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Genes to Cells (2005) 10, 333-344. doi:10.1111/j.1365-2443.2005.00837.x
© 2005 Blackwell Publishing or its licensors
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The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase

Fumi Kano1, Hisao Kondo2,3, Akitsugu Yamamoto4, Arowu R. Tanaka1, Nobuko Hosokawa5, Kazuhiro Nagata5 and Masayuki Murata1,*

1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan
2 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK
3 PRESTO, Japan Science and Technology Corporation, Japan
4 Department of Cell Biology, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan
5 Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
The endoplasmic reticulum (ER) comprises tubular networks and cisternae throughout the cytoplasm, which rearrange their configuration dynamically while maintaining lumenal continuity. The ER tubules occasionally detach and fuse to form the characteristic polygonal structures with three-way junctions (Lee & Chen 1988; Waterman-Storer & Salmon 1998). Even during mitosis, the continuity of the lumenal side of the organelle is maintained, as confirmed by fluorescence loss in photobleaching (FLIP) experiments (Ellenberg et al. 1997). How the ER maintains its network structure is an important question that has yet to be answered.

Several groups have reconstituted the process of ER membrane fusion and subsequent formation of three-way junctions using microsomal membrane vesicles prepared from Xenopus laevis eggs in vitro (Allan & Vale 1991; Dreier & Rapoport 2000). Dreier and Rapoport (2000) found that this process was dependent upon N-ethylmaleimide (NEM)-sensitive proteins, but not upon microtubule integrity. Subsequently, Hetzer et al. (2001) demonstrated that a NEM-sensitive AAA ATPase, p97 (Cdc48p in yeast), and its cofactor p47 were involved in forming three-way junctions in isolated ER membranes. These in vitro reconstitution systems made it possible to study the biochemical requirements for de novo formation of the ER network from membrane vesicles. However, to our knowledge, no assay system has been developed to investigate the factors that induce structural changes in preformed ER in situ.

Previously, we used a green fluorescent protein (GFP)-tagged, Golgi-resident membrane protein, galactosyltransferase, as a probe to study Golgi morphology in intact or ‘semi-intact’ cells (Kano et al. 2000a, 2000b). By ‘semi-intact’, we mean cells whose plasma membranes have been permeabilized with the bacterial pore-forming toxin, streptolysin O (SLO). Because both organelles and the cytoskeleton remain largely intact in such cells, this system—coupled with techniques for GFP-tagged organelle visualization—readily allows observation of the morphological consequences of various experimental conditions on organelles in situ while simultaneously allowing biochemical manipulation of the system.

In the present study, we visualized the dynamic behavior of the ER network in Chinese hamster ovary (CHO) cells, which constitutively express GFP fused to heat shock protein-47 (GFP-HSP47), a molecular chaperone that is ER-resident (the cells are referred to as CHO-HSP cells). Furthermore, we investigated the biochemical requirements for maintenance and morphological change in the ER network in SLO-permeabilized CHO-HSP cells (referred to as semi-intact CHO-HSP cells). Interestingly, the presence of mitotic cytosol disrupted the ER network in semi-intact cells. This process was shown to be dependent upon the depolymerization of microtubules. In addition, incubation with interphase cytosol restored the disrupted network. We found that p47 is important for the maintenance of the ER network, and that the disruption of the network by mitotic cytosol was dependent upon cdc2 kinase-dependent phosphorylation of p47.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary material
 References
 
Dynamics of the ER network in intact cells

To investigate the morphological changes of the ER network in mammalian cells, we created a clonal CHO-derived cell line that constitutively expressed GFP-HSP47 (CHO-HSP) and which had flat morphology when grown in culture, such that the cytoplasm was easy to visualize. Using confocal microscopy, we found that the fluorescence of GFP-HSP47 in CHO-HSP cells during interphase was associated with polygonal structures with three-way junctions at the periphery of the cells and in the cisternae in the perinuclear region (Fig. 1Aa). Using indirect immunofluorescence, we observed extensive overlap of the distribution of GFP-HSP47 with that of protein disulfide isomerase (PDI), a specific ER marker protein, during each stage of the cell cycle (Fig. 1B). As has been reported previously (Lee & Chen 1988; Terasaki 2000), ER tubules were observed to rearrange their configuration dynamically, often detaching and fusing together, and exhibited characteristic polygonal structures with three-way junctions (Fig. 1Ab). Despite these dynamic configurational changes, the lumenal continuity of the ER remained largely intact in both mitotic and interphase cells. This was confirmed by FLIP experiments (Fig. 6D and unpublished data) as previously reported (Ellenberg et al. 1997; Terasaki 2000).



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  		Figure 1  Dynamic behavior of the GFP-labelled ER network in CHO-HSP cells and the localization of GFP-HSP47 throughout the cell cycle. (A) A CHO-HSP cell viewed by confocal microscopy. Two domains of the ER were observed, the perinuclear cisternae and the peripheral network structure connected by three-way junctions (a). The ER tubules frequently extended and fused with other tubules, resulting in formation of three-way junctions (b). Arrows indicate the tips of the extending tubules. The time (in seconds) is indicated in the lower right corner. Bar = 10 µm (a), 5 µm (b). (B) Co-localization of GFP-HSP47 and PDI, an ER-specific marker, throughout the cell cycle. Bar = 10 µm.

