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A mechanism for nuclear positioning in fission yeast based on microtubule pushing.

Tran PT, Marsh L, Doye V, Inoué S, Chang F - J. Cell Biol. (2001)

Bottom Line: The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites.After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip.Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University, New York, New York 10032, USA. pt143@columbia.edu

ABSTRACT
The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

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MT bundles give transient pushes on the nuclear membrane. Cells expressing both GFP-tubulin and nup107-GFP (PT.65) were imaged in a single optical plane in time-lapse. (A) Representative images are shown, with arrows pointing to regions of bundled MTs that appear to be attached to the nucleus. Tracings of these images are shown on the right, with the MTs (green), nuclear envelope (blue), and regions of bundled MTs (red). Note that the nuclear envelope and the bundled MT region (red) move away from the cell tip only during the period when the MT contacts the cell tip. (B and C) Plots of MT dynamics (top) and displacement of these medial MT-bundled regions (bottom) in the cell shown in A. The double arrow lines indicate the period during which each MT end maintained contact with the cell tip and which tip it contacted; arrow on the left denotes that the left MT contacted the left cell tip. Numbers show the rates of growth or shrinkage during each period in μm min−1. Note the rates and periods of MT polymerization at the tip correspond to movement of the central-bundled region and of the nuclear envelope. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
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Figure 7: MT bundles give transient pushes on the nuclear membrane. Cells expressing both GFP-tubulin and nup107-GFP (PT.65) were imaged in a single optical plane in time-lapse. (A) Representative images are shown, with arrows pointing to regions of bundled MTs that appear to be attached to the nucleus. Tracings of these images are shown on the right, with the MTs (green), nuclear envelope (blue), and regions of bundled MTs (red). Note that the nuclear envelope and the bundled MT region (red) move away from the cell tip only during the period when the MT contacts the cell tip. (B and C) Plots of MT dynamics (top) and displacement of these medial MT-bundled regions (bottom) in the cell shown in A. The double arrow lines indicate the period during which each MT end maintained contact with the cell tip and which tip it contacted; arrow on the left denotes that the left MT contacted the left cell tip. Numbers show the rates of growth or shrinkage during each period in μm min−1. Note the rates and periods of MT polymerization at the tip correspond to movement of the central-bundled region and of the nuclear envelope. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.

Mentions: To visualize how MTs may move the nuclear envelope, we next imaged cells expressing both nup107-GFP and GFP-tubulin. MTs and the nuclear envelope exhibited dynamics as observed above. Videos 6, 7, and 8 (available at http://www.jcb.org/cgi/content/full/153/2/397/DC1) show the dynamic behavior of MTs and nuclear pushing events. Fig. 7 presents the analysis of a representative cell in which two MT bundles in the focal plane were closely associated with the nuclear membrane at the central-bundled MT region (Fig. 7 A, color arrows). Positions of the MT tip, central-bundled region, and nuclear envelope were analyzed. In the top MT bundle, the central-bundled region moved to the right when the left MT, MT#1, touched the left cell tip and continued to grow for 1.5 min (Fig. 7 B). MT length increased steadily at a velocity of 1.03 μm min−1, whereas the central-bundled region and the nuclear envelope moved with a similar velocity of 0.98 μm min−1, leading to ∼1.5 μm of MT polymerization giving 1.4 μm displacement of the nuclear envelope. These movements suggested that the MT pushed the nucleus at rate of MT polymerization. Progressive buckling of the MT (MT#1 in Fig. 7 A) further demonstrated that the MT exerted a pushing force and that MT must be attached to the nucleus, which exerted forces resisting movement. After 1.5 min at the cell tip, the MT (MT#1 in Fig. 7A and Fig. B) started to shrink. The MT stopped buckling and the MT central-bundled region and nuclear envelope immediately began to move back to the right in a less directed fashion. The nuclear membrane reformed into a more spherical shape, possibly from elastic recoil in the nuclear envelope or from other forces from opposite MT bundles not present in this focal plane. Similar pushing events were found at the right half of the same MT bundle (MT#2 in Fig. 7 A) that pushed the nuclear envelope to the left.


