© 2019. Published by The Company of Biologists Ltd | Journal of Science (2019) 132, jcs227611. doi:10.1242/jcs.227611

REVIEW SPECIAL ISSUE: RECONSTITUTING CELL BIOLOGY Reconstituting the reticular ER network – mechanistic implications and open questions Ning Wang and Tom A. Rapoport*

ABSTRACT in metazoans, the disassembles and The (ER) is a major membrane-bound peripheral ER tubules appear to be transformed into sheets (Lu in all eukaryotic cells. This organelle comprises et al., 2009; Wang et al., 2013). How the network is generated and morphologically distinct domains, including the nuclear envelope maintained, and how its morphology changes during the cell cycle, and peripheral sheets and tubules. The tubules are connected by is poorly understood. three-way junctions into a network. Several membrane have The tubules themselves are shaped by two evolutionarily been implicated in network formation; curvature-stabilizing proteins conserved families, the reticulons (Rtns) and REEPs generate the tubules themselves, and membrane-anchored (Yop1 in ) (Voeltz et al., 2006). These are ubiquitous GTPases fuse tubules into a network. Recent experiments have membrane proteins that are both necessary and sufficient to shown that a tubular network can be formed with reconstituted generate tubules (Hu et al., 2008). They belong to the ten most- ∼ proteoliposomes containing the yeast membrane-fusing GTPase abundant membrane proteins in a cell and together occupy 10% of Sey1 and a curvature-stabilizing protein of either the reticulon or the membrane surface of the reticular ER in Saccharomyces REEP protein families. The network forms in the presence of GTP cerevisiae (Hu et al., 2008). The Rtns and REEPS stabilize the high and is rapidly disassembled when GTP hydrolysis of Sey1 is membrane curvature seen in cross-sections of tubules and sheet inhibited, indicating that continuous membrane fusion is required for edges. How these proteins generate and stabilize membrane its maintenance. Atlastin, the ortholog of Sey1 in metazoans, forms a curvature is uncertain, as there are no structures available, but network on its own, serving both as a fusion and curvature-stabilizing they all contain pairs of closely spaced transmembrane (TM) protein. These results show that the reticular ER can be generated by segments and have an amphipathic helix (Fig. 1A). a surprisingly small set of proteins, and represents an energy- Connecting tubules into a network requires membrane fusion, dependent steady state between formation and disassembly. Models which is mediated by membrane-anchored GTPases, the atlastins for the molecular mechanism by which curvature-stabilizing proteins (ATLs) in metazoans and Sey1 and related proteins in yeast and cooperate with fusion GTPases to form a reticular network have been (Hu et al., 2009; Orso et al., 2009). have three proposed, but many aspects remain speculative, including the closely related ATLs, with ATL1 being prominently expressed in function of additional proteins, such as the lunapark protein, and neurons, and ATL2 and ATL3 more broadly in different cell types the mechanism by which the ER interacts with the cytoskeleton. How (Zhu et al., 2003). These proteins contain a cytoplasmic GTPase the nuclear envelope and peripheral ER sheets are formed remain domain, followed by a helical bundle, a membrane-embedded major unresolved questions in the field. Here, we review reconstitution region and an amphipathic helix (Fig. 1B). Until recently, the experiments with purified curvature-stabilizing proteins and fusion membrane-bound region was thought to comprise two closely GTPases, discuss mechanistic implications and point out open spaced TM segments, but it may actually consist of two questions. intramembrane hairpin loops (Betancourt-Solis et al., 2018), similar to those in the Rtns and REEPs. Crystal structures and KEY WORDS: Cell biology, Endoplasmic reticulum, Reconstitution, biochemical experiments have led to a model in which ATL Reticulon, Atlastin, Lunapark molecules localized to different membranes dimerize through their GTPase domains, and undergo a conformational change during the Introduction GTPase cycle, thereby pulling the two membranes together and All have characteristic shapes, but how their morphology fusing them (for a review, see Hu and Rapoport, 2016). Mutations in is generated is largely unknown. The endoplasmic reticulum (ER) is ATLs and REEPs can cause hereditary spastic paraplegia (Hübner a particularly intriguing organelle, as it consists of morphologically and Kurth, 2014; Salinas et al., 2008; Westrate et al., 2015), a distinct domains that change during differentiation and the cell neurodegenerative disease that is characterized by length-dependent cycle. In interphase, the ER consists of the nuclear envelope and a axonal dysfunction or degeneration. connected peripheral network of tubules and interspersed sheets The fusion of membrane tubules generates three-way junctions, (Baumann and Walz, 2001; Shibata et al., 2009; Terasaki et al., which are small, triangular sheets with negatively curved edge lines 1984; Voeltz et al., 2006). The tubules continuously form, retract (Shemesh et al., 2014). It has been suggested that these junctions are and slide along one another, a process facilitated by associated stabilized by the lunapark protein (Lnp, also known as LNPK) molecular motors and dynamic changes of the cytoskeleton. During (Chen et al., 2012, 2015; Shemesh et al., 2014), a conserved membrane protein containing two closely spaced TM segments and Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical several other domains (Fig. 1C). School, 240 Longwood Ave, Boston, MA 02115, USA. Peripheral ER sheets might be generated by the Rtns and REEPs stabilizing the highly curved membrane edges. Consistent with *Author for correspondence ([email protected]) this model, overexpression of these proteins in S. cerevisiae and

