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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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Local disruption of MTs in a cell by the local application of nocodazole. (Center) low magnification live fluorescence image of a cell with MTs labeled by injecting fluorescently tagged tubulin subunits. Image was obtained just before application through the micropipette of a nocodazole solution in the area depicted by the dashed line. Micrographs on the left and right are high magnification images of MTs in the boxed regions shown in the central panel acquired at the cell edge proximal to (left) or distal from (right) the micropipette tip, before (top) or after (bottom) the application of nocodazole. Time-lapsed series of MT dynamics corresponding to the micrographs on left and right are shown in Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. The graphs show kinetics of changes in the levels of tubulin monomer (plots shown in gray) or polymer (plots shown in black) at the cell edges proximal (left) or distal (right) to the micropipette tip. Bar, 20 μm.
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fig1: Local disruption of MTs in a cell by the local application of nocodazole. (Center) low magnification live fluorescence image of a cell with MTs labeled by injecting fluorescently tagged tubulin subunits. Image was obtained just before application through the micropipette of a nocodazole solution in the area depicted by the dashed line. Micrographs on the left and right are high magnification images of MTs in the boxed regions shown in the central panel acquired at the cell edge proximal to (left) or distal from (right) the micropipette tip, before (top) or after (bottom) the application of nocodazole. Time-lapsed series of MT dynamics corresponding to the micrographs on left and right are shown in Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. The graphs show kinetics of changes in the levels of tubulin monomer (plots shown in gray) or polymer (plots shown in black) at the cell edges proximal (left) or distal (right) to the micropipette tip. Bar, 20 μm.

Mentions: Organization of the centrosome–MT complex in BS-C-1 cells was examined by injecting them with Cy-3 labeled tubulin and acquiring images of fluorescent MTs (Fig. 1, center). The position of the centrosome was easily traceable as the focal point of converging MTs. Immunostaining for ɛ-, γ-, and α-tubulins confirmed that such a focal point corresponded to the actual position of the centrosome and indicated that, similar to other cell types, MTs were attached to the less motile mother centriole (unpublished data), which we will refer to as the centrosome here.


Centrosome positioning in interphase cells.

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

Local disruption of MTs in a cell by the local application of nocodazole. (Center) low magnification live fluorescence image of a cell with MTs labeled by injecting fluorescently tagged tubulin subunits. Image was obtained just before application through the micropipette of a nocodazole solution in the area depicted by the dashed line. Micrographs on the left and right are high magnification images of MTs in the boxed regions shown in the central panel acquired at the cell edge proximal to (left) or distal from (right) the micropipette tip, before (top) or after (bottom) the application of nocodazole. Time-lapsed series of MT dynamics corresponding to the micrographs on left and right are shown in Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. The graphs show kinetics of changes in the levels of tubulin monomer (plots shown in gray) or polymer (plots shown in black) at the cell edges proximal (left) or distal (right) to the micropipette tip. Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Local disruption of MTs in a cell by the local application of nocodazole. (Center) low magnification live fluorescence image of a cell with MTs labeled by injecting fluorescently tagged tubulin subunits. Image was obtained just before application through the micropipette of a nocodazole solution in the area depicted by the dashed line. Micrographs on the left and right are high magnification images of MTs in the boxed regions shown in the central panel acquired at the cell edge proximal to (left) or distal from (right) the micropipette tip, before (top) or after (bottom) the application of nocodazole. Time-lapsed series of MT dynamics corresponding to the micrographs on left and right are shown in Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200305082/DC1. The graphs show kinetics of changes in the levels of tubulin monomer (plots shown in gray) or polymer (plots shown in black) at the cell edges proximal (left) or distal (right) to the micropipette tip. Bar, 20 μm.
Mentions: Organization of the centrosome–MT complex in BS-C-1 cells was examined by injecting them with Cy-3 labeled tubulin and acquiring images of fluorescent MTs (Fig. 1, center). The position of the centrosome was easily traceable as the focal point of converging MTs. Immunostaining for ɛ-, γ-, and α-tubulins confirmed that such a focal point corresponded to the actual position of the centrosome and indicated that, similar to other cell types, MTs were attached to the less motile mother centriole (unpublished data), which we will refer to as the centrosome here.

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