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Centrosome positioning in interphase cells.

Burakov A, Nadezhdina E, Slepchenko B, Rodionov V - J. Cell Biol. (2003)

Bottom Line: It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors.It has also been suggested that actomyosin can play a direct or indirect role in this process.Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Center for Biomedical Imaging, University of Connecticut Health Center, Technology, Farmington, CT 06032-1507, USA.

ABSTRACT
The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

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Centrosome position is maintained through a MT mediated pulling force. (A and D) Pairs of live fluorescence images of the centrosome acquired before (top image of each pair) and after (bottom image of each pair) the local application of nocodazole. Insets show the position of the centrosome in the same cells at higher magnification. The initial positions of the centrosome are indicated by black arrows. See also the corresponding movies (Videos 3 and 4) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (A) Noninjected cell. Local application of nocodazole induces centrosome movement toward the application site. (D) Cell injected with a Rho inhibitor C3 transferase (0.1 mg/ml). Inhibition of RhoA activity reverses the direction of the centrosome movement upon application of nocodazole. (B) Method for the quantification of the centrosome movement. The centrosome position (C) was determined as the focus of the MT fluorescence. Centroid position (Ct) was calculated by Metamorph software as the point equidistant from all the cell margins. Centrosome movement was calculated as the distance between the initial (C0) and final (C1) positions of the centrosome, projected onto a straight line connecting the nocodazole pipette tip (N) and the centrosome at time point zero. Observations were made in stationary cells, but cell shape changes during the time of the experiment (6–15 min) sometimes led to small changes in the calculated position of the centroid. In such cases, centroid displacement was projected onto the same straight line and the vector sum of the centroid, and centrosome displacement was calculated to obtain the final value of the centrosome movement. (C) Quantification of the centrosome movement. Positive value of movement was considered when the centrosome moved from the initial position (C0) toward the nocodazole pipette tip (N). Open bar shows centrosome displacement in cytoplasts. (E) Kymograph analysis of rhodamine F-actin speckles in control (left) and C3-transferase–injected (right) cell. The absence of the centripetal flow after the injection of C3-transferase is evident from the lack of directional movement of the fluorescent speckles (right), compared with the diagonal pattern of movement in the control cells (left). Bars, 20 μm.
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fig2: Centrosome position is maintained through a MT mediated pulling force. (A and D) Pairs of live fluorescence images of the centrosome acquired before (top image of each pair) and after (bottom image of each pair) the local application of nocodazole. Insets show the position of the centrosome in the same cells at higher magnification. The initial positions of the centrosome are indicated by black arrows. See also the corresponding movies (Videos 3 and 4) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (A) Noninjected cell. Local application of nocodazole induces centrosome movement toward the application site. (D) Cell injected with a Rho inhibitor C3 transferase (0.1 mg/ml). Inhibition of RhoA activity reverses the direction of the centrosome movement upon application of nocodazole. (B) Method for the quantification of the centrosome movement. The centrosome position (C) was determined as the focus of the MT fluorescence. Centroid position (Ct) was calculated by Metamorph software as the point equidistant from all the cell margins. Centrosome movement was calculated as the distance between the initial (C0) and final (C1) positions of the centrosome, projected onto a straight line connecting the nocodazole pipette tip (N) and the centrosome at time point zero. Observations were made in stationary cells, but cell shape changes during the time of the experiment (6–15 min) sometimes led to small changes in the calculated position of the centroid. In such cases, centroid displacement was projected onto the same straight line and the vector sum of the centroid, and centrosome displacement was calculated to obtain the final value of the centrosome movement. (C) Quantification of the centrosome movement. Positive value of movement was considered when the centrosome moved from the initial position (C0) toward the nocodazole pipette tip (N). Open bar shows centrosome displacement in cytoplasts. (E) Kymograph analysis of rhodamine F-actin speckles in control (left) and C3-transferase–injected (right) cell. The absence of the centripetal flow after the injection of C3-transferase is evident from the lack of directional movement of the fluorescent speckles (right), compared with the diagonal pattern of movement in the control cells (left). Bars, 20 μm.

Mentions: Local application of nocodazole solution at the cell edge resulted in rapid (∼0.3 μm/min) movement of the centrosome toward the micropipette tip in all examined cells (n = 9) (Fig. 2, A and C; Video 3). In control experiments, local application of a drug-free medium did not induce significant changes in the centrosome position. The observed displacement was independent of the nucleus, since the same experiment in cell cytoplasts resulted in a similar centrosome behavior (Fig. 1 C). Thus, local disruption of MTs induces centrosome movement toward the site of nocodazole application.


Centrosome positioning in interphase cells.