T.A.R., 0000-0001-9911-4216 mammalian cells favors the formation of tubules over sheets (Shibata Journal of Cell Science

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A Reticulons/REEPs B ATL/Sey1 morphologies. As a first step, we concentrated on the formation of a tubular ER network. In this Review, we will first discuss the N GTPase reconstitution of membrane tubules by the curvature-stabilizing Amphipathic proteins of the Rtn and REEP families, then cover the reconstitution helix of membrane fusion by the ATL/Sey1 GTPases, and finally the Helix bundle C formation of a reticular membrane network by co-reconstitution of Cytosol N C the curvature-stabilizing proteins and fusion GTPases. At the end of this Review, we point out open questions and future directions.

Tubule formation by reconstituted Rtns or REEPs

Lumen Lumen Our initial reconstitution experiments focused on the role of the Rtns and REEPs in tubule formation (Hu et al., 2008). Yeast Yop1 C Lunapark D (the single REEP family member in S. cerevisiae) and Rtn1 were expressed in Escherichia coli or S. cerevisiae and purified to near P Zn homogeneity in detergent. After mixing with phospholipids, the N detergent was removed and the samples were analyzed by negative- Coiled-coil stain electron microscopy (EM). At early time points (a few hours) domain Cytosol during detergent removal, small particles were observed. These might be small vesicles, but their small size and the lack of an obvious luminal space raises the possibility that they are micelles containing a mixture of protein, lipids and detergent. Ultimately, after longer times of detergent removal, membrane tubules were Lumen observed that reached several hundred nanometers in length, Fig. 1. Schemes for the structures of ER-shaping proteins. (A) Membrane indicating that the Rtns and REEPs alone can generate the high topology of the curvature-stabilizing proteins of the reticulon (Rtn) and REEP membrane curvature that is characteristic of tubules when viewed in families. The double-hairpin trans-membrane (TM) segments (pink), together cross-section. The tubules had a luminal space, but were much with the C-terminal amphipathic helix (hydrophobic and hydrophilic surfaces narrower than ER tubules observed in yeast cells (17 nm versus in blue and yellow, respectively) are thought to induce positive curvature of 30 nm). This can be explained by the significantly higher protein- the ER membrane. Based on NMR studies, the TM segments are thought to to-lipid ratio (by a factor of ∼20). Indeed, when Rtn or REEPs were fully span the membrane (Brady et al., 2015). (B) Domains of the fusion ATL GTPase. The GTPase Sey1 has a similar topology, but the helix bundle is more overexpressed in yeast, or mammalian cells, ER tubules complex (Yan et al., 2015). The proteins are depicted with two fully spanning became so narrow that luminal proteins were forced to move to other TM segments, but recent experiments suggest that they have two areas of the ER (Hu et al., 2008; Tolley et al., 2008). Interestingly, intramembrane hairpin loops (Betancourt-Solis et al., 2018). (C) Domains of ER tubules with a diameter of ∼20 nm are common in neuronal the lunapark protein. P is a domain that is phosphorylated during mitosis in (Terasaki, 2018), indicating that narrow tubules can be 2+ higher . Zn is a Zn -finger domain