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Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum.

Jokitalo E, Cabrera-Poch N, Warren G, Shima DT - J. Cell Biol. (2001)

Bottom Line: Presley, T.H.Roberts, E.Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, Electron Microscopy Unit, University of Helsinki, 00014 Helsinki, Finland.

ABSTRACT
We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

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Video microscopy of partitioning Golgi clusters during early mitosis in NAGFP-PtK cells. (A) NAGFP-PtK cells were imaged by serial confocal microscopy at intervals of 30–50 s/Z-stack, and still images from the time-lapse experiments, at the indicated times (′, minutes; ″, seconds) and/or phase of mitosis, are presented together with phase (top and bottom) images. The distribution of Golgi fluorescence on either side of the presumptive midline is shown as a ratio above the cell (0′). The NAGFP in the right hand cell is present in a juxtanuclear reticulum characteristic of the Golgi apparatus in interphase cells with little if any present in the ER. The left-hand cell has entered prophase, and the Golgi apparatus is breaking down into small fragments that become distributed around the disassembling nuclear envelope (3′). As the mitotic spindle is being assembled (6′), more clusters appear to associate with the right-hand spindle pole and this imbalance appears to be corrected by shuttling of clusters (red arrowhead) and smaller Golgi membranes (white arrow) to the left-hand pole (11′15″ to 16′). Bar, 10 μm. Animations (Videos 1–3) are available at http://www.jcb.org/cgi/content/full/200104073/DC1. (B) The bulk of Golgi membrane partitioning occurs during the prophase-to-metaphase period. Individual cells were followed during mitosis, and the amount of membrane on each side of the metaphase plate (or on either side of a line drawn half-way between the spindle poles) was measured using GFP fluorescence (see Materials and methods). The results are presented as the change in partitioning accuracy during the prophase-to-metaphase period and the metaphase-to-telophase period. The values for individual cells are shown on the left; and the mean values for all cells (± SEM, n = 7), on the right. (C) For the cells shown in Figs. 3 A and 4 A, fluorescence intensity was measured as a function of fragment size. The data are displayed graphically as the percentage of cellular fluorescence in structures ≥300 nm in major axis diameter throughout mitosis. A diameter of 300 nm was estimated to be the working limit of x–y resolution under the experimental conditions used for live cell experiments. The results from Fig. 3 A are shown on the left; and from Fig. 4 A, on the right.
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fig3: Video microscopy of partitioning Golgi clusters during early mitosis in NAGFP-PtK cells. (A) NAGFP-PtK cells were imaged by serial confocal microscopy at intervals of 30–50 s/Z-stack, and still images from the time-lapse experiments, at the indicated times (′, minutes; ″, seconds) and/or phase of mitosis, are presented together with phase (top and bottom) images. The distribution of Golgi fluorescence on either side of the presumptive midline is shown as a ratio above the cell (0′). The NAGFP in the right hand cell is present in a juxtanuclear reticulum characteristic of the Golgi apparatus in interphase cells with little if any present in the ER. The left-hand cell has entered prophase, and the Golgi apparatus is breaking down into small fragments that become distributed around the disassembling nuclear envelope (3′). As the mitotic spindle is being assembled (6′), more clusters appear to associate with the right-hand spindle pole and this imbalance appears to be corrected by shuttling of clusters (red arrowhead) and smaller Golgi membranes (white arrow) to the left-hand pole (11′15″ to 16′). Bar, 10 μm. Animations (Videos 1–3) are available at http://www.jcb.org/cgi/content/full/200104073/DC1. (B) The bulk of Golgi membrane partitioning occurs during the prophase-to-metaphase period. Individual cells were followed during mitosis, and the amount of membrane on each side of the metaphase plate (or on either side of a line drawn half-way between the spindle poles) was measured using GFP fluorescence (see Materials and methods). The results are presented as the change in partitioning accuracy during the prophase-to-metaphase period and the metaphase-to-telophase period. The values for individual cells are shown on the left; and the mean values for all cells (± SEM, n = 7), on the right. (C) For the cells shown in Figs. 3 A and 4 A, fluorescence intensity was measured as a function of fragment size. The data are displayed graphically as the percentage of cellular fluorescence in structures ≥300 nm in major axis diameter throughout mitosis. A diameter of 300 nm was estimated to be the working limit of x–y resolution under the experimental conditions used for live cell experiments. The results from Fig. 3 A are shown on the left; and from Fig. 4 A, on the right.

Mentions: The fragmentation of the Golgi apparatus during the early phases of mitosis is shown in Fig. 3 and the accompanying animation in supplemental Videos 1–3 (available at http://www.jcb.org/cgi/content/full/200104073/DC1). Fig. 3 A shows an interphase cell on the right with a characteristic Golgi ribbon and a cell on the left that has just entered prophase. The Golgi apparatus undergoes rapid and extensive fragmentation, the Golgi membranes associating with the forming spindle poles. By metaphase, two sets of Golgi clusters could be readily visualized, one at each pole. An example of the Golgi membrane distribution is seen at the bottom of Fig. 3 A where a single phase image of the metaphase cell has been superimposed on the fluorescence image.


Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum.

