Correspondence to: Tom A. Rapoport, Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115-6091. Tel:(617) 432-0637 Fax:(617) 432-1190 E-mail:tom_rapoport{at}hms.harvard.edu.
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Abstract |
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We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca2+ concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.
Key Words: membrane tubules, endoplasmic reticulum, membrane fusion, microtubules, Xenopus
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Introduction |
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Cellular organelles have characteristic shapes. Some organelles, such as lysosomes, vacuoles, endosomes, peroxisomes, synaptic vesicles, and various transport vesicles resemble spheres, likely the thermodynamically most stable form of a lipid vesicle. Other organelles have more complex shapes in which two membranes are closely apposed to one another. These include the nuclear envelope, the ER, the Golgi cisternae, and the mitochondrial cristae. How these structures are generated and maintained is largely unknown.
The ER forms an elaborate tubular and cisternal network that is continuous with the outer nuclear membrane. This network is dynamic; new membrane tubules are continuously formed, fuse with other tubules to form three-way junctions, and move relative to one another (
In vitro experiments suggest a direct role of microtubules in the formation of an ER network. In these experiments, membranes and cytosol from chicken embryo fibroblasts or Xenopus laevis eggs were placed in a flow chamber, and video-enhanced differential interference contrast (DIC) microscopy was used to follow the movement of membrane tubules on a glass surface (
The formation of membrane tubules or tubular networks has also been observed with Golgi membranes in vivo and in vitro. As with the ER, Golgi membrane tubules often coalign with microtubules, and formation in vitro depends on prepolymerized microtubules as well as motor proteins (
An excellent system to study the de novo formation of an ER network is egg extracts from the frog Xenopus laevis (
Using Xenopus egg extracts, we have established an in vitro system for the formation of an ER network. Surprisingly, we find that microtubules or an actin scaffold are not required for the process. Rather, other cytosolic factors serve to incorporate a basic fusion reaction, which itself generates only large round vesicles, into a controlled reaction that results in a tubular network.
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Materials and Methods |
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Preparation of Xenopus Egg Extract, Membranes, and Cytosol
Xenopus egg extract was prepared as described with minor modifications (
To prepare membrane and cytosol fractions, the egg extract was centrifuged in an SW40 rotor (Beckman) for 1 h at 40,000 rpm and 2°C, resulting in sedimentation of a heavy membrane fraction. The supernatant was centrifuged in a 100.4 rotor (Beckman) for 1.5 h at 100,000 rpm and 2°C. The resulting supernatant contained the cytosol and was almost completely free of membranes. The pellet consisted of a clear layer with light membranes on top. The latter fraction was resuspended with very little buffer A, frozen in liquid nitrogen, and stored at -80°C. For some experiments, the light membranes were washed twice with 15 vol of buffer A to remove residual cytosol, and resuspended in buffer A. The absorption of the light membrane suspension in 1% SDS was between 20 and 40 OD (280 nm).
Mitotic egg extract was prepared from interphase extract by the addition of an energy regenerating system and 0.1 mg/ml glutathione S-transferase (GST)cyclin B1 90, that has GST fused to cyclin B1 with a deletion of the destruction box, followed by an incubation for 30 min at room temperature (
90 was kindly provided by P. Stein (Harvard Medical School, Boston, MA). The histone H1 kinase activity of the extract was determined as described (
Preparation of Various Membranes and Cytosols
Dog pancreas rough ER microsomes, yeast membranes and cytosol, and wheat germ cytosol were prepared as described by
Formation of ER Networks In Vitro
To form membrane networks, 10 µl of cytosol, 0.51 µl of the light membranes, and 0.5 µl of an energy regenerating system (1 mM ATP, 0.5 mM GTP, 20 mM creatine phosphate, 0.1 mg/ml creatine kinase) were mixed and incubated at room temperature for the indicated times (1090 min). Afterwards, membranes were stained by pipetting 1 µl of the reaction mixture into a 2-µl drop of 0.1% (vol/vol) octadecyl rhodamine (Molecular Probes) in buffer A, and observed by fluorescence microscopy using an Axioplan II microscope (Zeiss) equipped with an Orca 12-bit cooled CCD camera (Hamamatsu Photonics). In the basic fusion reaction, cytosol was replaced with buffer A.