 


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  		Figure 6  Biochemical requirements for ER disruption by mitotic cytosol. (A) Nocodazole-treated semi-intact cells were incubated with mitotic cytosol with an ATP-depletion system (M-ATP), mitotic cytosol in the presence of 1 mM AMP-PNP (M+AMP-PNP), 0.2 mM GTP{gamma}S (M+GTP{gamma}S) or aluminum fluoride (30 mM NaF and 50 µM AlCl3; M+AlF), respectively, at 32 °C for 40 min. The cells were subjected to a three-way junction assay. (B) Cdc2 kinase activity correlated with the disruption of ER network. Nocodazole-treated semi-intact CHO-HSP cells were incubated with interphase cytosol (I, cdc2 kinase activity was 0.27 units/µL), mitotic cytosol (M, 5.68 units/µL), mitotic cytosol containing 30 µM butyrolactone 1 (M+BL, 1.86 units/µL), mock mitotic cytosol (mock (M), 6.55 units/µL), cdc2-depleted mitotic cytosol (cdc2 dep.1.66 units/µL), mock interphase cytosol (mock (I), 0.52 units/µL), or interphase cytosol treated with cyclin A (I+cycA, 5.18 units/µL). After the incubation, the cells were subjected to a three-way junction assay. Cdc2 kinase activity in each reaction mixture (means from two independent measurements) is shown in the right hand column, where 100% represents the value of cdc2 kinase activity in mitotic cytosol. (C) Nocodazole-treated semi-intact CHO-HSP cells were incubated with interphase (I) or mitotic (M) cytosol at 32 °C for 30 min, followed by a FLIP experiment performed as described in Experimental procedures. The boxed area was bleached by scanning with a high power laser. The number of bleaching cycles is shown in the upper right corner. Bar = 10 µm. (D) The fluorescence intensities of the semi-intact cells in the presence of interphase cytosol (semi I), or mitotic cytosol (semi M) or intact interphase cells (intact I) were plotted against numbers of photobleaching cycles, and the rate of fluorescence loss was compared.

 
Biochemical requirements for the maintenance of the ER network in semi-intact cells

In semi-intact CHO-HSP cells treated with interphase cytosol for 30 min in the presence of ATP, both the morphology and the number of three-way junctions per unit area were nearly indistinguishable from those in intact cells (see Supplementary Fig. 1 at http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC837/GTC837.htm). The number of three-way junctions per unit area was 214.5 ± 14 in intact cells and 195.7 ± 18.8 in semi-intact cells incubated with interphase cytosol and ATP. To examine factors that affect morphological change of the ER network, semi-intact CHO-HSP cells were incubated under different conditions and then subjected to a three-way junction assay as described in Experimental procedures. In the presence of AMP-PNP, a non-hydrolysable ATP homolog, the ER tubules were observed as fragmented different sized vesicle-like fluorescence (Fig. 2Ab; 2B, AMP-PNP). Incubation in the presence of GTP{gamma}S, but not aluminum fluoride, reduced the number of three-way junctions, suggesting the possible involvement of small GTP-binding proteins in the maintenance of the junctions (Fig. 2Ac: 2B, GTP{gamma}S and AlF). Dreier and Rapoport (2000) have previously reported that microtubules are not necessary for the de novo formation of the ER network. We found that the disruption of microtubules or actin filaments by nocodazole or cytochalasin B had no effect on the maintenance of three-way junctions (Fig. 2Ad and e: 2B, CCB and Noc).



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  		Figure 2  Biochemical requirements for the maintenance of the ER network in semi-intact CHO-HSP cells. (A) CHO-HSP cells were permeabilized with SLO, and incubated with HeLa cytosol containing ATP (a), 1 mM AMP-PNP (b), and 0.2 mM GTP{gamma}S (c) at 32 °C for 40 min. In d and e, the cells were pretreated with 25 µg/mL of cytochalaxin B and 4 µg/mL of nocodazole, respectively, and then permeabilized and incubated with HeLa cytosol and ATP. After incubation, the cells were viewed with a confocal microscope. Bar = 10 µm. (B) CHO-HSP cells were treated as described in (A), and were subjected to a three-way junction assay. (C) CHO-HSP cells were pretreated with (+Noc) or without (–Noc) nocodazole and then permeabilized with SLO. In the KCl wash, the permeabilized cells were washed with 2 M KCl and then incubated with NEM-treated cytosol or mock cytosol, to which NEM and DTT were simultaneously added, at 32 °C for 40 min. After the incubation, the samples were subjected to a three-way junction assay.