involved in dimerization of the physiological. However, it is unknown whether these tubules protein. (D) The Rtns and REEPs are proposed to form arc-shaped contain a particularly high concentration of curvature-stabilizing oligomers (shown in shades of pink) that bend the membrane (gray). proteins. The reconstitution experiments are consistent with results et al., 2010; Voeltz et al., 2006). Conversely, sheets become more obtained with more physiological systems. For example, abundant at the expense of tubules when these proteins are absent to an Rtn isoform (Rtn4a) block the formation of a (Shibata et al., 2010; Voeltz et al., 2006) or when phosphopholipid tubular ER network in egg extracts (Voeltz et al., 2006). synthesis is increased (Shibata et al., 2010). In higher eukaryotes, Although the exact mode of action of the antibodies remains sheet formation is stimulated by coiled-coil containing membrane unclear, those experiments provided the first evidence that the Rtns proteins, such as p180 (also known as RRBP1), kinectin (also are involved in tubule formation. Further evidence was provided by known as KTN1) and CLIMP-63 (also known as CKAP4), with the experiments with intact cells. Deletion of the Rtns and Yop1 in S. latter forming luminal bridges between the apposing membranes cerevisiae, and depletion of the proteins in mammalian cells, (Shibata et al., 2010). resulted in the conversion of ER tubules into sheets (Anderson and How the nuclear envelope is formed remains unclear. Hetzer, 2008; Voeltz et al., 2006; Wang et al., 2016); and Experiments in Xenopus egg extracts have suggested that both overexpression of these proteins led to long tubules (Voeltz et al., ATL- and ER SNARE-mediated membrane fusion steps are 2006; Wang et al., 2016). Finally, all these proteins localize to required to form the double membrane around chromatin (Wang tubules or sheet edges and avoid the low curvature areas of sheets et al., 2013), but how exactly membranes bind and fuse on the (Shemesh et al., 2014; Shibata et al., 2010; Voeltz et al., 2006; surface of chromatin at the end of mitosis remains to be elucidated. Wang et al., 2016). Targeting to the high-curvature regions appears Reconstituted systems containing purified proteins have been to be mediated by the TM hairpins, which can also be found in all instrumental in understanding the mechanism of many biological the other proteins that specifically localize to these areas, such as processes, including transcription (Ge et al., 1996; Sayre et al., ATLs, Sey1 and Lnp. Taken together with the reconstitution 1992), replication (Waga and Stillman, 1994; Yeeles et al., 2015), experiments, these results indicate that the Rtns and REEPs stabilize protein translocation (Akimaru et al., 1991; Brundage et al., 1990; high membrane curvature. Görlich and Rapoport, 1993) and ER-associated protein degradation The mechanism by which these proteins induce and stabilize high (ERAD) (Baldridge and Rapoport, 2016). This Review focuses on membrane curvature remains unclear. It has been proposed that they the ongoing efforts to reconstitute the ER. Our goal is to establish utilize two cooperating mechanisms, hydrophobic insertion reconstituted systems that recapitulate the formation of different ER (wedging) and scaffolding (Fig. 1D) (reviewed in Shibata et al., Journal of Cell Science

