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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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MTs are organized from multiple medial stable dots near the nucleus. (A) Cells expressing GFP-tubulin (PT.47) were treated with 25 μg ml−1 MBC for 5 min at 25°C. Interphase cells exhibited discrete dots or short fragments of GFP-tubulin staining. The bottom left cell is in cytokinesis/septation, and the GFP-tubulin labeled the postanaphase MTOC at the septum and faint GFP-tubulin dots near the two nuclei located near the cell tips. (B) The number of MT dots per interphase cell after MBC treatment (n = 96 cells). (C) Time-lapse sequences of MT repolymerization after depolymerization from cold treatment. PT.47 (GFP-tubulin) cells were incubated on ice for 1 h and then placed on a slide and imaged at room temperature (23–25°C). Time denotes time after temperature shift. MTs rapidly repolymerized from both ends of each of the medial MT fragments. Bar, 5 μm.
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Figure 4: MTs are organized from multiple medial stable dots near the nucleus. (A) Cells expressing GFP-tubulin (PT.47) were treated with 25 μg ml−1 MBC for 5 min at 25°C. Interphase cells exhibited discrete dots or short fragments of GFP-tubulin staining. The bottom left cell is in cytokinesis/septation, and the GFP-tubulin labeled the postanaphase MTOC at the septum and faint GFP-tubulin dots near the two nuclei located near the cell tips. (B) The number of MT dots per interphase cell after MBC treatment (n = 96 cells). (C) Time-lapse sequences of MT repolymerization after depolymerization from cold treatment. PT.47 (GFP-tubulin) cells were incubated on ice for 1 h and then placed on a slide and imaged at room temperature (23–25°C). Time denotes time after temperature shift. MTs rapidly repolymerized from both ends of each of the medial MT fragments. Bar, 5 μm.

Mentions: Our analysis of MT dynamics showed that they grew from and shrank to multiple sites near the nucleus. To define these regions further, cells were treated with 25 μg ml−1 MBC, an MT-depolymerizing drug, for 5 min. MBC-treated cells exhibited multiple GFP-tubulin dots or stubs near or on the nucleus (Fig. 4 A). In cells not expressing GFP-tubulin, similar tubulin spots were seen by immunofluorescence of MBC-treated or cold-treated cells (our unpublished observations) (Mata and Nurse 1997; Chen et al. 1999). Fig. 4 B shows that interphase cells contain two to four stable MT dots. The number of stable MT dots per cell was similar to but slightly less than the number of MT bundles per cell (compare Fig. 1 F with Fig. 4 B), suggesting that each MT bundle contained one medially located stable region. The difference in the number of dots in MBC-treated cells compared with the number of MT bundles may be caused by the ability of MBC to depolymerize some of the stable regions, by clustering of some of these spots upon MBC treatment, or by the possibility that some MTs do not have a stable region.


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)

MTs are organized from multiple medial stable dots near the nucleus. (A) Cells expressing GFP-tubulin (PT.47) were treated with 25 μg ml−1 MBC for 5 min at 25°C. Interphase cells exhibited discrete dots or short fragments of GFP-tubulin staining. The bottom left cell is in cytokinesis/septation, and the GFP-tubulin labeled the postanaphase MTOC at the septum and faint GFP-tubulin dots near the two nuclei located near the cell tips. (B) The number of MT dots per interphase cell after MBC treatment (n = 96 cells). (C) Time-lapse sequences of MT repolymerization after depolymerization from cold treatment. PT.47 (GFP-tubulin) cells were incubated on ice for 1 h and then placed on a slide and imaged at room temperature (23–25°C). Time denotes time after temperature shift. MTs rapidly repolymerized from both ends of each of the medial MT fragments. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 4: MTs are organized from multiple medial stable dots near the nucleus. (A) Cells expressing GFP-tubulin (PT.47) were treated with 25 μg ml−1 MBC for 5 min at 25°C. Interphase cells exhibited discrete dots or short fragments of GFP-tubulin staining. The bottom left cell is in cytokinesis/septation, and the GFP-tubulin labeled the postanaphase MTOC at the septum and faint GFP-tubulin dots near the two nuclei located near the cell tips. (B) The number of MT dots per interphase cell after MBC treatment (n = 96 cells). (C) Time-lapse sequences of MT repolymerization after depolymerization from cold treatment. PT.47 (GFP-tubulin) cells were incubated on ice for 1 h and then placed on a slide and imaged at room temperature (23–25°C). Time denotes time after temperature shift. MTs rapidly repolymerized from both ends of each of the medial MT fragments. Bar, 5 μm.
Mentions: Our analysis of MT dynamics showed that they grew from and shrank to multiple sites near the nucleus. To define these regions further, cells were treated with 25 μg ml−1 MBC, an MT-depolymerizing drug, for 5 min. MBC-treated cells exhibited multiple GFP-tubulin dots or stubs near or on the nucleus (Fig. 4 A). In cells not expressing GFP-tubulin, similar tubulin spots were seen by immunofluorescence of MBC-treated or cold-treated cells (our unpublished observations) (Mata and Nurse 1997; Chen et al. 1999). Fig. 4 B shows that interphase cells contain two to four stable MT dots. The number of stable MT dots per cell was similar to but slightly less than the number of MT bundles per cell (compare Fig. 1 F with Fig. 4 B), suggesting that each MT bundle contained one medially located stable region. The difference in the number of dots in MBC-treated cells compared with the number of MT bundles may be caused by the ability of MBC to depolymerize some of the stable regions, by clustering of some of these spots upon MBC treatment, or by the possibility that some MTs do not have a stable region.

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