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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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Organization of interphase MTs in multiple bundles with a medial overlap region. Wild-type cells expressing GFP-tubulin (PT.47) were imaged for GFP fluorescence using a confocal microscope. (A and B) Optical sections through a cell with three MT bundles. Each MT bundle contained a region of higher fluorescence intensity located near the cell center. (C–E) Intensity line scans along the length of each MT bundle labeled in A and B (see Materials and Methods). (F) Plot of the number of MT bundles per cell (n = 128 cells). Video available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
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Figure 1: Organization of interphase MTs in multiple bundles with a medial overlap region. Wild-type cells expressing GFP-tubulin (PT.47) were imaged for GFP fluorescence using a confocal microscope. (A and B) Optical sections through a cell with three MT bundles. Each MT bundle contained a region of higher fluorescence intensity located near the cell center. (C–E) Intensity line scans along the length of each MT bundle labeled in A and B (see Materials and Methods). (F) Plot of the number of MT bundles per cell (n = 128 cells). Video available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.

Mentions: Intensity scans in Fig. 1 were done with the shareware NIH Image (http://rsb.info.nih.gov/nih-image/download.html). MT bundles in Fig. 1A and Fig. B, were imaged with the real-time confocal microscope, thereby minimizing out-of-focus fluorescence. Digital images had an 8-bit dynamic range or 255 grey levels (0 = black, 255 = white). Outside the cell, the background had an average intensity of 65. The MT flanking the central higher fluorescent region had an average intensity of 130. The central higher fluorescent region had an average intensity between 170 and 190. Correcting for background contribution, the medial higher fluorescence region had approximately two times greater intensity than the flanking MT regions.


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)

Organization of interphase MTs in multiple bundles with a medial overlap region. Wild-type cells expressing GFP-tubulin (PT.47) were imaged for GFP fluorescence using a confocal microscope. (A and B) Optical sections through a cell with three MT bundles. Each MT bundle contained a region of higher fluorescence intensity located near the cell center. (C–E) Intensity line scans along the length of each MT bundle labeled in A and B (see Materials and Methods). (F) Plot of the number of MT bundles per cell (n = 128 cells). Video available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Organization of interphase MTs in multiple bundles with a medial overlap region. Wild-type cells expressing GFP-tubulin (PT.47) were imaged for GFP fluorescence using a confocal microscope. (A and B) Optical sections through a cell with three MT bundles. Each MT bundle contained a region of higher fluorescence intensity located near the cell center. (C–E) Intensity line scans along the length of each MT bundle labeled in A and B (see Materials and Methods). (F) Plot of the number of MT bundles per cell (n = 128 cells). Video available at http://www.jcb.org/cgi/content/full/153/2/397/DC1. Bar, 5 μm.
Mentions: Intensity scans in Fig. 1 were done with the shareware NIH Image (http://rsb.info.nih.gov/nih-image/download.html). MT bundles in Fig. 1A and Fig. B, were imaged with the real-time confocal microscope, thereby minimizing out-of-focus fluorescence. Digital images had an 8-bit dynamic range or 255 grey levels (0 = black, 255 = white). Outside the cell, the background had an average intensity of 65. The MT flanking the central higher fluorescent region had an average intensity of 130. The central higher fluorescent region had an average intensity between 170 and 190. Correcting for background contribution, the medial higher fluorescence region had approximately two times greater intensity than the flanking MT regions.

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