 
Time-lapse observations of the ER network by confocal microscopy revealed the frequent deformation/formation of three-way junctions by detached ER tubules, and the fusion of detached ER tubules to other tubules. We hypothesized that fusion promoting factors, such as the NEM-sensitive fusion protein (NSF) and/or p97, might mediate these events. Both proteins are sensitive to NEM and are involved in various processes of intracellular membrane fusion (for reviews, see Mayer 2002; Shorter & Warren 2002). To test the involvement of fusion-promoting factors on ER network integrity, we prepared and tested two types of cytosol, NEM-treated interphase cytosol (NEM-cytosol), in which ATPases such as NSF and p97 are inactivated, and cytosol treated with NEM and dithiothreitol (DTT) simultaneously as a control cytosol (NEM/DTT). Addition of either cytosol to semi-intact CHO-HSP cells had no effect on the integrity of the ER network (Fig. 2C, NEM-cyto and NEM/DTT in the column –Noc). Because membrane-bound, NEM-sensitive proteins might remain in semi-intact cells, we washed the semi-intact cells with transport buffer (TB) containing 2 M KCl. We confirmed by Western blotting that > 90% of membrane-associated NSF or p97 was released in KCl-washed, semi-intact cells (data not shown). However, NEM-treated, interphase cytosol had no effect, even in KCl-washed, semi-intact cells (Fig. 2C, KCl wash > NEM-cyto, NEM/DTT in the column –Noc). Interestingly, when the cells were pretreated with nocodazole to depolymerize the microtubles, a high-salt wash followed by incubation with NEM-cytosol caused the disruption of three-way junctions (Fig. 2C, KCl wash > NEM-cyto and NEM/DTT in the column +Noc). Without the high-salt wash, NEM-cytosol had no effect on nocodazole-treated, semi-intact cells (unpublished data). These results suggest that, when microtubule integrity was perturbed and semi-intact cells were washed with high-salt solution, NEM-sensitive factors were necessary for the maintenance of the ER network. In other words, dysfunction of NEM-sensitive fusion promoting factors in cells with perturbed microtubules may cause disruption of the ER network.

Disruption of the ER networks by mitotic cytosol

Various processes involved in membrane fusion, vesicular transport, and organelle reassembly is inhibited at the onset of mitosis (Tuomikoski et al. 1989; Thomas et al. 1992; Lowe et al. 1998). If fusion promoting factors in the mitotic cytosol were dysfunctional, the cytosol should cause the disruption of the ER network in nocodazole-treated, semi-intact cells, as we observed in the case of the NEM-treated, interphase cytosol (Fig. 2C, KCl > NEM-cyto in the column +Noc). To test this, we examined the effect of mitotic cytosol from HeLa cells on the maintenance of ER networks in semi-intact cells. Without nocodazole treatment, neither interphase nor mitotic cytosol affected ER morphology, and the number of three-way junctions was largely unchanged (Fig. 3B,I,M in the column –Noc). In contrast, in nocodazole-treated, semi-intact cells, mitotic cytosol caused disruption of the network (Fig. 3Ac and d) and the number of three-way junctions was greatly decreased, whereas the ER network was maintained in the presence of interphase cytosol (Fig. 3B,I,M in the column +Noc). In the presence of interphase cytosol, the ER network was indistinguishable from that in intact cells (Fig. 3Aa and b). We found that the morphology of the ER network disrupted by mitotic cytosol was indistinguishable from that observed in cells treated with NEM-cytosol after the high-salt wash (Fig. 3C).



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  		Figure 3  Disruption of the ER network by mitotic cytosol in semi-intact CHO-HSP cells. (A) CHO-HSP cells were pretreated with nocodazole and permeabilized with SLO. The cells were incubated with interphase (a, b) or mitotic (c, d) cytosol at 32 °C for 40 min, and observed under a confocal microscope at low (a, c) or high (b, d) magnification. Bar = 10 µm (low mag.), 5 µm (high mag.). (B) Nocodazole-treated (+Noc) or untreated (–Noc) semi-intact cells were incubated with interphase (I) or mitotic (M) cytosol. After the incubation, the cells were subjected to a three-way junction assay. (C) Morphology of the ER network in semi-intact CHO-HSP cells incubated with NEM/DTT-treated cytosol, NEM-treated cytosol after 2 M KCl washing, or mitotic cytosol. Bar = 5 µm.

 
Next, we investigated the kinetics of ER network disruption in nocodazole-treated or untreated semi-intact cells in the presence of interphase or mitotic cytosol. As shown in Fig. 4, the number of three-way junctions decreased from 100% to 20% in 60 min in the presence of mitotic cytosol in nocodazole-treated cells (Fig. 4M[+Noc]), whereas the number of three-way junctions decreased to 70% in the presence of mitotic cytosol in nocodazole-untreated cells (Fig. 4,M[–Noc]), and 80% in the presence of interphase cytosol in nocodazole-treated cells (Fig. 4,I[+Noc]). We confirmed that microtubules did not fragment in the presence of the mitotic cytosol (F.K., unpublished data). These results suggest that microtubules strengthen the integrity of the ER network, and that both depolymerization of microtubules and exposure to mitotic cytosol are necessary for the complete disruption of the ER network.



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  		Figure 4  The kinetics of ER disruption in the presence of interphase or mitotic cytosol. Nocodazole-treated (+Noc) or nocodazole-untreated (–Noc) semi-intact cells were incubated with interphase (I) or mitotic (M) cytosol at 32 °C for 0, 10, 20, 30, 40, 60 min, and then subjected to a three-way junction assay. To compare the efficiencies of disruption in each case, the fraction of disrupted three-way junctions are shown. The means and deviations are plotted in the graph.