In experiments in which the membrane network was allowed to settle onto a glass surface, the reaction mixture was transferred to one well of a 10-well slide (ICN Pharmaceuticals) that was precoated for 510 min with 20 µl 5% BSA in buffer A. After incubation for 1 h at room temperature in a humidified chamber, bound membranes were carefully washed with buffer A, stained with 0.1% (vol/vol) octadecyl rhodamine in buffer A, and washed again with buffer A. A Delta Vision microscope system (Applied Precision Instruments) with a Zeiss Axiovert microscope and a PXL CCD camera (Photometrics Ltd.) was used to take images.
Simple flow chambers with a volume of ~10 µl were built with a slide, an 18-mm2 No. 1.5 coverslip, and two strips of double stick Scotch tape as a spacer (
Quantitation of network formation was done by counting and averaging the number of three-way junctions in 10 randomly selected fields with a size of 65 x 65 µm2 or 31 x 31 µm2 for networks on 10-well slides or in flow chambers, respectively (
To confirm that the various inhibitors of microtubule polymerization prevent the formation of microtubules in our system, we visualized microtubules with Oregon greenlabeled taxol (10 µM; a gift of Tim Mitchison, Harvard Medical School) in reactions that were preincubated with or without the inhibitors for 15 min on ice.
Colchicine, nocodazole, ionomycin, A23187, thapsigargin, cytochalasin D, and latrunculin A were dissolved in DMSO and added to the reaction mixture so that the final DMSO concentration was 1% (vol/vol) or less. DMSO at this concentration does not affect the in vitro reactions. Vinblastine was dissolved in water. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)1 and derivatives were purchased from Molecular Probes.
To deplete ATP, hexokinase (0.2 U/µl) and glucose (50 mM) were added to the reaction mixture and incubated on ice for 5 min. GTPS (Boehringer Mannheim) was added to the reaction mixture at a concentration of 1 mM. Treatment of cytosol or membranes with N-ethylmaleimide (NEM; 10 mM) was done for 15 min on ice, and unreacted NEM was quenched by the addition of 20 mM DTT. NEM-treated cytosol was used directly and NEM-treated membranes were pelleted and resuspended in buffer A. ATP
S (1 mM) was added to a basic fusion reaction with washed light membranes and buffer A, or to an ER formation reaction with washed light membranes and dialyzed cytosol. In both cases, an energy regenerating system with only 0.1 mM ATP was added, and the reaction mixture was incubated for 30 min on ice before transfer to room temperature. This procedure enhanced the inhibitory effect of ATP
S on ER formation. Proteinase K (2 mg/ml) and CaCl2 (2 mM) were included in the staining solution and thereby added to preformed networks. Proteinase K was gel-filtered into buffer A before use.
Depletion of Tubulin
Tubulin was removed from the cytosol in two different ways. Microtubules with a length of 2 µm were prepared by incubating 5 mg/ml bovine brain tubulin, 1 mM GTP, 30% glycerol in BRB80 for 10 min at 35°C, and then stabilized by the addition of 20 µM taxol and stored at room temperature. 1 µl of the microtubules was added to 100 µl cytosol together with 1 mM GTP/Mg2+, 5 µM taxol, and an energy regenerating system and incubated for 5 min at 25°C. The concentration of taxol was increased to 20 µM, followed by further incubation for 25 min at 25°C. The microtubules were then sedimented for 20 min at 75,000 rpm and 22°C in a 100.3 rotor (Beckman).
The second method to deplete tubulin used vinblastine, which forms aggregates with tubulin that can be separated from the cytosol by centrifugation. 50 µl cytosol was incubated with 100 µM vinblastine for 30 min on ice and then centrifuged as above. Depletion of tubulin from the supernatant was analyzed by quantitative immunoblotting using the DM1A antibody against -tubulin (Sigma Chemical Co.).