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2009). The hydrophobic insertion mechanism postulates that the hydrolyzed by the dimer, the two helical bundle domains associate two hydrophobic TM hairpins in these proteins, together with the with one another, generating a tight dimer in the transition state (step amphipathic helix, form a wedge-like structure that displaces 2). This interaction likely pulls the two membranes together so that the lipids preferentially in the outer leaflet of the , thus they can fuse. The C-terminal amphipathic helix may facilitate causing local curvature. The scaffolding mechanism assumes that membrane fusion, likely by perturbing the phospholipid bilayer the Rtns and REEPs form arc-like oligomers that mold the lipid (Liu et al., 2012). Finally, phosphate (Pi) and GDP are released in a bilayer into tubules (Fig. 1D). Arc-like scaffolds, rather than rings or sequential manner, causing the dissociation of the dimer into spirals that wrap around tubules, are consistent with the observation monomers (step 3). All these reactions can occur with ATL that the Rtns also localize to sheet edges and that the diffusion of ER molecules located either in different or the same membranes (trans- proteins along tubules is not restricted (Shibata et al., 2008, 2010; and cis-interactions, respectively), but membrane tethering and Shemesh et al., 2014). Structural information on the Rtns and fusion happen only with trans-interactions (Liu et al., 2015). The REEPs are required to test these ideas. formation and disassembly of cis-dimers is thus a futile cycle. ATLs also form trans-dimers in a futile manner (Liu et al., 2015; Membrane fusion in systems reconstituted with ATL or Sey1 Saini et al., 2014). Experiments with reconstituted proteoliposomes The fusion of ER membranes could also be recapitulated with have shown that multiple GTPase cycles are required for a purified proteins. ATL was successfully purified after successful fusion event, as the transition state of trans-dimers expression in E. coli and reconstituted with phospholipids into often converts them back into monomers without having caused proteoliposomes; the subsequent addition of GTP resulted in the fusion (Liu et al., 2015). Other experiments have indicated that the fusion of these vesicles, as initially demonstrated by lipid mixing and efficiency of tethering and fusion increases with the density of ATL size increase of the vesicles (Orso et al., 2009), and later by content molecules in the two membranes (Liu et al., 2015). ATL molecules mixing (Liu et al., 2012). The lipid composition of the membranes appear to associate with one another through their TM segments. In does not seem to play a major role (Orso et al., 2009). GTPase- fact, the TM segments cannot be replaced by those from other competent ATL molecules need to be present in both vesicles for their proteins, and point mutations in these regions can reduce the fusion to happen (Orso et al., 2009). Similar results were obtained efficiency of fusion (Liu et al., 2012). Recent experiments suggest with Sey1, also purified after expression in E. coli (Anwar et al., that the TMs form two intramembrane hairpin loops, rather than two 2012; Powers et al., 2017). These results indicate that these GTPases segments that completely span the membrane (Betancourt-Solis alone can mediate the homotypic fusion of membranes. et al., 2018), which may explain their unique role in fusion. Taken The reconstitution experiments are again in agreement with together, it appears that a successful fusion event requires the experiments performed in physiological systems, as the depletion of cooperation of multiple trans-dimerization events: as one dimer ATLs or the expression of dominant-negative mutants leads to ER reaches the transition state, other dimers forming nearby help to tubule fragmentation and the appearance of long, unbranched ER maintain the tethered state and add to the pulling force exerted on tubules in tissue culture cells (Hu et al., 2009; Wang et al., 2016) or the opposing membranes. The futile formation of trans-dimers (Orso et al., 2009). These phenotypes are suggests that a fraction of three-way junctions in cells may actually likely caused by insufficient fusion between the tubules. Similarly, consist of tethered, rather than fused, tubules, and may explain, at in Xenopus egg extracts, the addition of a dominant-negative ATL least in part, why the ER network rapidly disassembles upon mutant or of non-hydrolysable GTP analogs prevents ER network inhibition of the GTPase activity of ATL (Wang et al., 2013). formation (Wang et al., 2013). Surprisingly, these reagents also cause a pre-formed ER network to disintegrate into smaller Reconstitution of a reticular ER network membrane structures within a few minutes (Wang et al., 2013), The next step in reconstituting the ER is to generate a reticular ER indicating that continuous ATL function is required to maintain the network. We therefore tested whether using a curvature-stabilizing integrity of an ER network. protein, an Rtn or REEP protein, and a fusion GTPase, either ATL or The molecular mechanism of ATL function has been elucidated Sey1, is sufficient (Powers et al., 2017). To this end, we first purified from crystal structures of the cytosolic domain (Bian et al., 2011; full-length S. cerevisiae Yop1 and Sey1 after expression in E. coli Byrnes and Sondermann, 2011) and biochemical studies (Bian and reconstituted both proteins together into proteoliposomes. et al., 2011; Byrnes and Sondermann, 2011; Liu et al., 2012, 2015). Essentially all molecules were incorporated into the vesicles with These have led to a plausible model for ATL-mediated membrane their cytosolic domains on the outside, as shown by protease fusion, which begins with the binding of GTP to ATL monomers protection experiments. The reconstituted vesicles converted into a (Fig. 2; step 1). The monomers then rapidly form a dimer, with the reticular network upon addition of GTP, which could be visualized bound GTP molecules buried at the interface. When GTP is with a hydrophobic fluorescent dye in a light microscope (Fig. 3) or