 
An interesting question is whether mitotic cytosol has an effect on the morphology of specific ER subdomains, rough ER and smooth ER. Although HSP47-GFP appeared to diffuse freely in the ER, we did not know whether HSP47-GFP was present in rough and/or smooth ER or not. To address this question, we first tried to observe the fine structure of the disrupted ER in semi-intact cells by immunoEM using anti-GFP antibodies. However, it was technically difficult for us to distinguish precisely between rough and smooth ER in semi-intact cells by immunoEM, because the semi-intact treatment causes dissociation of membrane-bound ribosomes. Therefore, we used BODIPY TR-ceramide, a lipidic probe of the Golgi apparatus and the ER. The lipidic probe was expected to label both rough and smooth ER. Under our experimental conditions (see Experimental procedures), the ER was clearly labelled with BODIPY TR-ceramide, with faint staining of the Golgi apparatus. CHO-HSP cells were first treated with BODIPY TR-ceramide, and then permeabilized with SLO. The resulting semi-intact cells were incubated with mitotic cytosol and viewed with a confocal microscope. In the presence of mitotic cytosol, ER labelled with GFP-HSP47 was disrupted and had merged with ER labelled with BODIPY TR-ceramide (Fig. 5A). This indicated that both the rough and smooth ER appeared to be disrupted by mitotic cytosol. However, confirmation of this point requires more extensive morphological evidence using immunoelectron microscopy.



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  		Figure 5  Effect of mitotic cytosol on the morphology of smooth and rough ER. (A) Double staining of the ER network with GFP-HSP47 and BODIPY TR-ceramide in the presence of mitotic cytosol. CHO-HSP cells were incubated with BODIPY TR-ceramide, a lipid marker of the Golgi and the ER, and then permeabilized with SLO. The semi-intact CHO-HSP cells were incubated with mitotic cytosol, and viewed with confocal microscopy. Images at high magnification are shown to present the overlap of two signals. Bar = 10 µm. (B) Conventional electron microscopic observation of the ER network in semi-intact CHO-HSP cells, which were incubated with interphase (a) or mitotic (b) cytosol. Arrows indicate the ER. Bar = 1 µm.

 
A conventional EM observation revealed that ER network in semi-intact cells was clearly seen as electron-dense tubules with three-way junctions in the presence of interphase cytosol (Fig. 5Ba). In contrast, in the presence of mitotic cytosol, the severed ER network was seen and they appeared to be swollen as shown in Fig. 5Bb. It is probably because mitotic cytosol may inhibit the exit of proteins from the ER to the downstream and result in the accumulation of the secreted proteins in the ER.

We frequently observed dynamic tubulation/bifurcation of ER tubules in the presence of either mitotic or interphase cytosol, suggesting that partial disruption of the ER network by mitotic cytosol resulted from inhibition of the fusion process, rather than the process of tubulation/bifurcation (unpublished data). If fusion processes are inhibited in mitotic cytosol, then the disruption of the ER network should occur even in nocodazole-untreated cells. As shown in Fig. 4, the number of three-way junctions decreased to ~70% in response to incubation with mitotic cytosol for 60 min when microtubule integrity was not disrupted by nocodazole treatment. This result suggests that the contribution of microtubule integrity to the maintenance of the ER network might be so great that the effect of mitotic cytosol on ER disruption could be masked. Therefore, we investigated what factor(s) in mitotic cytosol caused the disruption of the ER network when microtubules were disrupted by nocodazole.

Biochemical requirements for the disruption of the ER network induced by mitotic cytosol

We found that mitotic cytosol did not cause disruption of the ER network in either the absence of ATP, or in the presence of AMP-PMP (Fig. 6A,M–ATP, M+AMP-PNP). No GTP-binding proteins were involved in the disruption process (Fig. 6A,M+GTP{gamma}S, M+AlF). We next investigated whether the requirement for ATP was the result of the involvement of a kinase in the disruption of the network. We examined the effect of cdc2 kinase, one of the master regulatory kinases for mitotic events, on this disruption. Butyrolactone I (BL), a specific inhibitor of cdc2 kinase, inhibited disruption of the ER network (Fig. 6B,M+BL). In contrast, map kinase kinase 1 (MEK1) inhibitor PD98059 did not inhibit ER disruption (unpublished data), although it inhibited mitotic Golgi disassembly in semi-intact cells (Kano et al. 2000a). The immunodepletion of cdc2 kinase from mitotic cytosol also resulted in retention of network integrity (Fig. 6Ba, cdc2 dep. and mock [M]). Cdc2 depletion was confirmed by Western blotting (Fig. 6Bb), and the loss of kinase activity in the depleted cytosol was confirmed by assay of cdc2 kinase activity (Fig. 6Ba, right column). We next tested whether an increase in cdc2 kinase activity in interphase cytosol could cause ER disruption. We incubated interphase cytosol with cyclin A for 60 min to activate the kinase (Mackay et al. 1993). Such treatment increased the kinase activity more than 10-fold (Fig. 6Ba, right column), and the resulting cytosol induced substantial disruption of the ER network (Fig. 6Ba, I+cycA and mock [I]). The mitotic cytosol used in the experiments possessed high levels of cdc2 kinase activity (about 10-fold higher than that of interphase cytosol) and led to chromosomal condensation and Golgi disassembly, but did not cause microtubule rearrangement (F. K., unpublished data). These results suggest that the disruption of the ER network is regulated by cdc2 kinase.

The continuity of the ER network following disruption by mitotic cytosol was next evaluated using FLIP. Unexpectedly, the continuity of the network was similar in cells treated with either mitotic or interphase cytosol (Fig. 6C,D, semi I and semi M). In addition, the rate of fluorescence loss in intact interphase CHO-HSP cells was similar to that in semi-intact cells incubated with either interphase or mitotic cytosol (Fig. 6D, intact I). These results suggest that mitotic cytosol did not cause the fragmentation of the ER network, and that the lumenal continuity of the disrupted ER remained intact.

