd) or other cross-linking factors that may operate at minus ends (e.g., Asp or NuMA).


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

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

Computer simulation of pole focusing by minus end–directed motor proteins. (A) Five K fibers and one aster of dynamic microtubules were simulated to study the pole formation in the mitotic spindle by dynein (green), Ncd (red), and EB1 (yellow) proteins. The K fibers were 10 times stiffer than single astral microtubule, and arranged in a parallel manner with the plus end upward, from which they were held in place firmly, fixed both in position, and rotation (e.g., immobile chromosomes, large white discs). The interaction of K fibers and microtubules was mediated by complexes able to cross-link any two fibers together (see zoomed regions a–c). The time sequence shows the process of K fiber focusing during this simulation. (B) Definition of K fiber distance in the simulation. (C) Influence of dynein dwell-time after reaching the microtubule minus end. Dynein is simulated alone, varying its detachment rate after reaching the minus end, and the other characteristics of the motor (Table I). Because these different parameters are chosen independently, the random sampling can be used to assess the influence of the dwell-time on the focusing. The quality of the focusing is measured likewise experimentally, and varies between 5 μm for nonfocused K fibers to ∼2 μm for a good focusing (see Materials and methods). The points are derived from 2,135 individual simulations and collected in bins based upon different minus end dwell times. The average and standard deviation of the data from these simulations are shown with open symbols (in order to simplify the graph, at dwell-time below 50 ms, circles represent the mean of more than one dwell time). These simulations reveal that the dwell-time has a strong influence on focusing, with a smooth transition around 100 ms. Dynein with a dwell time of 80 ms or more were able to focus the ends very well in some runs. See also Video 9. (D and E) Effect of plus end–localized Ncd–EB1 complexes on K fiber distance (see Table I for each parameter). In the simulations presented in D, the motor domain of Ncd–EB1 complex binds to C-MTs and the nonmotor domain to the K fiber, whereas it is reversed in E (Ncd motor domain binds to K fibers). Each point represents results of K fiber focusing from two simulations: one for dynein alone and one in which dynein was augmented by Ncd–EB1 (various motor parameters used, but the same set of dynein parameter was compared between plus and minus Ncd–EB1). The K fiber focusing result obtained for dynein alone defines the x-axis coordinate, while the focusing obtained after the addition of Ncd–EB1 defines the y-axis coordinate. Points on the diagonal show that Ncd–EB1 did not affect the outcome of dynein-mediated K fiber focusing. Points below the diagonal reveal a positive contribution of Ncd–EB1 to the focusing, while points above reflect a detrimental effect. These simulation reveal that Ncd–EB1, in a configuration where the Ncd motor domain interacts with C-MTs and the nonmotor domain interacts with K fibers, contributes positively to dynein-mediated K fiber focusing (points tend to be below the diagonal), and that this effect is particularly dramatic for simulations in which dynein was less effective for focusing on its own (larger values on the x-axis). In contrast, Ncd–EB1 complex has a strong negative effect on K fiber focusing in the configuration where the motor domain binds to K fibers. See also Video 10.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171195&req=5

