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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 positioning is regulated by Cdc42-dependent signaling pathway. (A) Kinetics of the centrosome movement in cells injected with Rho inhibitor C3 transferase (gray), or a combination of C3-transferase and Cdc42 dominant–negative recombinant protein N17Cdc42 (black). Arrowheads on the curves show the time points where images were taken in B and C. (B and C) Time series of the fluorescence images of MTs showing centrosome movement quantified in A, injected with C3 transferase (B) or double injected with C3 transferase and N17Cdc42 (C). See also the corresponding movies (Videos 5 and 6) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (B) In the absence of actin centripetal flow, the centrosome (black arrowhead) moves continuously away from the nocodazole pipette tip, driven by the pulling force on the MTs at the cortex. The pulling force on the end distal to the pipette tip is strong enough to induce breakage of the MTs at the proximal end (white arrowhead shows the position of the nascent end of the broken MT), accelerating the centrosome movement away from the pipette tip (as shown in A). (C) In the absence of both actin centripetal flow and the activity of Cdc42, centrosome moves back and forth around the central point. Pulling forces applied at the cortex are not enough to induce MT breakage. A single MT is enough to anchor the centrosome and pull it back toward the pipette tip, causing reversal of the direction of the movement (as shown in A). White arrowhead shows the position of the anchored MT end. Bars, 5 μm.
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fig4: Centrosome positioning is regulated by Cdc42-dependent signaling pathway. (A) Kinetics of the centrosome movement in cells injected with Rho inhibitor C3 transferase (gray), or a combination of C3-transferase and Cdc42 dominant–negative recombinant protein N17Cdc42 (black). Arrowheads on the curves show the time points where images were taken in B and C. (B and C) Time series of the fluorescence images of MTs showing centrosome movement quantified in A, injected with C3 transferase (B) or double injected with C3 transferase and N17Cdc42 (C). See also the corresponding movies (Videos 5 and 6) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (B) In the absence of actin centripetal flow, the centrosome (black arrowhead) moves continuously away from the nocodazole pipette tip, driven by the pulling force on the MTs at the cortex. The pulling force on the end distal to the pipette tip is strong enough to induce breakage of the MTs at the proximal end (white arrowhead shows the position of the nascent end of the broken MT), accelerating the centrosome movement away from the pipette tip (as shown in A). (C) In the absence of both actin centripetal flow and the activity of Cdc42, centrosome moves back and forth around the central point. Pulling forces applied at the cortex are not enough to induce MT breakage. A single MT is enough to anchor the centrosome and pull it back toward the pipette tip, causing reversal of the direction of the movement (as shown in A). White arrowhead shows the position of the anchored MT end. Bars, 5 μm.

Mentions: It has been shown that dynein-dependent centrosome repositioning during cell polarization requires the activity of Cdc42, a Rho family small GTPase (Etienne-Manneville and Hall, 2001; Palazzo et al., 2001). To test whether Cdc42 is involved in the MT pulling force applied to the centrosome, we have examined nocodazole-induced centrosome movement in cells coinjected with C3 transferase and N17Cdc42 dominant–negative recombinant protein to inhibit both actomyosin contractility and the activity of Cdc42 (Fig. 4). In C3 transferase–injected cells, the centrosome moved continuously away from the pipette tip. Close observation showed that the pulling force applied to the centrosome from the distal side was strong enough to induce breakage of the proximal MTs anchored at the cortex, often resulting in an acceleration of the centrosome toward the distal end of the cell (Fig. 4, A and B; Video 5). In contrast, in cells coinjected with C3 transferase and Cdc42 inhibitor the centrosome wobbled around the central position, apparently held in place by MTs anchored at the cortex. Close observation revealed that the force of a few remaining MTs at the proximal end (often just one MT) was enough to hold the centrosome in place and/or reverse the direction of its movement (Fig. 4, A and C; Video 6), indicating the absence of a strong pull at the distal end. Thus, inhibition of the Cdc42 activity causes the reduction of the MT pulling force applied to the centrosome.


Centrosome positioning in interphase cells.