Phosphorylation of p47 triggers the disruption of the ER network by mitotic cytosol

With regard to the fusion process, some cytosolic proteins or their regulators are thought to be inactive downstream of cdc2 kinase signaling in mitotic cytosol (Lowe et al. 1998; Kano et al. 2000a). Extrapolating from these findings, the disruption of the ER network by mitotic cytosol in vitro could also result from the blocking of fusion events by cdc2 kinase-mediated phosphorylation. One of the candidates for this process is p47, a cofactor of p97 that mediates membrane fusion of Golgi membranes (Kondo et al. 1997). More recently, Uchiyama et al. (2003) found that Ser140 of p47 was selectively phosphorylated by cdc2 kinase and that this phosphorylation was involved in mitotic Golgi disassembly. They also found that a non-phosphorylated form of p47, p47 (S140A), referred to as p47NP, inhibited mitotic Golgi disassembly in vitro and in vivo. p47 is also reported to play a crucial role in the formation of the ER network from microsomal membrane vesicles (Hetzer et al. 2001). From these results, we hypothesized that p47 might also be required for the maintenance of the ER network and that activation of cdc2 kinase in mitotic cytosol might cause the disruption of ER networks through phosphorylation of p47. To test this, we first investigated whether p47NP inhibited disruption of the ER network caused by mitotic cytosol. Semi-intact cells were incubated with mitotic cytosol/ATP in the presence of either p97/p47 or p97/p47NP complexes, and a three-way junction assay was performed. As shown in Fig. 7, in the presence of p47NP, but not wild-type p47, mitotic disruption of the ER network was completely inhibited (Fig. 7A and B,M+p97/p47 and M+p97/p47NP). These data suggest that cdc2 kinase-dependent phosphorylation of p47 plays a crucial role in the disruption of a ER network.



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  		Figure 7  Phosphorylation of p47 by cdc2 results in the disruption of the ER network. Nocodazole-treated semi-intact CHO-HSP cells were incubated with interphase cytosol (I), mitotic cytosol (M), mitotic cytosol +recombinant p97 and p47 (M+p97/p47), mitotic cytosol +p97 and mutated p47S140 A (M+p97/p47NP), interphase cytosol +anti-EEA1 antibodies (I+anti-EEA1 Ab), interphase cytosol +anti-p47 antibodies (I+anti-p47 Ab), interphase cytosol +anti-p97 antibodies (I+anti-p97 Ab), p97-depleted interphase cytosol +anti-EEA1 antibodies (p97 depleted I+anti-EEA1 Ab), or p97-depleted interphase cytosol +anti-p97 antibodies (p97 depleted I+anti-p97Ab) at 32 °C for 40 min. The cells were observed by confocal microscopy (A) and were subjected to a three-way junction assay (B). Bar = 10 µm.

 
We next examined whether inhibition of p47 and p97 activity in interphase cytosol could induce disruption of the ER network. Nocodazole-treated, semi-intact CHO-HSP cells were incubated with interphase cytosol in the presence of antibodies against p47, p97, or EEA1 (an endosome marker protein as a control) at 32 °C for 30 min, and a three-way junction assay was performed. Addition of anti-p47 antibody induced the disruption of the ER network (Fig. 7B,I+anti-p47 Ab), while addition of anti-EEA1 antibody had no effect (Fig. 7B,I+anti-EEA1 Ab). Unexpectedly, antibody against p97 had no effect on the maintenance of the ER network (Fig. 7B,I+anti-p97 Ab). Next, we immunodepleted p97 from interphase cytosol, and then incubated semi-intact CHO-HSP cells with p97-depleted cytosol containing anti-p97 antibodies, and viewed the cells with confocal microscopy. In the presence of p97-depleted cytosol and antibodies against EEA1, the number of three-way junctions did not change (Fig. 7B, p97 depleted I+anti-EEA1 Ab). However, in the presence of p97-depleted cytosol containing antibodies against p97, the ER network was disrupted and the number of three-way junctions substantially decreased (Fig. 7B, p97 depleted I+anti-p97 Ab). We noted that flattened membranous structures and fragmented, different-sized vesicles were frequently formed when cells were incubated with p97-depleted cytosol containing anti-p97 antibodies (unpublished data). These results clearly indicate that p97/p47 is required for the maintenance of the ER network.

Interphase cytosol induces reformation of the disrupted ER network

We next reconstituted the ER network from disrupted ER tubules. We first prepared semi-intact cells whose ER network had been disrupted by mitotic cytosol. After washing away the mitotic cytosol, the semi-intact cells were incubated with interphase or mitotic cytosol for 60 min at 32 °C, the reaction was stopped by washing with cold TB. Interphase cytosol induced reformation of the ER network (Fig. 8A,M->I) and the number of three-way junctions/area increased from about 50/area to about 170/area (Fig. 8B,M->I). In contrast, mitotic cytosol did not induce network reformation (Fig. 8A and 8B,M->M). Incubation with interphase cytosol thereafter had no effect on the morphology of the ER network (Fig. 8A and 8B,I->I). As shown in Fig. 8A(M->I), the reformed network was indistinguishable from the intact network. These data indicate that interphase cytosol can reconstruct or maintain three-way junctions and can restore ER network continuity. We also found that reformation was inhibited by 0.2 mM GTP{gamma}S or 0.2–1.0 mM Ca2+, and that microtubules were not reformed during the incubation with interphase cytosol at a level detectable by indirect immunofluorescence (data not shown). The fact that interphase cytosol restored the network structure of the ER might indicate that mitotic cytosol-induced disruption of the ER network is not artificial, but rather that it has physiological relevance.