Immunofluorescence Microscopy
For immunofluorescence microscopy, membrane networks were allowed to settle on the glass surface of a coverslip (see above), washed, and fixed with 1% glutaraldehyde in buffer A without ß-mercaptoethanol for 15 min. An antibody against translocon-associated protein (TRAP
) (
Fluorescence Microscopy
BHK cells that stably express a fusion of the ER protein Sec61ß to the green fluorescent protein were used to analyze the ER in cells. This cell line was generated as described and kindly provided by M. Rolls (Harvard Medical School) and P. Stein (
Electron Microscopy (EM)
ER formation reactions were carried out in 200-µl reactions, the membrane structures were fixed for 15 min in 2 ml 1% glutaraldehyde in buffer A without sucrose and ß-mercaptoethanol, and sedimented for 5 min at 10,000 rpm. The pellet was washed four times with 100 mM Hepes/KOH, pH 7.7, postfixed with 1% OsO4, and prepared for thin sectioning. Alternatively, ER formation reactions were carried out in a humidified chamber on EM grids. The grids were then briefly dipped into buffer A without sucrose and ß-mercaptoethanol, fixed for 15 min in 1% glutaraldehyde in buffer A without sucrose and ß-mercaptoethanol, washed with 200 mM Hepes/KOH, pH 7.7, and negatively stained with uranyl acetate.
Formation of Nuclei
Sperm chromatin was prepared as described by
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Results |
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In Vitro Formation of a Membrane Network
To establish an in vitro ER formation system, we fractionated an interphase Xenopus egg extract into cytosol and membrane fractions. The eggs were crushed in a centrifuge at low speed, and the extract was further centrifuged to sediment a heavy membrane fraction. The supernatant was centrifuged again to pellet a light membrane fraction and to obtain cytosol that was almost completely free of residual membranes. When the light membranes were placed on a coverslip and stained with the hydrophobic fluorescent dye octadecyl rhodamine, a relatively homogeneous bright area was seen in the fluorescence microscope (Fig 1 A). When they were viewed in the electron microscope after thin sectioning, they appeared as vesicles with a diameter of 100200 nm (Fig 2 B).
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We first developed an assay in which the behavior of membranes could be followed in bulk solution. The light membrane fraction was mixed with cytosol and incubated at room temperature in the presence of ATP, GTP, and an ATP-regenerating system. Then, octadecyl rhodamine was added and the stained membranes were viewed with a fluorescence microscope. An elaborate network of interconnected membrane tubules was seen (Fig 1 B). The network could also be stained with another hydrophobic fluorescent dye (3,3'-dihexyloxacarbocyanine iodide) and was sensitive to detergents. It was not seen if the membranes were omitted, if energy was depleted, or if the reaction mixture was kept on ice (data not shown). The network was very delicate; it was disrupted by repeated pipetting up and down or by centrifugation (data not shown).
When the light membrane fraction was extensively washed with buffer and incubated in the absence of cytosol, the membranes fused to form large vesicles rather than a tubular network (Fig 1 C). This process was also temperature- and energy-dependent (data not shown), and will be referred to as the basic fusion reaction. Washing of the membranes with buffers containing high salt concentrations did not abolish their basic fusion activity (data not shown), indicating that the fusion machinery is tightly bound to the membranes. Small amounts of cytosol, e.g., the residual cytosol after washing the light membranes only once with buffer, were sufficient to obtain at least some network formation. Cytosol had to be present during the fusion reaction for networks to be formed; it was without effect if added to the large vesicles that had been formed in the basic fusion reaction (data not shown).
To better visualize the different membrane structures, we allowed the membranes in the reaction mixture to settle onto the glass surface of a 10-well microscope slide. After washing away unbound membranes, the attached structures were stained with octadecyl rhodamine and visualized with a fluorescence microscope. The network that formed when membranes and cytosol were present was seen as a polygonal structure with tubules several micrometers long connected by three-way junctions (Fig 1 E). A very similar network was seen with an unfractionated egg extract after settling onto a glass surface, even though in bulk solution it was difficult to visualize, possibly because of the presence of other stained membranes that did not form networks (data not shown). Only small membrane structures, likely small vesicles, were seen when the membranes were allowed to settle onto a glass surface without having been incubated at elevated temperature (Fig 1 D). When membranes were incubated in the absence of cytosol and allowed to settle onto a glass surface, large spherical vesicles with a diameter of up to a few micrometers were seen (Fig 1 F), in agreement with the results obtained in bulk solution (Fig 1 C).