Fig. 2. Scheme for ATL-mediated membrane fusion. In step 1, ATL molecules sitting in different membranes bind GTP and associate with one another. In step 2, GTP is hydrolyzed and the proteins undergo GTP Step 1 Step 2 GDP+P Step 3 GDP i conformational changes that allow the helical bundles to interact. The two membranes are pulled together so

that they can fuse. In step 3, Pi and GDP are released, and the ATL dimer dissociates into monomers. Journal of Cell Science

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Yop1 + Sey1 of tubules may preferentially occur at free ends, as the tip of a −GTP +GTP membrane tubule already corresponds to half a vesicle. However, such free ends are infrequent in mammalian cells and in Xenopus extracts (Friedman et al., 2010; Wang et al., 2013); most tubules are PE

− anchored at three-way junctions, and free ends are often associated with the tip of a growing microtubule or molecular motor (Friedman et al., 2010). Fusion of the tips of ER tubules with other tubules continuously generates new three-way junctions (Lee and Chen, Rhodamine 1988) and thus increases the number of polygons in the tubular network. This process is counteracted by ‘ring closure’ (Lee and Chen, 1988), a process in which three-way junctions merge and thus Fig. 3. Generation of a tubular membrane network with purified proteins. abolish polygons. The dynamics of the ER network is different from Purified Yop1, a curvature-stabilizing protein from S. cerevisiae that belongs to the REEP family, was mixed with purified Sey1, the fusion that of mitochondria, which maintain their structure by a balance of GTPase in S. cerevisiae. Phospholipids, including Rhodamine-labeled fusion and fission reactions (Shaw and Nunnari, 2002). phosphoethanolamine (PE) were added, and the detergent was subsequently The Rtns and REEPs have been shown to form oligomers, which removed to generate proteoliposomes. The vesicles were incubated without could contribute to tubule formation (Shibata et al., 2008). or with GTP (left and right panel, respectively). The samples were placed on However, whether their oligomeric state is different in tubules and a coverslip and visualized in a fluorescence microscope. Scale bars: 20 µm. vesicles and affected by the fusion GTPases remains to be tested. The figure is reproduced from Powers et al., (2017) with permission from Springer Nature. The roles of the amphipathic helices of the curvature-stabilizing proteins and GTPases in network formation also needs to be clarified. by EM. Network formation was not observed with the individual proteins. In these experiments, Yop1 could be replaced with a Reconstitution of Lnp and its implications number of other curvature-stabilizing proteins of the Rtn and REEP Another protein that is important for ER network formation is Lnp, a families, such as Rtns from S. cerevisiae or Drosophila and REEPs conserved membrane protein containing two closely spaced TM from Drosophila and Xenopus. Our titration experiments showed segments, two coiled-coil regions and a Zn2+-finger domain that the network forms when a physiological molar ratio of Rtn or involved in dimerization (Fig. 1C) (Wang et al., 2018). The REEP to Sey1 was used, which is ∼10:1 (Kulak et al., 2014). mammalian protein also has a domain that is phosphorylated during However, the total protein concentration required was ∼10 times mitosis. The protein was originally discovered in a genetic screen in higher than in vivo. As was the case in Xenopus extracts (Wang S. cerevisiae for ER morphology defects and recognized as a et al., 2013), the network rapidly disassembled when a non- counteracting factor of the fusion GTPase Sey1 (Chen et