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  		Figure 8  Reformation of the ER network from the disrupted ER tubules by interphase cytosol. Nocodazole-treated, semi-intact CHO-HSP cells were incubated with interphase (I->) or mitotic (M->) cytosol at 32 °C for 40 min. After washing out the cytosol with cold TB, the cells were further incubated with interphase (I->I, M->I), or mitotic cytosol (M->M). The cells were fixed, and images were acquired by confocal microscopy (A) or were subjected to a three-way junction assay (B). Bar = 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary material
 References
 
In many cell types, the ER network is characterized by a polygonal structure with tubules several micrometers long, connected by three-way junctions. In living cells, the ER network is structurally dynamic, characterized by continual fusion and detachment of ER tubules with one another. Several in vitro systems have been developed to study the de novo formation of the ER network and to determine the biochemical requirements of this process (Allan & Vale 1991; Dreier & Rapoport 2000). An in vitro formation assay developed by Dreier and Rapoport (2000) revealed that the characteristic polygonal structure was formed from microsomal membranes. However, the in vitro network produced in their assay appeared to be slightly different from the ER network in CHO-HSP cells. The length of one side of the three-way junctions was about 5 µm in their reconstituted network, compared to 1–1.5 µm in our CHO-HSP cells. We have frequently observed that the lengths vary with the cellular conditions. For example, following serum starvation, the length appears to be greater than 5 µm (F. K., unpublished data).

Morphological configuration and dynamics of the ER network in intact CHO-HSP cells

We have documented the dynamic behavior of the ER network and the rearrangement of three-way junctions during interphase in CHO-HSP cells (Fig. 1). ER tubules were often observed to detach and refuse with one another during interphase, so as to maintain the continuity of the network. Even during mitosis, the fluorescent ER network appeared to be continuous. This is consistent with several previous reports that suggested that network continuity is maintained throughout the cell cycle. These include studies using GFP-tagged lamin B receptor in COS cells (Ellenberg et al. 1997), GFP-tagged POM121 in NRK cells (Daigle et al. 2001), or GFP-tagged KDEL-receptor during cleavage of early sea urchin egg development (Terasaki 2000). However, we were unable to observe the precise structural features of the ER network in mitotic cells as a result of both the dynamic movements of the tubules and the round shape of mitotic cells.

Assay for examining factors that affect the configuration of the ER network in semi-intact CHO-HSP cells

We have established an ER disruption assay using SLO-mediated semi-intact CHO-HSP cells to investigate the biochemical requirements for the maintenance of the ER network. Using the assay, we found that in the presence of interphase cytosol, the hydrolysis of ATP and GTP was required for maintenance of the junctions (Fig. 2A,b,c: 2B, AMP-PNP, GTP{gamma}S, and AlF), but the integrity of the cytoskeleton (microtubules and actin filaments) had little effect (Fig. 2A,d,e: 2B, CCB and Noc). We also found that NEM-treated or mitotic cytosol caused disruption of the ER network in nocodazole-treated, semi-intact cells but did not have this effect in nocodazole-untreated, semi-intact cells (Figs 2C and 3A and 3B). This indicates that both inhibition of fusion processes and disturbance of the microtubules were required for the disruption of the ER network. FLIP experiments revealed that the continuity of the network in mitotic cytosol-treated, semi-intact cells was indistinguishable from that of interphase cytosol-treated cells (Fig. 6C,D, semi I and semi M) and intact interphase cells (Fig. 6D, intact I). These data suggest that the ER network disrupted by mitotic cytosol was not completely fragmented, but that it retained its lumenal continuity, which is consistent with previous reports in which the continuity of the ER appeared to remain intact during mitosis (Ellenberg et al. 1997).

Interestingly, our ER disassembly assay revealed that a p97/p47-mediated fusion process played a crucial role in the maintenance of the ER network when microtubules were disrupted by nocodazole (Fig. 7). When the microtubules were intact, the contribution of the fusion process to the maintenance of ER network structure appeared to be masked. As shown in Fig. 4, the fraction of three-way junctions decreased to ~70% when semi-intact CHO-HSP cells were incubated with mitotic cytosol at 32 °C for 60 min in the absence of nocodazole, whereas ER tubules were completely disrupted by mitotic cytosol in cells treated with nocodazole. Microtubules might contribute to the network structure and strengthen its integrity. Possible mechanisms for maintenance of the network structure by microtubules would be strengthening the structure by associating ER membranes to microtubules. Indeed, various reports have demonstrated an association of the ER with microtubules (Allan & Vale 1991; Dabora & Sheetz 1988) and the association might be executed by microtubule-dependent motor proteins, such as kinesin and dynein (Lane & Allan 1999) with their unknown partners. One of the data which supports this hypothesis is that pre-treatment of high-salt wash (2 M KCl wash) was required for the ER disruption by NEM-treated cytosol (Fig. 2C). Microtubule-binding domain of kinesin and dynein is positively charged and their binding to microtubules is sensitive to salt (Vallee 1986). High-salt wash may facilitate to dissociate the microtubule-dependent motor protein from microtubules. Another support is that these proteins are modified during mitosis, which may lead to dysfunction of the proteins. Dynein intermediate chain is phosphorylated by cdc2 kinase, a key kinase during mitosis (Dell et al. 2000). Despite some implications as described above, additional experiments are required to confirm this hypothesis.