The network was also examined by EM. When allowed to settle onto an electron microscope grid and negatively stained, the membrane tubules had a diameter of ~100 nm (Fig 2 A). The distance between the two membrane surfaces is about the same as observed in previous in vitro experiments and in ER membranes of mammalian cells (
The Network Contains ER Membranes
To further test whether the network is formed from ER membranes, we determined if it contained ER proteins. Immunofluorescence indicated that the network contains the rough ER protein TRAP (Fig 3 A) (
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Formation of the Network Does Not Require Microtubules or Actin Filaments
Next, we tested whether cytoskeletal elements are required for ER network formation. Various inhibitors of microtubule polymerization were added to our in vitro reaction containing membranes and cytosol. After incubation on ice for 15 min to promote microtubule depolymerization, the mixture was further incubated at room temperature. Nocodazole (up to 300 µM), colchicine (up to 200 µM), or vinblastine (up to 200 µM) did not inhibit formation of the network in bulk solution or on a glass surface (Fig 4A and Fig B; data not shown). Identical results were obtained when unfractionated egg extract was used (Fig 5 A). Quantitation of network formation by counting the number of three-way junctions in a given area confirmed that the drugs have no inhibitory effect (Fig 5 A). Experiments with colchicine demonstrated that the time course of network formation did not change either (Fig 5 B). The highest concentrations of microtubule inhibitors in our assay were much higher than commonly used to prevent the formation of microtubules. Indeed, upon addition of colchicine, no microtubules could be seen when labeling was performed with fluorescently labeled taxol (Fig 5 D; control shown in Fig 5 C). Similar results were obtained with the other drugs (data not shown).
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To provide further evidence that microtubules are not required for ER network formation, tubulin was depleted from the cytosol. Although the membranes contain a small amount of tubulin that is not removed by salt washing, the cytosol contributes >95% of the total tubulin in the reaction mixture (data not shown). In one set of experiments, we added short microtubules as seeds for microtubule polymerization and taxol to stabilize the microtubules, and sedimented the polymerized microtubules. Quantitative immunoblotting with an antibody to -tubulin showed that 9095% of the protein was removed from the cytosol (data not shown). When the depleted cytosol was mixed with washed membranes, the network was formed as efficiently as in controls (Fig 4 C). Even when vinblastine was added to this reaction to prevent polymerization of the residual tubulin, there was no sign of a reduction in network formation (Fig 4 D). Similar results were obtained when tubulin was removed from the cytosol by the addition of vinblastine; this drug leads to the formation of large complexes with tubulin that can be removed by centrifugation (
Actin filaments also did not seem to be required for the formation of the ER network. Neither latrunculin A nor cytochalasin D had any detectable effect, even when used at very high concentrations (Fig 4 E and Fig 5 A). Quantitation of the number of three-way junctions demonstrated that neither the extent nor the kinetics of network formation were altered (Fig 5A and Fig B).
Previously, it had been suggested that microtubules are required for the tubular ER network formation. To reconcile this discrepancy, we performed experiments in a similar way as described in previous studies. A simple flow chamber consisting of a closely spaced microscope slide and cover glass was precoated with taxol-stabilized microtubules, and an unfractionated egg extract was flowed into the chamber. As observed by others before, when microtubules were present, a dense membrane network was seen attached to the glass surface, in contrast to the situation without microtubules (Fig 6 A). When the nonattached membranes were recovered from the flow chamber, no difference in the extent of network formation was detected with and without microtubules (data not shown). Interestingly, when the isolated light membrane and cytosol fractions were mixed and analyzed in an analogous manner, microtubules had only little effect on the extent of the network attached to the glass surface (Fig 6 B). These results indicate that microtubules do not affect network formation per se, but may rather stimulate the attachment of membranes from unfractionated extracts onto a glass surface.
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Taken together, these results lead to the surprising conclusion that an elaborate network of ER membranes can form without a cytoskeletal scaffold.
Protein Factors Are Required for Generation and Maintenance of the Network
To begin to analyze the mechanism responsible for network formation, we first asked whether the membrane and cytosol fractions could be replaced with material from other sources. Among the membranes tested, only the light membrane fraction from Xenopus eggs gave networks (Table 1). These data indicate that the Xenopus egg membranes are specifically primed for network formation, consistent with their behavior in vivo during early development. In contrast, the cytosol from Xenopus eggs could be replaced with that from bovine liver, bovine pancreas, rat liver, or rabbit reticulocyte lysate; wheat germ extract and yeast cytosol were inactive (Table 1). Thus, the cytosolic factor(s) appear to be common.
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At least one of the cytosolic factors required for network formation is a protein. When the cytosol was dialyzed or gel-filtered, activity was found in the high molecular weight fraction. In addition, heat treatment of this material (5 min at 95°C) led to a drastic reduction of the activity (data not shown).