Jokitalo E, Cabrera-Poch N, Warren G, Shima DT - J. Cell Biol. (2001)

Video microscopy of partitioning Golgi clusters during early mitosis in NAGFP-PtK cells. (A) NAGFP-PtK cells were imaged by serial confocal microscopy at intervals of 30–50 s/Z-stack, and still images from the time-lapse experiments, at the indicated times (′, minutes; ″, seconds) and/or phase of mitosis, are presented together with phase (top and bottom) images. The distribution of Golgi fluorescence on either side of the presumptive midline is shown as a ratio above the cell (0′). The NAGFP in the right hand cell is present in a juxtanuclear reticulum characteristic of the Golgi apparatus in interphase cells with little if any present in the ER. The left-hand cell has entered prophase, and the Golgi apparatus is breaking down into small fragments that become distributed around the disassembling nuclear envelope (3′). As the mitotic spindle is being assembled (6′), more clusters appear to associate with the right-hand spindle pole and this imbalance appears to be corrected by shuttling of clusters (red arrowhead) and smaller Golgi membranes (white arrow) to the left-hand pole (11′15″ to 16′). Bar, 10 μm. Animations (Videos 1–3) are available at http://www.jcb.org/cgi/content/full/200104073/DC1. (B) The bulk of Golgi membrane partitioning occurs during the prophase-to-metaphase period. Individual cells were followed during mitosis, and the amount of membrane on each side of the metaphase plate (or on either side of a line drawn half-way between the spindle poles) was measured using GFP fluorescence (see Materials and methods). The results are presented as the change in partitioning accuracy during the prophase-to-metaphase period and the metaphase-to-telophase period. The values for individual cells are shown on the left; and the mean values for all cells (± SEM, n = 7), on the right. (C) For the cells shown in Figs. 3 A and 4 A, fluorescence intensity was measured as a function of fragment size. The data are displayed graphically as the percentage of cellular fluorescence in structures ≥300 nm in major axis diameter throughout mitosis. A diameter of 300 nm was estimated to be the working limit of x–y resolution under the experimental conditions used for live cell experiments. The results from Fig. 3 A are shown on the left; and from Fig. 4 A, on the right.
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Related In: Results  -  Collection

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fig3: Video microscopy of partitioning Golgi clusters during early mitosis in NAGFP-PtK cells. (A) NAGFP-PtK cells were imaged by serial confocal microscopy at intervals of 30–50 s/Z-stack, and still images from the time-lapse experiments, at the indicated times (′, minutes; ″, seconds) and/or phase of mitosis, are presented together with phase (top and bottom) images. The distribution of Golgi fluorescence on either side of the presumptive midline is shown as a ratio above the cell (0′). The NAGFP in the right hand cell is present in a juxtanuclear reticulum characteristic of the Golgi apparatus in interphase cells with little if any present in the ER. The left-hand cell has entered prophase, and the Golgi apparatus is breaking down into small fragments that become distributed around the disassembling nuclear envelope (3′). As the mitotic spindle is being assembled (6′), more clusters appear to associate with the right-hand spindle pole and this imbalance appears to be corrected by shuttling of clusters (red arrowhead) and smaller Golgi membranes (white arrow) to the left-hand pole (11′15″ to 16′). Bar, 10 μm. Animations (Videos 1–3) are available at http://www.jcb.org/cgi/content/full/200104073/DC1. (B) The bulk of Golgi membrane partitioning occurs during the prophase-to-metaphase period. Individual cells were followed during mitosis, and the amount of membrane on each side of the metaphase plate (or on either side of a line drawn half-way between the spindle poles) was measured using GFP fluorescence (see Materials and methods). The results are presented as the change in partitioning accuracy during the prophase-to-metaphase period and the metaphase-to-telophase period. The values for individual cells are shown on the left; and the mean values for all cells (± SEM, n = 7), on the right. (C) For the cells shown in Figs. 3 A and 4 A, fluorescence intensity was measured as a function of fragment size. The data are displayed graphically as the percentage of cellular fluorescence in structures ≥300 nm in major axis diameter throughout mitosis. A diameter of 300 nm was estimated to be the working limit of x–y resolution under the experimental conditions used for live cell experiments. The results from Fig. 3 A are shown on the left; and from Fig. 4 A, on the right.
Mentions: The fragmentation of the Golgi apparatus during the early phases of mitosis is shown in Fig. 3 and the accompanying animation in supplemental Videos 1–3 (available at http://www.jcb.org/cgi/content/full/200104073/DC1). Fig. 3 A shows an interphase cell on the right with a characteristic Golgi ribbon and a cell on the left that has just entered prophase. The Golgi apparatus undergoes rapid and extensive fragmentation, the Golgi membranes associating with the forming spindle poles. By metaphase, two sets of Golgi clusters could be readily visualized, one at each pole. An example of the Golgi membrane distribution is seen at the bottom of Fig. 3 A where a single phase image of the metaphase cell has been superimposed on the fluorescence image.

Bottom Line: Presley, T.H.Roberts, E.Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, Electron Microscopy Unit, University of Helsinki, 00014 Helsinki, Finland.

ABSTRACT
We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

Show MeSH
Related in: MedlinePlus