It is important to note that the ER network is not uniform. It has been reported that the assembly of smooth and rough ER in vitro are differentially affected by depletion of ATP or addition of anti-p97 antibody (Roy et al. 2000). Therefore, different fusion machinery might be required for the formation of each subdomain of the ER network. However, we were unable to distinguish smooth and rough ER, even using immunoEM, because of technical difficulties. Instead, we performed an ER disruption assay using the lipidic probe BODIPY TR-ceramide as a marker of both smooth and rough ER. As shown in Fig. 5A, the ER network labelled with BODIPY-ceramide was disrupted and the signal overlapped with the GFP-HSP47 signal in the disrupted ER network. We considered that the ER network disrupted by mitotic cytosol contained both rough and smooth ER. However, more sophisticated reconstitution assays will be needed to elucidate whether specific subdomain was recognized during ER disruption/reformation process or not.

Involvement of p47-phosphorylation in the disruption of the ER network by mitotic cytosol

We focused on the role of a cofactor of the NEM-sensitive fusion protein p97, and its cofactor p47, in this process. p97 is a multifunctional protein involved in homotypic fusion of the ER membranes in yeast karyogamy (Latterich et al. 1995), transitional ER assembly (Roy et al. 2000; Kano et al. 2004), assembly of the nuclear envelope and ER network formation from isolated vesicles (Hetzer et al. 2001), extraction of polyubiquitinated proteins from the ER to cytosol (Ye et al. 2001), and spindle pole disassembly (Cao et al. 2003). Recently, Uchiyama et al. (2003) reported that p47 was phosphorylated by cdc2 kinase and that it regulated the disassembly of mitotic Golgi. In the current study, we found that inhibition of p47 by an anti-p47 antibody led to disruption of the ER (Fig. 7B,I+anti-p47 Ab). This result might be unexpected as p47 is primarily localized to the nucleus during interphase, as reported by Uchiyama et al. (2003). We believe that a small amount of p47 is sufficient to maintain the ER network. We also demonstrated that a non-phosphorylated form of p47, p47S140 A, inhibited disruption of the ER by mitotic cytosol (Fig. 7B,M+ p97/p47NP), and that the ER network was disrupted in the presence of p97-depleted interphase cytosol containing anti-p97 antibodies (Fig. 7B, p97 depleted I+anti-p97 Ab). These results strongly suggest that a component of the intracellular fusion machinery, the p97/p47 complex, is involved in the maintenance of the ER network. In contrast, disruption of the ER network induced by NEM-treated cytosol was not restored by either p97/p47 or p97/p47NP (data not shown). We therefore propose that NEM treatment leads to the disruption of the activity of other factors required for the maintenance of the network.

Morphological changes of the ER network are regulated by mitotic and interphase cytosol in semi-intact cells

The ER network that has been disrupted by incubation with mitotic cytosol can be reformed by incubation with interphase cytosol/ATP (Fig. 8). This process requires the hydrolysis of ATP and GTP, and is inhibited by Ca2+. Interestingly, microtubule integrity does not appear to be necessary for ER reformation, as was previously reported for the de novo formation of the ER network by Dreier and Rapoport (2000). Recently, Wang et al. (2004) reported that the deubiquitinating enzyme activity of VCIP135, a cofactor of p97/p47, was required for p97/p47-mediated reassembly of mitotic Golgi fragments. An interesting question is whether fusion of the disrupted ER was dependent on the same ubiquitinating system. Because we have been able to reconstitute the reformation of the ER network and have confirmed that p97/p47 is necessary for this process (F. K., unpublished data), our reconstitution system may allow us to address this problem in the future.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary material
 References
 
Materials and antibodies

Nocodazole and N-ethylmaleimide (NEM) were purchased from Sigma. Butyrolactone I (BL, Affiniti Research Products) was stored at 100 mg/mL in DMSO. BODIPY TR-ceramide was from Molecular Probes. All other chemicals were from Wako Pure Chemical Industries. The following antibodies were used: mouse anti-cdc2 antibody (Santa Cruz Biotechnology); mouse anti-VCP (p97) antibody (Abcam); mouse anti-EEA1 antibody (Transduction Laboratories); normal mouse IgG (Santa Cruz Biotechnology); rat anti-protein disulfide isomerase (PDI) antibody from Dr Ryuichi Masaki (Kansai Medical University). All other antibodies, proteins, and the HeLa cytosols were prepared as described in Uchiyama et al. (2003).

Establishment of CHO-HSP cells

Complementary DNA encoding mouse HSP47 (except for the C-terminal RDEL ER retention signal) was cloned upstream of the EGFP cDNA in the EGFP-N1 mammalian expression vector (Clontech). The HSP47 RDEL ER retention signal sequence was inserted at the end of the EGFP cDNA sequence. The recombinant plasmid was introduced into CHO-K1 cells by transfection using LipofectAMINE plus (Invitrogen). We selected positive clones in complete medium containing 300 µg/mL of Geneticin (Gibco BRL). CHO-HSP cells, constitutively expressing the GFP-tagged mouse HSP47, were cultured as described in Kano et al. (2000b). To count the number of three-way junctions, we selected one cell line, CHO-HSP, which exhibited a flat shape and a large cytoplasmic region.