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

Centrosome positioning is regulated by Cdc42-dependent signaling pathway. (A) Kinetics of the centrosome movement in cells injected with Rho inhibitor C3 transferase (gray), or a combination of C3-transferase and Cdc42 dominant–negative recombinant protein N17Cdc42 (black). Arrowheads on the curves show the time points where images were taken in B and C. (B and C) Time series of the fluorescence images of MTs showing centrosome movement quantified in A, injected with C3 transferase (B) or double injected with C3 transferase and N17Cdc42 (C). See also the corresponding movies (Videos 5 and 6) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (B) In the absence of actin centripetal flow, the centrosome (black arrowhead) moves continuously away from the nocodazole pipette tip, driven by the pulling force on the MTs at the cortex. The pulling force on the end distal to the pipette tip is strong enough to induce breakage of the MTs at the proximal end (white arrowhead shows the position of the nascent end of the broken MT), accelerating the centrosome movement away from the pipette tip (as shown in A). (C) In the absence of both actin centripetal flow and the activity of Cdc42, centrosome moves back and forth around the central point. Pulling forces applied at the cortex are not enough to induce MT breakage. A single MT is enough to anchor the centrosome and pull it back toward the pipette tip, causing reversal of the direction of the movement (as shown in A). White arrowhead shows the position of the anchored MT end. Bars, 5 μm.
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Related In: Results  -  Collection

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fig4: Centrosome positioning is regulated by Cdc42-dependent signaling pathway. (A) Kinetics of the centrosome movement in cells injected with Rho inhibitor C3 transferase (gray), or a combination of C3-transferase and Cdc42 dominant–negative recombinant protein N17Cdc42 (black). Arrowheads on the curves show the time points where images were taken in B and C. (B and C) Time series of the fluorescence images of MTs showing centrosome movement quantified in A, injected with C3 transferase (B) or double injected with C3 transferase and N17Cdc42 (C). See also the corresponding movies (Videos 5 and 6) for full time-lapse series, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. (B) In the absence of actin centripetal flow, the centrosome (black arrowhead) moves continuously away from the nocodazole pipette tip, driven by the pulling force on the MTs at the cortex. The pulling force on the end distal to the pipette tip is strong enough to induce breakage of the MTs at the proximal end (white arrowhead shows the position of the nascent end of the broken MT), accelerating the centrosome movement away from the pipette tip (as shown in A). (C) In the absence of both actin centripetal flow and the activity of Cdc42, centrosome moves back and forth around the central point. Pulling forces applied at the cortex are not enough to induce MT breakage. A single MT is enough to anchor the centrosome and pull it back toward the pipette tip, causing reversal of the direction of the movement (as shown in A). White arrowhead shows the position of the anchored MT end. Bars, 5 μm.
Mentions: It has been shown that dynein-dependent centrosome repositioning during cell polarization requires the activity of Cdc42, a Rho family small GTPase (Etienne-Manneville and Hall, 2001; Palazzo et al., 2001). To test whether Cdc42 is involved in the MT pulling force applied to the centrosome, we have examined nocodazole-induced centrosome movement in cells coinjected with C3 transferase and N17Cdc42 dominant–negative recombinant protein to inhibit both actomyosin contractility and the activity of Cdc42 (Fig. 4). In C3 transferase–injected cells, the centrosome moved continuously away from the pipette tip. Close observation showed that the pulling force applied to the centrosome from the distal side was strong enough to induce breakage of the proximal MTs anchored at the cortex, often resulting in an acceleration of the centrosome toward the distal end of the cell (Fig. 4, A and B; Video 5). In contrast, in cells coinjected with C3 transferase and Cdc42 inhibitor the centrosome wobbled around the central position, apparently held in place by MTs anchored at the cortex. Close observation revealed that the force of a few remaining MTs at the proximal end (often just one MT) was enough to hold the centrosome in place and/or reverse the direction of its movement (Fig. 4, A and C; Video 6), indicating the absence of a strong pull at the distal end. Thus, inhibition of the Cdc42 activity causes the reduction of the MT pulling force applied to the centrosome.

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