An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle - PubMed (original) (raw)

An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle

D T Shima et al. J Cell Biol. 1998.

Abstract

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1-3-micron fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

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Figures

Figure 1

Transport inhibition by a dominant mutant of Sar1p (mSar1p). (a) Schematic representation of the Golgi-to-ER recycling model for Golgi disassembly, and the experimental approach using mSar1p to test this model. (b–e) Vero cells were microinjected with 2 mg/ml purified mSar1p mixed with 2 mg/ml cascade blue-conjugated BSA which served as a coinjection marker (not shown). After incubation of cells for 20 min at 37°C, they were injected a second time with plasmids encoding the plasma membrane marker CD8 or NAGFP and incubated as described in experimental procedures. (b) NAGFP-specific fluorescence; (c) staining for CD8; (d) staining for endogenous ERGIC 53. In cells injected with mSar1p (asterisks) all markers analyzed accumulated in the ER. In noninjected cells CD8 and NAGFP were transported to the plasma membrane or Golgi complex (marked by an arrow in b), respectively, and ERGIC 53 showed a typical distribution of the intermediate compartment. Arrowheads in b and c point to the nuclear envelope staining which is characteristic for ER localization. In e, mSar1p-injected (asterisk) and control cells were treated with 5 μg/ml BFA for 20 min, the BFA was washed out and cells fixed 2 h later and stained for the endogenous Golgi marker, giantin. In the presence of mSar1p, exit from the ER was inhibited for the Golgi resident giantin. Bars: (b, c, and e) 15 μm; (d) 10 μm.

Figure 1

Figure 2

Analysis of mitotic Golgi fragmentation in mSar1p injected cells. HeLa cells stably expressing NAGFP were enriched in late G2, and cells that had visibly not entered mitosis were microinjected with mSar1p and cascade blue conjugated BSA (b and d) or with cascade blue conjugated BSA only (a and c). 1 h after injection cells were fixed and mitotic cells analyzed by confocal microscopy. Projections of 10–25 z-sections (0.5-μm steps) ranging from the bottom to the top of the cells are shown. In e, cells were treated with 2 μg/ml BFA at the time of injection; in f staining for p62, an ER membrane protein is shown. Bar, 10 μm.

Figure 3

Direct visualization of Golgi membranes during mitosis. (A) Images were acquired every 5 min to see changes in Golgi morphology indicating that cells were in late G2 or the beginning of prophase. Thereafter images were acquired every minute, and cells were followed through all phases of mitosis. Images in a–d show early prophase cells before the breakdown of the nuclear envelope. In e–h the prophase to metaphase transition is shown, and i shows the same cell in telophase. Arrows in a point to Golgi fragments organized around the position of the emerging spindle poles. (B) A series of images (1 min interval) acquired at the prophase to metaphase transition are shown. 02:00 marks the beginning of the second stage of fragmentation and a period of extensive Golgi scattering. Mitotic Golgi fragments are designated by arrows. A quicktime movie comprising the images acquired will be available on the internet. Bar, 15 μm.

Figure 4

Analysis of HeLa cell mitotic Golgi fragment dynamics and subcellular localization. (A) Images were acquired every 10 s to track Golgi fragment movements in the future polar regions during the prophase to metaphase transition. Asterisks in 0 indicate Golgi fragments associated with the spindle poles (identified by phase microscopy and subsequent location of metaphase chromosomes). Dotted lines indicate the emergence of a stable, non-mobile population of Golgi (group 1), and a population that reorganizes and disperses during the period of observation (group 2). Arrowheads indicate Golgi fragments stably associated with the spindle poles during the entire period of observation. (B) Dynamics of Golgi fragments in metaphase cells were analyzed by confocal microscopy acquiring five z-sections every 20 s. Shown is a two-dimensional projection of the z-series of images. Metaphase Golgi fragments are stable (see arrowheads) and undergo only insignificant redistribution. (C) Double label analysis of the Golgi marker GM130 (green) and microtubules (red) in HeLa cells. (i) Golgi membranes associate with microtubules of the separating centrosomes (asterisks). Arrows designate membranes that appear to be distributing between the two separating centrosomes. (ii) By late prophase Golgi membranes are situated around the developing asters on either side of the nucleus. (iii) An en face view of the long axis of the metaphase spindle pole demonstrates the organization of a subset of Golgi membranes in a circular array surrounding the aster. Bar: (i and ii) 5 μm; (iii) 1 μm.

