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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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Fluorescent speckle microscopy analysis of an MT bundle. PT.47 cells expressing low levels of GFP-tubulin were imaged for GFP fluorescence (see Materials and Methods). (A) Time-lapse sequences of a single MT bundle with GFP-tubulin speckles. Arrows and lines mark the tips of the cell. (B) Trace of the images in A. Color arrows label regions of speckles, and the red rectangle denotes the medial-bundled MT region. (C) Plot of changes in MT lengths over time. Position zero indicates the position of the region of the medial MT-bundled region. (D) Plot of the position of the higher fluorescence intensity region over time. Position zero indicates the mean position. The double arrow lines indicate the period over which each MT end contacted with the cell tip. Numbers show the rate of MT growth or shrinkage in each period in μm min−1. The MT exhibited growth and shrinkage at the distal tips of the MT bundle with little change at the medial portion of the bundle. The whole MT lattice along with the medial MT-bundled region moved away from the cell tip when the MT touched the cell tip.
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Figure 3: Fluorescent speckle microscopy analysis of an MT bundle. PT.47 cells expressing low levels of GFP-tubulin were imaged for GFP fluorescence (see Materials and Methods). (A) Time-lapse sequences of a single MT bundle with GFP-tubulin speckles. Arrows and lines mark the tips of the cell. (B) Trace of the images in A. Color arrows label regions of speckles, and the red rectangle denotes the medial-bundled MT region. (C) Plot of changes in MT lengths over time. Position zero indicates the position of the region of the medial MT-bundled region. (D) Plot of the position of the higher fluorescence intensity region over time. Position zero indicates the mean position. The double arrow lines indicate the period over which each MT end contacted with the cell tip. Numbers show the rate of MT growth or shrinkage in each period in μm min−1. The MT exhibited growth and shrinkage at the distal tips of the MT bundle with little change at the medial portion of the bundle. The whole MT lattice along with the medial MT-bundled region moved away from the cell tip when the MT touched the cell tip.

Mentions: The interphase MTs were highly dynamic. Cells were imaged at 2–5-s intervals in single focal planes to obtain high temporal resolution; however, similar results on MT dynamics were also obtained in three-dimensional sections (our unpublished observations). Table summarizes the measured parameters of MT dynamics in the fission yeast. Growth rates of ∼2 μm min−1 and shrinkage rates of ∼9 μm min−1 were comparable to rates for MT plus ends measured in vitro with purified tubulin (Horio and Hotani 1986; Walker et al. 1988; Gildersleeve et al. 1992) or in vivo in other eukaryotes (Verde et al. 1992; Carminati and Stearns 1997; Shaw et al. 1997; Adames and Cooper 2000; Maddox et al. 2000). To test whether expression of GFP-tubulin may alter MT dynamics, we analyzed MT dynamics in cells expressing high and low levels of GFP-tubulin fluorescence. At low levels, speckles of GFP fluorescence were seen (see Fig. 3 A), suggesting that only ∼1–5% of the total MT polymer was composed of GFP-tubulin (Waterman-Storer and Salmon 1998; Waterman-Storer et al. 1998). No significant differences in MT dynamics were found at these different expression levels (Table ), suggesting that our use of GFP-tubulin at these expression levels did not perturb MT dynamics.


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)

Fluorescent speckle microscopy analysis of an MT bundle. PT.47 cells expressing low levels of GFP-tubulin were imaged for GFP fluorescence (see Materials and Methods). (A) Time-lapse sequences of a single MT bundle with GFP-tubulin speckles. Arrows and lines mark the tips of the cell. (B) Trace of the images in A. Color arrows label regions of speckles, and the red rectangle denotes the medial-bundled MT region. (C) Plot of changes in MT lengths over time. Position zero indicates the position of the region of the medial MT-bundled region. (D) Plot of the position of the higher fluorescence intensity region over time. Position zero indicates the mean position. The double arrow lines indicate the period over which each MT end contacted with the cell tip. Numbers show the rate of MT growth or shrinkage in each period in μm min−1. The MT exhibited growth and shrinkage at the distal tips of the MT bundle with little change at the medial portion of the bundle. The whole MT lattice along with the medial MT-bundled region moved away from the cell tip when the MT touched the cell tip.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169469&req=5

Figure 3: Fluorescent speckle microscopy analysis of an MT bundle. PT.47 cells expressing low levels of GFP-tubulin were imaged for GFP fluorescence (see Materials and Methods). (A) Time-lapse sequences of a single MT bundle with GFP-tubulin speckles. Arrows and lines mark the tips of the cell. (B) Trace of the images in A. Color arrows label regions of speckles, and the red rectangle denotes the medial-bundled MT region. (C) Plot of changes in MT lengths over time. Position zero indicates the position of the region of the medial MT-bundled region. (D) Plot of the position of the higher fluorescence intensity region over time. Position zero indicates the mean position. The double arrow lines indicate the period over which each MT end contacted with the cell tip. Numbers show the rate of MT growth or shrinkage in each period in μm min−1. The MT exhibited growth and shrinkage at the distal tips of the MT bundle with little change at the medial portion of the bundle. The whole MT lattice along with the medial MT-bundled region moved away from the cell tip when the MT touched the cell tip.
Mentions: The interphase MTs were highly dynamic. Cells were imaged at 2–5-s intervals in single focal planes to obtain high temporal resolution; however, similar results on MT dynamics were also obtained in three-dimensional sections (our unpublished observations). Table summarizes the measured parameters of MT dynamics in the fission yeast. Growth rates of ∼2 μm min−1 and shrinkage rates of ∼9 μm min−1 were comparable to rates for MT plus ends measured in vitro with purified tubulin (Horio and Hotani 1986; Walker et al. 1988; Gildersleeve et al. 1992) or in vivo in other eukaryotes (Verde et al. 1992; Carminati and Stearns 1997; Shaw et al. 1997; Adames and Cooper 2000; Maddox et al. 2000). To test whether expression of GFP-tubulin may alter MT dynamics, we analyzed MT dynamics in cells expressing high and low levels of GFP-tubulin fluorescence. At low levels, speckles of GFP fluorescence were seen (see Fig. 3 A), suggesting that only ∼1–5% of the total MT polymer was composed of GFP-tubulin (Waterman-Storer and Salmon 1998; Waterman-Storer et al. 1998). No significant differences in MT dynamics were found at these different expression levels (Table ), suggesting that our use of GFP-tubulin at these expression levels did not perturb MT dynamics.

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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