Burakov A, Nadezhdina E, Slepchenko B, Rodionov V - J. Cell Biol. (2003)

Centrosome position is maintained through a MT mediated pulling force. (A and D) Pairs of live fluorescence images of the centrosome acquired before (top image of each pair) and after (bottom image of each pair) the local application of nocodazole. Insets show the position of the centrosome in the same cells at higher magnification. The initial positions of the centrosome are indicated by black arrows. See also the corresponding movies (Videos 3 and 4) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (A) Noninjected cell. Local application of nocodazole induces centrosome movement toward the application site. (D) Cell injected with a Rho inhibitor C3 transferase (0.1 mg/ml). Inhibition of RhoA activity reverses the direction of the centrosome movement upon application of nocodazole. (B) Method for the quantification of the centrosome movement. The centrosome position (C) was determined as the focus of the MT fluorescence. Centroid position (Ct) was calculated by Metamorph software as the point equidistant from all the cell margins. Centrosome movement was calculated as the distance between the initial (C0) and final (C1) positions of the centrosome, projected onto a straight line connecting the nocodazole pipette tip (N) and the centrosome at time point zero. Observations were made in stationary cells, but cell shape changes during the time of the experiment (6–15 min) sometimes led to small changes in the calculated position of the centroid. In such cases, centroid displacement was projected onto the same straight line and the vector sum of the centroid, and centrosome displacement was calculated to obtain the final value of the centrosome movement. (C) Quantification of the centrosome movement. Positive value of movement was considered when the centrosome moved from the initial position (C0) toward the nocodazole pipette tip (N). Open bar shows centrosome displacement in cytoplasts. (E) Kymograph analysis of rhodamine F-actin speckles in control (left) and C3-transferase–injected (right) cell. The absence of the centripetal flow after the injection of C3-transferase is evident from the lack of directional movement of the fluorescent speckles (right), compared with the diagonal pattern of movement in the control cells (left). Bars, 20 μm.
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Related In: Results  -  Collection

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fig2: Centrosome position is maintained through a MT mediated pulling force. (A and D) Pairs of live fluorescence images of the centrosome acquired before (top image of each pair) and after (bottom image of each pair) the local application of nocodazole. Insets show the position of the centrosome in the same cells at higher magnification. The initial positions of the centrosome are indicated by black arrows. See also the corresponding movies (Videos 3 and 4) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (A) Noninjected cell. Local application of nocodazole induces centrosome movement toward the application site. (D) Cell injected with a Rho inhibitor C3 transferase (0.1 mg/ml). Inhibition of RhoA activity reverses the direction of the centrosome movement upon application of nocodazole. (B) Method for the quantification of the centrosome movement. The centrosome position (C) was determined as the focus of the MT fluorescence. Centroid position (Ct) was calculated by Metamorph software as the point equidistant from all the cell margins. Centrosome movement was calculated as the distance between the initial (C0) and final (C1) positions of the centrosome, projected onto a straight line connecting the nocodazole pipette tip (N) and the centrosome at time point zero. Observations were made in stationary cells, but cell shape changes during the time of the experiment (6–15 min) sometimes led to small changes in the calculated position of the centroid. In such cases, centroid displacement was projected onto the same straight line and the vector sum of the centroid, and centrosome displacement was calculated to obtain the final value of the centrosome movement. (C) Quantification of the centrosome movement. Positive value of movement was considered when the centrosome moved from the initial position (C0) toward the nocodazole pipette tip (N). Open bar shows centrosome displacement in cytoplasts. (E) Kymograph analysis of rhodamine F-actin speckles in control (left) and C3-transferase–injected (right) cell. The absence of the centripetal flow after the injection of C3-transferase is evident from the lack of directional movement of the fluorescent speckles (right), compared with the diagonal pattern of movement in the control cells (left). Bars, 20 μm.
Mentions: Local application of nocodazole solution at the cell edge resulted in rapid (∼0.3 μm/min) movement of the centrosome toward the micropipette tip in all examined cells (n = 9) (Fig. 2, A and C; Video 3). In control experiments, local application of a drug-free medium did not induce significant changes in the centrosome position. The observed displacement was independent of the nucleus, since the same experiment in cell cytoplasts resulted in a similar centrosome behavior (Fig. 1 C). Thus, local disruption of MTs induces centrosome movement toward the site of nocodazole application.

Bottom Line: It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors.It has also been suggested that actomyosin can play a direct or indirect role in this process.Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Center for Biomedical Imaging, University of Connecticut Health Center, Technology, Farmington, CT 06032-1507, USA.

ABSTRACT
The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.

Show MeSH
Related in: MedlinePlus