Proteins are not only required for the formation of the network, but also for its maintenance. When proteinase K was added to preformed ER networks, the diameter of the tubules dramatically increased up to a few micrometers (Fig 7A vs. B), indicating that protease-sensitive components are required to maintain the narrow diameter of the membrane tubules.
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Network Formation Requires Energy and Is Sensitive to Fusion Inhibitors
In other systems, steps preceding the actual membrane fusion reaction can be inhibited by the sulfhydryl-modifying reagent NEM and the poorly hydrolyzable GTP analogue GTPS (
S inhibited both the basic fusion reaction (data not shown) and network formation (Fig 8 B). Treatment of the cytosol with NEM had no effect on network formation (data not shown). These data confirm that the machinery leading to fusion is located on the membranes and suggest that some steps in the reactions with and without cytosol are similar.
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Energy is required for both the basic fusion reaction in the absence of cytosol and for network formation. Addition of an energy regenerating system with ATP and GTP gave optimal results for the network formation, whereas the basic fusion reaction was almost as efficient with 1 mM GTP alone as with a complete energy regenerating system (data not shown). Although we cannot exclude that ATP is also required because some GTP could have been converted into ATP, the poorly hydrolyzable analogue ATPS did not inhibit the basic fusion reaction (Fig 8C and Fig D). In contrast, network formation in the presence of cytosol required ATP and was inhibited by ATP
S (Fig 8E and Fig F). Interestingly, ATP
S blocked network formation at a stage distinct from that seen in the presence of GTP
S. While in the presence of GTP
S, the membranes appeared as a homogeneously stained area, essentially like at the beginning of the reaction, with ATP
S the vesicles appeared to cluster (Fig 8A vs. E). Other ATP analogues (AMP-PNP and AMP-PCP) had similar, but weaker effects (data not shown). Taken together, these results suggest that network formation includes an ATP-dependent step that is not required for the basic fusion reaction.
An Intermediate Stage in Network Formation
The apparent clustering of vesicles in the presence of ATPS raised the possibility that this may represent an intermediate stage in network formation, similar to the intermediates of docked or tethered membrane vesicles described for other fusion reactions (
S-sensitive reaction, and that further progression towards a network requires an ATP
S-inhibitable step.
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The Network Is Sensitive to High Cytosolic Ca2+ Concentrations
The ER network in mammalian cells has been reported to disassemble in the presence of high cytosolic Ca2+ concentrations (
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High cytosolic Ca2+ concentrations also inhibited the de novo formation of the network. At 200 µM Ca2+, only a few tubules were seen and instead membrane clusters formed. After longer incubations (1.5 h), larger vesicles were also seen (Fig 11A and Fig B). Similar effects were observed when Ca2+ was released from the interior of the vesicles by the addition of ionomycin or A23187 (Fig 11 C; data not shown). The inhibitory effect of ionomycin could be reversed by the addition of the Ca2+ chelator EGTA (Fig 11 D), indicating that ionomycin was indeed acting by increasing the cytosolic Ca2+ concentration. Similarly, inhibition of the Ca2+ pumps in the membrane vesicles by thapsigargin (25 µM), which is also expected to increase the cytosolic Ca2+ concentration, inhibited network formation and caused the appearance of membrane clusters (data not shown). It should be noted that neither the addition of high Ca2+ concentrations nor of Ca2+ ionophores or Ca2+ pump inhibitors prevented the formation of large vesicles in the absence of cytosol (data not shown). Thus, ER network formation, but not the basic fusion reaction, requires a Ca2+-sensitive step.
