Limits...
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.

Show MeSH

Related in: MedlinePlus

Positioning of the centrosome in the cell center requires the activity of cytoplasmic dynein. (A) Live fluorescence images of MTs in a cell before (top) or 45 min after (bottom) the injection of a dynein blocking antibody 74.1. After the injection of dynein blocking antibody, the centrosome moved to the cell margin. (B) Quantification of the displacement of the centrosome in cells injected with C3 transferase, antibody 74.1, or the p50 subunit of dynactin. Relative displacement of the centrosome was determined as the ratio between the initial (before injection) and the final (after injection) positions of the centrosome, calculated as the percentage of the cell radius drawn from the centroid through the centrosome. In control and C3-injected cells, where the centrosome remained in place, the ratios were close to 1. In cells injected with antibody 74.1 or the p50 subunit of dynactin, the centrosome traveled significantly toward the nearest cell margin, causing the greatly increased displacement values. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172857&req=5

fig3: Positioning of the centrosome in the cell center requires the activity of cytoplasmic dynein. (A) Live fluorescence images of MTs in a cell before (top) or 45 min after (bottom) the injection of a dynein blocking antibody 74.1. After the injection of dynein blocking antibody, the centrosome moved to the cell margin. (B) Quantification of the displacement of the centrosome in cells injected with C3 transferase, antibody 74.1, or the p50 subunit of dynactin. Relative displacement of the centrosome was determined as the ratio between the initial (before injection) and the final (after injection) positions of the centrosome, calculated as the percentage of the cell radius drawn from the centroid through the centrosome. In control and C3-injected cells, where the centrosome remained in place, the ratios were close to 1. In cells injected with antibody 74.1 or the p50 subunit of dynactin, the centrosome traveled significantly toward the nearest cell margin, causing the greatly increased displacement values. Bar, 20 μm.

Mentions: The nocodazole-induced centrosome displacement may be caused by the pushing force produced by MT growth at the edge distal to the MT disruption site. However, MT depolymerization can also activate RhoA-dependent signaling pathway that is known to affect actomyosin contractility (for review see Wittmann and Waterman-Storer, 2001). Therefore, another explanation of the observed effect is that MT depolymerization by nocodazole mediates the induction of the local contraction of the actin cytoskeleton, and therefore facilitates the movement of the actin cytoskeleton with the attached MT aster toward the site of nocodazole application. To determine the role of actomyosin contractility in the movement of the centrosome, we either inhibited the activity of Rho A by injecting C3 transferase, or suppressed myosin activity by treating cells with myosin light chain kinase inhibitor ML7. Fluorescent speckle microscopy of actin in cells with intact MTs indicated that, as expected, C3 transferase (Fig. 2 E) or ML7 (unpublished data) completely suppressed actin centripetal flow. Inhibitors of actomyosin contractility did not significantly affect the position of the centrosome in intact cells, indicating that actomyosin contractility alone does not play a role in maintaining the centrosome position at the cell center (Fig. 3 B). However, local disruption of MTs in cells injected with C3 transferase or treated with ML7 induced profound displacement of the centrosome away from the micropipette tip (Fig. 2, C and D; Video 4). This result indicates that the displacement toward the pipette tip observed in the previous experiment was likely caused by the increase in actomyosin contractility, and that in its absence, when the forces applied to the centrosome are purely MT dependent, local depolymerization of MTs causes the pull on the centrosome from the distal end. We therefore conclude that the force exerted on the centrosome by MTs is of a pulling rather than a pushing nature.


Centrosome positioning in interphase cells.

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

Positioning of the centrosome in the cell center requires the activity of cytoplasmic dynein. (A) Live fluorescence images of MTs in a cell before (top) or 45 min after (bottom) the injection of a dynein blocking antibody 74.1. After the injection of dynein blocking antibody, the centrosome moved to the cell margin. (B) Quantification of the displacement of the centrosome in cells injected with C3 transferase, antibody 74.1, or the p50 subunit of dynactin. Relative displacement of the centrosome was determined as the ratio between the initial (before injection) and the final (after injection) positions of the centrosome, calculated as the percentage of the cell radius drawn from the centroid through the centrosome. In control and C3-injected cells, where the centrosome remained in place, the ratios were close to 1. In cells injected with antibody 74.1 or the p50 subunit of dynactin, the centrosome traveled significantly toward the nearest cell margin, causing the greatly increased displacement values. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172857&req=5

fig3: Positioning of the centrosome in the cell center requires the activity of cytoplasmic dynein. (A) Live fluorescence images of MTs in a cell before (top) or 45 min after (bottom) the injection of a dynein blocking antibody 74.1. After the injection of dynein blocking antibody, the centrosome moved to the cell margin. (B) Quantification of the displacement of the centrosome in cells injected with C3 transferase, antibody 74.1, or the p50 subunit of dynactin. Relative displacement of the centrosome was determined as the ratio between the initial (before injection) and the final (after injection) positions of the centrosome, calculated as the percentage of the cell radius drawn from the centroid through the centrosome. In control and C3-injected cells, where the centrosome remained in place, the ratios were close to 1. In cells injected with antibody 74.1 or the p50 subunit of dynactin, the centrosome traveled significantly toward the nearest cell margin, causing the greatly increased displacement values. Bar, 20 μm.
Mentions: The nocodazole-induced centrosome displacement may be caused by the pushing force produced by MT growth at the edge distal to the MT disruption site. However, MT depolymerization can also activate RhoA-dependent signaling pathway that is known to affect actomyosin contractility (for review see Wittmann and Waterman-Storer, 2001). Therefore, another explanation of the observed effect is that MT depolymerization by nocodazole mediates the induction of the local contraction of the actin cytoskeleton, and therefore facilitates the movement of the actin cytoskeleton with the attached MT aster toward the site of nocodazole application. To determine the role of actomyosin contractility in the movement of the centrosome, we either inhibited the activity of Rho A by injecting C3 transferase, or suppressed myosin activity by treating cells with myosin light chain kinase inhibitor ML7. Fluorescent speckle microscopy of actin in cells with intact MTs indicated that, as expected, C3 transferase (Fig. 2 E) or ML7 (unpublished data) completely suppressed actin centripetal flow. Inhibitors of actomyosin contractility did not significantly affect the position of the centrosome in intact cells, indicating that actomyosin contractility alone does not play a role in maintaining the centrosome position at the cell center (Fig. 3 B). However, local disruption of MTs in cells injected with C3 transferase or treated with ML7 induced profound displacement of the centrosome away from the micropipette tip (Fig. 2, C and D; Video 4). This result indicates that the displacement toward the pipette tip observed in the previous experiment was likely caused by the increase in actomyosin contractility, and that in its absence, when the forces applied to the centrosome are purely MT dependent, local depolymerization of MTs causes the pull on the centrosome from the distal end. We therefore conclude that the force exerted on the centrosome by MTs is of a pulling rather than a pushing nature.

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