Figure 5

Appearance of Golgi membranes and microtubules during mitosis in PtK1 cells. Exponentially growing PtK1 cells were fixed and stained for the Golgi marker GM130 (green, a–g) or mitochondria (see Material and Methods; green, h) and microtubules (red). The cells shown in different images represent different phases of mitosis characterized by their organization of microtubules/ chromatin. Asterisks indicate the centrosomal/spindle pole regions. Arrowheads (b) designate Golgi fragments that were frequently detected on either side of the nuclear envelope by the earliest indications of entrance into mitosis. Arrows indicate Golgi fragments in proximity to aster microtubules (d), or concentrated around spindle poles (g). Bar: (a–g) 10 μm; (h), 5 μm.

Figure 6

Organization of mitotic Golgi membranes by microtubules in living PtK1 cells. (a) A series of images acquired during the metaphase-to-telophase progression in a PtK1 cell line stably expressing the NAGFP chimera. Images were taken at 20-s intervals. Still images highlight the concentration of mitotic Golgi (arrowheads) on either side of the metaphase plate (arrows in first panel), presumably in association with elements of the mitotic spindle. This peri-spindle distribution of mitotic Golgi was maintained while Golgi membranes were partitioned into the daughter cells. A quicktime movie comprising the images acquired will be available on the internet. (b–e) A living prometaphase cell stably expressing NAGFP (b and d) was stained with dihydroethidium (c and e) to label DNA as described in Materials and Methods, and visualized using confocal microscopy. 10 z-sections through the entire depth of the cell were taken either before or at different time-points (shown is 60 min) after the addition of nocodazole (10 μM) and cycloheximide (100 μg/ml). Two-dimensional projections of the z-sections are shown. After disruption of the mitotic spindle with nocodazole, the peri-spindle organization of Golgi clusters redistributes (d), with mitotic Golgi appearing more frequently at the cell periphery. Bar, 10 μm.

Figure 6

Figure 7

Analysis of nocodazole induced Golgi fragmentation in mSar1p injected cells. Vero cells were incubated for 30 min at 4°C to depolymerize microtubules. Cells were microinjected in the cold with 2 mg/ml purified mSar1p mixed with 2 mg/ml cascade blue conjugated BSA. Immediately after injection, cells were incubated for 1 h (a and b) or 2 h (c and d) at 37°C in the presence of nocodazole (10 μM) before they were fixed and stained for ERGIC 53 (a, c, and e) or the Golgi marker giantin (b, d, and f). In e and f, cells were first treated with nocodazole for 2 h, microinjected and subsequently incubated in the presence of nocodazole for 1 h. Microinjected cells (marked by asterisks) were identified by the coinjected BSA (not shown). Microinjection of mSar1p results in the accumulation of ERGIC 53 in the ER but does not interfere with the formation and distribution of the Golgi fragments (marked by arrowheads in b); in noninjected cells ERGIC 53 and giantin often appear to colocalize in peripheral Golgi fragments (marked by arrows). Bar, 15 μm.

Figure 8

Model for Golgi apparatus disassembly and partitioning during mitosis. (A) In most cells, the Golgi apparatus exhibits a polarised position in interphase, at one side of the nucleus and next to the centrosome. (B) At the G2/M transition, the Golgi ribbon reorganizes and adopts a more perinuclear localization. This reorganization may be coordinated by the separation of centrosomes, and the association of Golgi membranes with the two microtubules organizing arrays. (C) Fragmentation into Golgi stacks continues throughout prophase, and coincident with nuclear envelope break down, Golgi stacks rapidly fragment to yield mitotic Golgi clusters that are repositioned around the spindle poles and by astral microtubules. (D) As cell division ensues, Golgi clusters associate with microtubules, forming ring-like structures that are partitioned along with each spindle pole and one complement of sister chromatids. (E) At the end of cytokinesis, reformed Golgi stacks are positioned both close to the midbody and to the daughter cell centrosome, then slowly converge to reform the Golgi ribbon as shown in F (Moskalewski and Thyberg, 1990).

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An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle - PubMed (original) (raw)