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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-dependent movement of the nuclear membrane. PT.53 cells expressing the nuclear pore marker nup107-GFP and PT.104 cells expressing sad1-GFP were either treated for 5 min with 100 μg ml−1 TBZ (B), 25 μg ml−1 MBC (D), or not treated (A and C) and then imaged for GFP fluorescence in time-lapse using wide-field (A and B) or confocal (C and D) microscopy. (A) The nuclear envelope exhibited frequent deformations or displacements at multiple locations (arrows). (B) Cells treated with TBZ, the nuclear envelope appeared rounder, without deformities. (C) PT.104 cells exhibited a major dot (SPB, green arrow) and often one to three other minor dots (red and blue arrows). Both the SPB and minor dots were associated with nuclear envelope deformations (for example, red arrow at 6 min and green arrow at 8 min). Time-lapse sequence showed that the dot labeled by the blue arrow was distinct from the other two dots. (D) PT.104 cells treated for 5 min with MBC exhibited a rounder nucleus without deformities. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. (E) Superimposed image of multiple time-lapse images in C, which shows the sum excursions of the SPB and a minor sad-GFP dot. (F) Superimposed image of multiple time-lapse images in D. Bars, 5 μm.
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Figure 6: MT-dependent movement of the nuclear membrane. PT.53 cells expressing the nuclear pore marker nup107-GFP and PT.104 cells expressing sad1-GFP were either treated for 5 min with 100 μg ml−1 TBZ (B), 25 μg ml−1 MBC (D), or not treated (A and C) and then imaged for GFP fluorescence in time-lapse using wide-field (A and B) or confocal (C and D) microscopy. (A) The nuclear envelope exhibited frequent deformations or displacements at multiple locations (arrows). (B) Cells treated with TBZ, the nuclear envelope appeared rounder, without deformities. (C) PT.104 cells exhibited a major dot (SPB, green arrow) and often one to three other minor dots (red and blue arrows). Both the SPB and minor dots were associated with nuclear envelope deformations (for example, red arrow at 6 min and green arrow at 8 min). Time-lapse sequence showed that the dot labeled by the blue arrow was distinct from the other two dots. (D) PT.104 cells treated for 5 min with MBC exhibited a rounder nucleus without deformities. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. (E) Superimposed image of multiple time-lapse images in C, which shows the sum excursions of the SPB and a minor sad-GFP dot. (F) Superimposed image of multiple time-lapse images in D. Bars, 5 μm.

Mentions: We used GFP-nup107 to examine the dynamics of the nuclear envelope. nup107-GFP labeled the nuclear envelope in a patchy manner. Time-lapse microscopy revealed that the nuclear membrane exhibited frequent and transient deformations, giving the nucleus a nonspherical shape (Fig. 6 A and video 2 available at http://www.jcb.org/cgi/content/full/153/2/397/DC1). In cells treated for 5 min with 100 μg ml−1 TBZ, the nuclear envelope did not exhibit these deformations and maintained a spherical shape (Fig. 6 B and video 3 available at http://www.jcb.org/cgi/content/full/153/2/397/DC1). These effects were seen in all the cells examined (30 TBZ− cells and 15 TBZ+ cells).


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-dependent movement of the nuclear membrane. PT.53 cells expressing the nuclear pore marker nup107-GFP and PT.104 cells expressing sad1-GFP were either treated for 5 min with 100 μg ml−1 TBZ (B), 25 μg ml−1 MBC (D), or not treated (A and C) and then imaged for GFP fluorescence in time-lapse using wide-field (A and B) or confocal (C and D) microscopy. (A) The nuclear envelope exhibited frequent deformations or displacements at multiple locations (arrows). (B) Cells treated with TBZ, the nuclear envelope appeared rounder, without deformities. (C) PT.104 cells exhibited a major dot (SPB, green arrow) and often one to three other minor dots (red and blue arrows). Both the SPB and minor dots were associated with nuclear envelope deformations (for example, red arrow at 6 min and green arrow at 8 min). Time-lapse sequence showed that the dot labeled by the blue arrow was distinct from the other two dots. (D) PT.104 cells treated for 5 min with MBC exhibited a rounder nucleus without deformities. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. (E) Superimposed image of multiple time-lapse images in C, which shows the sum excursions of the SPB and a minor sad-GFP dot. (F) Superimposed image of multiple time-lapse images in D. Bars, 5 μm.
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Figure 6: MT-dependent movement of the nuclear membrane. PT.53 cells expressing the nuclear pore marker nup107-GFP and PT.104 cells expressing sad1-GFP were either treated for 5 min with 100 μg ml−1 TBZ (B), 25 μg ml−1 MBC (D), or not treated (A and C) and then imaged for GFP fluorescence in time-lapse using wide-field (A and B) or confocal (C and D) microscopy. (A) The nuclear envelope exhibited frequent deformations or displacements at multiple locations (arrows). (B) Cells treated with TBZ, the nuclear envelope appeared rounder, without deformities. (C) PT.104 cells exhibited a major dot (SPB, green arrow) and often one to three other minor dots (red and blue arrows). Both the SPB and minor dots were associated with nuclear envelope deformations (for example, red arrow at 6 min and green arrow at 8 min). Time-lapse sequence showed that the dot labeled by the blue arrow was distinct from the other two dots. (D) PT.104 cells treated for 5 min with MBC exhibited a rounder nucleus without deformities. Videos available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. (E) Superimposed image of multiple time-lapse images in C, which shows the sum excursions of the SPB and a minor sad-GFP dot. (F) Superimposed image of multiple time-lapse images in D. Bars, 5 μm.
Mentions: We used GFP-nup107 to examine the dynamics of the nuclear envelope. nup107-GFP labeled the nuclear envelope in a patchy manner. Time-lapse microscopy revealed that the nuclear membrane exhibited frequent and transient deformations, giving the nucleus a nonspherical shape (Fig. 6 A and video 2 available at http://www.jcb.org/cgi/content/full/153/2/397/DC1). In cells treated for 5 min with 100 μg ml−1 TBZ, the nuclear envelope did not exhibit these deformations and maintained a spherical shape (Fig. 6 B and video 3 available at http://www.jcb.org/cgi/content/full/153/2/397/DC1). These effects were seen in all the cells examined (30 TBZ− cells and 15 TBZ+ cells).

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
Related in: MedlinePlus