fig5: Computer simulation of pole focusing by minus end–directed motor proteins. (A) Five K fibers and one aster of dynamic microtubules were simulated to study the pole formation in the mitotic spindle by dynein (green), Ncd (red), and EB1 (yellow) proteins. The K fibers were 10 times stiffer than single astral microtubule, and arranged in a parallel manner with the plus end upward, from which they were held in place firmly, fixed both in position, and rotation (e.g., immobile chromosomes, large white discs). The interaction of K fibers and microtubules was mediated by complexes able to cross-link any two fibers together (see zoomed regions a–c). The time sequence shows the process of K fiber focusing during this simulation. (B) Definition of K fiber distance in the simulation. (C) Influence of dynein dwell-time after reaching the microtubule minus end. Dynein is simulated alone, varying its detachment rate after reaching the minus end, and the other characteristics of the motor (Table I). Because these different parameters are chosen independently, the random sampling can be used to assess the influence of the dwell-time on the focusing. The quality of the focusing is measured likewise experimentally, and varies between 5 μm for nonfocused K fibers to ∼2 μm for a good focusing (see Materials and methods). The points are derived from 2,135 individual simulations and collected in bins based upon different minus end dwell times. The average and standard deviation of the data from these simulations are shown with open symbols (in order to simplify the graph, at dwell-time below 50 ms, circles represent the mean of more than one dwell time). These simulations reveal that the dwell-time has a strong influence on focusing, with a smooth transition around 100 ms. Dynein with a dwell time of 80 ms or more were able to focus the ends very well in some runs. See also Video 9. (D and E) Effect of plus end–localized Ncd–EB1 complexes on K fiber distance (see Table I for each parameter). In the simulations presented in D, the motor domain of Ncd–EB1 complex binds to C-MTs and the nonmotor domain to the K fiber, whereas it is reversed in E (Ncd motor domain binds to K fibers). Each point represents results of K fiber focusing from two simulations: one for dynein alone and one in which dynein was augmented by Ncd–EB1 (various motor parameters used, but the same set of dynein parameter was compared between plus and minus Ncd–EB1). The K fiber focusing result obtained for dynein alone defines the x-axis coordinate, while the focusing obtained after the addition of Ncd–EB1 defines the y-axis coordinate. Points on the diagonal show that Ncd–EB1 did not affect the outcome of dynein-mediated K fiber focusing. Points below the diagonal reveal a positive contribution of Ncd–EB1 to the focusing, while points above reflect a detrimental effect. These simulation reveal that Ncd–EB1, in a configuration where the Ncd motor domain interacts with C-MTs and the nonmotor domain interacts with K fibers, contributes positively to dynein-mediated K fiber focusing (points tend to be below the diagonal), and that this effect is particularly dramatic for simulations in which dynein was less effective for focusing on its own (larger values on the x-axis). In contrast, Ncd–EB1 complex has a strong negative effect on K fiber focusing in the configuration where the motor domain binds to K fibers. See also Video 10.
Mentions: To explore how minus end–directed motors and EB1 may contribute to the focusing of K fibers at the spindle pole, we applied computer simulations based upon our previous study that simulates establishment of bipolarity of two asters by varying properties of motors (Nedelec, 2002). Specifically, we sought to examine a possible role of an Ncd–EB1 complex at the plus ends of C-MTs. For simplicity, we used a half spindle in a two-dimensional simulation with five K fibers of constant length (6 μm) that have a 10-fold greater stiffness (result of microtubule bundling) compared with single C-MT (Fig. 5 A). The chromosomes to which the K fibers connect at the plus ends were not explicitly represented in the model; instead the fibers were immobilized at their plus end throughout the simulation (Fig. 5 A, white discs). Onto this microtubule array, we added a complex of minus end–directed motors (Nedelec, 2002), which can move toward the minus ends of K fibers and also can cross-bridge a K fiber to a C fiber microtubule thereby exerting forces between the centrosome and the K fiber (Fig. 5 A, green). Although we do not know if such a multiple motor complex exists in the spindle, it could be produced by multifunctional cargos such as Asp or NuMA that could bind multiple motors simultaneously. For our modeling, we endowed this motor complex with characteristics of dynein (e.g., speeds of 1 μm/s). This is consistent with our RNAi results indicating that dynein is the dominant motor for K fiber transport; however, also note that Ncd may also execute this function, albeit less efficiently, in the absence of dynein (e.g., after dynein RNAi). In some simulations, we also added Ncd–EB1 complex, in which EB1 (Fig. 5 A, yellow) is localized on the ∼1 μm distal plus ends of C-MTs to mimic our finding of microtubule plus end tracking of Ncd (Fig. 5 A, red). Because we do not know whether motor or tail domain of plus end–tracking Ncd binds to K fiber, we investigated the effect of each configuration. To evaluate the outcomes of these simulations, the distance between leftmost and rightmost K fibers was used (Fig. 5 B; analogous to the K fiber focusing measurement in Fig. 2 C). The simulation is described in more detail in the Materials and methods section. Note that we did not take into account other factors that contribute to pole focusing, such as inter-K fiber cross-linking (driven by Ncd) or other cross-linking factors that may operate at minus ends (e.g., Asp or NuMA).

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
Related in: MedlinePlus