Assay for investigating factors responsible for the maintenance of the ER-network (ER disruption and reformation assay)

Semi-intact CHO-HSP cells were prepared as described in Kano et al. (2000b). For the nocodazole treatment, the cells were incubated with 4 µg/mL of nocodazole at 0 °C for 20 min followed by 20 min at 37 °C to depolymerize microtubules before the SLO treatment. Destruction of microtubule networks was confirmed by an indirect immunofluorescence method using anti-{alpha}-tubulin antibody (Sigma). The semi-intact CHO-HSP cells were incubated under various conditions with an ATP-regenerating system (1 mM ATP, 8 mM creatine kinase, and 50 µg/mL of creatine phosphate), 1 mM GTP, 1 mg/mL of glucose, and HeLa mitotic or interphase cytosol (3–3.5 mg/mL protein concentration) at 32 °C for various periods of time. For reformation of the ER network, we first prepared semi-intact cells whose ER network had been disrupted by mitotic cytosol. These cells were then washed extensively with cold transport buffer (TB; 25 mM HEPES-KOH, pH 7.4, 115 mM potassium acetate, 2.5 mM MgCl2, 1 mM DTT, 2 mM EGTA) to remove the mitotic cytosol. Then, the cells were incubated with interphase cytosol and an ATP-regenerating system at 32 °C for 60 min. After the incubation, disruption or reformation reactions were stopped by the addition of TB containing ATP, and the cells were viewed with a confocal microscope (LSM510, Zeiss) or subjected to a three-way junction assay as described in Dreier and Rapoport (2000). Briefly, we counted the number of three-way junctions in randomly selected fields of 23 x 23 µm2 within the ER network of semi-intact cells. We performed three independent experiments and calculated the means and standard deviations (n = 30–50).

Immunodepletion

Cdc2 was depleted from mitotic cytosol as described in Kano et al. (2000a), except that we substituted p13suc1 conjugated to agarose (Upstate Biotechnology) for the anti-cdc2 antibody coupled to protein G-Sepharose. The kinase activity of Cdc2 was quantified with a MESACUP cdc2 kinase assay kit (Medical & Biological Laboratories Co.). For immunodepletion of p97 from interphase cytosol, we incubated interphase cytosol with antibody-coupled beads (5 µL of mouse anti-VCP [p97] antibodies or normal mouse IgG antibodies on 30 µL of protein G-Sepharose [Amersham Pharmacia Biotech]) at 4 °C for 1 h. The beads were removed by centrifugation and the supernatants were stored at –80 °C. The extent of depletion was confirmed by Western blotting using anti-cdc2 or anti-VCP antibodies.

FLIP experiment

We placed CHO-HSP cells grown on glass-based dishes (IWAKI) on the stage of a Zeiss LSM 510 confocal microscope. In FLIP experiments, the boxed area spanning the cell was repetitively photobleached (30 scans) using an argon laser (wave length = 488 nm) with 50 percent laser power. After bleaching, an image of the whole cell was acquired by scanning with low laser power (3%). The bleaching cycle was repeated at 10-second intervals. We normally performed 14 cycles of bleaching.

BODIPY TR-ceramide labeling of cells

CHO-HSP cells were incubated with 2,5 µM BODIPY TR-ceramide at 4 °C for 15 min, and then washed with serum-free Ham's F12 (Nissui). The cells were further incubated at 37 °C for 15 min and then subjected to the ER disruption assay using mitotic cytosol.

Conventional electron microscopy

CHO-HSP cells were cultured on plastic coverslips (Celldesk LF1, Sumitomo Bakelite Co. Ltd), were permeabilized with SLO, and were incubated with interphase or mitotic cytosol and ATP-regenerating system at 32 °C for 40 min. The cells were fixed in 2.5% glutalaldehyde in 0.1 M Na-phosphate buffer, pH 7.4 (PB), for 2 h. The cells were washed in the same buffer three times, and were post-fixed in 1% OsO in the same buffer for 1 h. After washing in distilled water, cells were incubated with 50% ethanol for 10 min, and block stained with 2% uranyl acetate in 70% ethanol for 2 h. The cells were further dehydrated with a graded series of ethanol, and were embedded in epoxy resin. Ultra-thin sections were doubly stained with uranyl acetate and lead citrate, and observed under a Hitachi H7600 electron microscope (Hitachi).


   Supplementary material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
References
 
The following supplementary material is available at: http://www.blackwellpublishing.com/products/journals/suppmat/gtc/gtc837/gtc837.htm

Supplementary Figure 1 The ER network in intact CHO-HSP cells and semi-intact CHO-HSP cells in the presence of interphase cytosol and ATP. CHO-HSP cells (intact) or semi-intact CHO-HSP cells incubated with interphase cytosol and ATP-regenerating system at 32 °C for 30 min (semi-intact) were viewed with confocal miscroscopy. Propidium iodide (PI) is an impermeable DNA dye, and staining of P1 indicated that the cells were permeable. Right panels are images that were obtained at high magnification.


   Acknowledgements
 
F. Kano and A.R. Tanaka are supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. H. Kondo is supported by a Wellcome Trust grant. This work was supported by a Grant from JSPS (15GS0310) (M. Murata).


   Footnotes
 
Communicated by: Yoshinori Ohsumi

* Correspondence: E-mail: mmurata{at}bio.c.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary material
 References
 
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Received: 20 September 2004
Accepted: 20 December 2004




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