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Similarities between Network and Nuclear Envelope Formation
Both the unfractionated Xenopus egg extract and a mixture of membranes and cytosol prepared from it are capable of forming nuclei when chromatin or DNA are present (
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When the formation of the nuclear envelope was followed over time, at early timepoints a network of tubular structures was seen on the chromatin surface, similar to the tubules in ER networks, as well as small flat patches (Fig 12A and Fig B), which have been described before (
Other data also indicate similarities between ER network and nuclear envelope formation. In agreement with results in the literature (S or treatment of nuclear membranes with NEM inhibited the fusion of chromatin-bound vesicles, whereas treatment of the cytosol with NEM did not (data not shown). As in the ER network formation system, high cytosolic Ca2+ concentrations are inhibitory, although in this case, the effect may be due to changes of the chromatin (
Further similarities are borne out by experiments with Ca2+ chelators. Nuclear envelope formation was maximally inhibited by chelators with a binding constant of ~1 µM, and less by chelators with higher or lower binding constants (
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Finally, both ER network and nuclear envelope formation only occur in interphase extracts. Mitotic egg extracts were generated by the addition of a cyclin B1 mutant protein that is made nondegradable by deletion of its destruction box (cyclin B1 90); this leads to constitutive activation of the mitotic CDC2 kinase (
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Discussion |
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We have established a system from Xenopus eggs in which the reticular network of the ER is formed in vitro. Our data lead to the surprising conclusion that an ER network can form independently of microtubules or actin filaments. Previously, a direct role for microtubules in the formation of the ER network was proposed based on studies of the interaction between the ER and microtubules in cells and in vitro (
Although not essential for the de novo formation, the cytoskeleton appears to play an important role in distributing the ER network throughout the cell (
If the ER membrane network is not formed along a cytoskeletal scaffold, how is it generated? An important hint to the answer is that, in the absence of cytosol, the membranes can fuse, but form large spherical vesicles rather than networks. Furthermore, cytosol has to be present during the fusion reaction to mediate tubular network formation. When cytosol is added to the large vesicles formed in a basic fusion reaction, networks are not formed. Since spheres are possibly the thermodynamically most stable result of fusion, one or more cytosolic factors could modify this default reaction to convert it into a fusion reaction that results in networks. At least one of the cytosolic factors is a protein. Both network formation and the basic fusion reaction could be inhibited by GTPS and by treatment of the membranes with NEM, suggesting that a similar fusion machinery is used. NEM and GTP
S are inhibitors of steps that in other systems precede the actual fusion reaction. Known targets are the NEM-sensitive fusion protein (NSF) (or related proteins, such as p97) and rab proteins (
Network formation may initially proceed similarly to the basic fusion reaction, but must then diverge. Our data show that during network formation the vesicles first form cluster-like structures in a reaction that can be inhibited by NEM and GTPS. This may correspond to the tethering or docking of membrane vesicles that have been described as early steps in other fusion reactions (
S-sensitive step is required. High cytosolic Ca2+ concentrations appear to interfere at this step as well, explaining why they do not affect the basic fusion reaction. Our data with various Ca2+ chelators also suggest that Ca2+ fluctuations of ~1 µM may be important for network formation. A chelator with a binding constant in this range had a stronger inhibitory effect than chelators with lower or higher binding constants. In other systems, similar observations were explained by the fact that cytosolic Ca2+ gradients can be most effectively dispersed by chelators with a binding constant in the range of the average Ca2+ concentration in the gradient (
The exact mechanism by which a tubular membrane network is generated without a cytoskeleton remains mysterious. Several models of how the shape of membranes can be determined have been described. A number of mechanisms are based on changes in the preferred curvature of the lipid bilayer (
Consistent with the fact that the outer nuclear membrane and the ER form a continuous membrane, our results indicate that the in vitro formation of the two structures shares similarities. Both processes were inhibited by NEM, GTPS, and high Ca2+ concentrations, and were affected in a similar manner by the various Ca2+ chelators. Our data also suggest that the nuclear envelope forms via ER-like tubular intermediates bound to chromatin, similar to observations in mammalian cells (J. Ellenberg, personal communication). The formation of chromatin-bound membrane sheets and of an intact nuclear envelope require cytosolic factors, and in the absence of cytosol, chromatin-bound vesicles fuse to form large vesicles with diameters of up to a few micrometers (
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Footnotes |
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1 Abbreviations used in this paper: BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; ConA, concanavalin A; NEM, N-ethylmaleimide; TRAP, translocon-associated protein
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Acknowledgements |
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We would like to thank Tanya Civco, Judy Chou, Maria Ericsson, Julie Huang, Tim Mitchison, Jan-Michael Peters, Melissa Rolls, Pascal Stein, Todd Stukenberg, and Sidney Whiteheart for help with experimental techniques and for materials, and Jean-Francois Menetret and Chris Akey for cryo-EM. We thank Kent Matlack, Kathrin Plath, Will Prinz, and Melissa Rolls for critical reading of the manuscript.
T.A. Rapoport is a Howard Hughes Medical Institute Investigator.
Submitted: 29 September 1999
Revised: 18 January 2000
Accepted: 3 February 2000
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References |
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