A mechanism for nuclear positioning in fission yeast based on microtubule pushing.

Tran PT, Marsh L, Doye V, Inoué S, Chang F - J. Cell Biol. (2001)

MT bundles give transient pushes on the nuclear membrane. Cells expressing both GFP-tubulin and nup107-GFP (PT.65) were imaged in a single optical plane in time-lapse. (A) Representative images are shown, with arrows pointing to regions of bundled MTs that appear to be attached to the nucleus. Tracings of these images are shown on the right, with the MTs (green), nuclear envelope (blue), and regions of bundled MTs (red). Note that the nuclear envelope and the bundled MT region (red) move away from the cell tip only during the period when the MT contacts the cell tip. (B and C) Plots of MT dynamics (top) and displacement of these medial MT-bundled regions (bottom) in the cell shown in A. The double arrow lines indicate the period during which each MT end maintained contact with the cell tip and which tip it contacted; arrow on the left denotes that the left MT contacted the left cell tip. Numbers show the rates of growth or shrinkage during each period in μm min−1. Note the rates and periods of MT polymerization at the tip correspond to movement of the central-bundled region and of the nuclear envelope. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
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Figure 7: MT bundles give transient pushes on the nuclear membrane. Cells expressing both GFP-tubulin and nup107-GFP (PT.65) were imaged in a single optical plane in time-lapse. (A) Representative images are shown, with arrows pointing to regions of bundled MTs that appear to be attached to the nucleus. Tracings of these images are shown on the right, with the MTs (green), nuclear envelope (blue), and regions of bundled MTs (red). Note that the nuclear envelope and the bundled MT region (red) move away from the cell tip only during the period when the MT contacts the cell tip. (B and C) Plots of MT dynamics (top) and displacement of these medial MT-bundled regions (bottom) in the cell shown in A. The double arrow lines indicate the period during which each MT end maintained contact with the cell tip and which tip it contacted; arrow on the left denotes that the left MT contacted the left cell tip. Numbers show the rates of growth or shrinkage during each period in μm min−1. Note the rates and periods of MT polymerization at the tip correspond to movement of the central-bundled region and of the nuclear envelope. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
Mentions: To visualize how MTs may move the nuclear envelope, we next imaged cells expressing both nup107-GFP and GFP-tubulin. MTs and the nuclear envelope exhibited dynamics as observed above. Videos 6, 7, and 8 (available at http://www.jcb.org/cgi/content/full/153/2/397/DC1) show the dynamic behavior of MTs and nuclear pushing events. Fig. 7 presents the analysis of a representative cell in which two MT bundles in the focal plane were closely associated with the nuclear membrane at the central-bundled MT region (Fig. 7 A, color arrows). Positions of the MT tip, central-bundled region, and nuclear envelope were analyzed. In the top MT bundle, the central-bundled region moved to the right when the left MT, MT#1, touched the left cell tip and continued to grow for 1.5 min (Fig. 7 B). MT length increased steadily at a velocity of 1.03 μm min−1, whereas the central-bundled region and the nuclear envelope moved with a similar velocity of 0.98 μm min−1, leading to ∼1.5 μm of MT polymerization giving 1.4 μm displacement of the nuclear envelope. These movements suggested that the MT pushed the nucleus at rate of MT polymerization. Progressive buckling of the MT (MT#1 in Fig. 7 A) further demonstrated that the MT exerted a pushing force and that MT must be attached to the nucleus, which exerted forces resisting movement. After 1.5 min at the cell tip, the MT (MT#1 in Fig. 7A and Fig. B) started to shrink. The MT stopped buckling and the MT central-bundled region and nuclear envelope immediately began to move back to the right in a less directed fashion. The nuclear membrane reformed into a more spherical shape, possibly from elastic recoil in the nuclear envelope or from other forces from opposite MT bundles not present in this focal plane. Similar pushing events were found at the right half of the same MT bundle (MT#2 in Fig. 7 A) that pushed the nuclear envelope to the left.

Bottom Line: The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites.After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip.Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Columbia University, New York, New York 10032, USA. pt143@columbia.edu

ABSTRACT
The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

Show MeSH