al., 2012). hydrolysable GTP analog was added to inhibit Sey1 activity It was later proposed to stabilize three-way tubular junctions (Chen (Powers et al., 2017). Surprisingly, the entire network was dynamic, et al., 2015; Shemesh et al., 2014). However, although Lnp localizes with tubules sliding passed each other. As a cytoskeleton, which in preferentially to three-way junctions of the ER, it is not essential for cells is typically required for ER dynamics, is missing in these their formation. Indeed, mammalian cells lacking Lnp have more reconstitutions, it is possible that thermal convection on the ER sheets, but the tubular network does not completely disappear, coverslip provided the energy for the morphological changes. particularly remaining at the periphery of the cells (Wang et al., Taken together, these results indicated that an ER network can be 2016; Zhou et al., 2018). generated with a small set of proteins, a curvature-stabilizing protein To better understand the function of Lnp, we purified Xenopus or and a membrane-fusing GTPase. Lnp after expression in E. coli, before mixing the protein with Surprisingly, reconstituted Drosophila ATL was capable of lipid and removing the detergent (Wang et al., 2018). Surprisingly, forming a reticular network on its own (Powers et al., 2017). This negative-stain EM showed that the resulting structures were stacked can be explained by the fact that ATL has both curvature-stabilizing membrane discs (bicelles) (Fig. 4A), as confirmed by EM cryo- and fusion activities. Indeed, a GTP-dependent network was formed tomography. Mutagenesis indicated that the Lnp protein is when a GTPase-defective ATL mutant, which retained its curvature- incorporated into the membrane discs in both orientations; the stabilizing activity, was co-reconstituted with Sey1 (Powers et al., discs are linked with one another through the cytosolic domains of 2017). It is currently unclear whether Sey1 also has a curvature- Lnp (Fig. 4B). The domain phosphorylated during mitosis is stabilizing activity, as vesicle-flotation experiments showed that it important for trans-interactions between Lnp molecules, and a was less efficiently incorporated into proteoliposomes than ATL. phosphomimetic Lnp mutant showed reduced bicelle stacking (Wang et al., 2018). Inactivation of Lnp during mitosis is also suggested by What is the mechanism of ER network formation? experiments with Xenopus egg extracts: treatment with dominant- Both the in vivo and in vitro results indicate that the ER network negative Lnp fragments converts the tubular ER into a network of corresponds to a steady-state of continuous membrane fusion and small sheets that are connected by short tubules, which resembles the fragmentation. Fusion is mediated by the GTPases ATL or Sey1 and ER morphology seen during mitosis (Wang et al., 2016). fragmentation is caused by the curvature-stabilizing proteins of the We have proposed that Lnp localizes preferentially to three-way Rtn and REEP/Yop1 families, which, in the absence of ATL junctions because it engages in trans-interactions when junctions activity, appear to prefer the higher membrane curvature of small come into close proximity within a 3D network (Wang et al., 2018). vesicles to that of tubules. In a steady-state network, fusion mediated Three-way junctions are small, triangular membrane sheets and by the GTPases appears to be faster than the breakage of tubules or offer a better geometry for trans-interactions between multiple Lnp the shedding of small vesicles mediated by the curvature-stabilizing molecules than tubules. Lnp-containing three-way junctions may proteins, explaining why tubule fission is rarely observed in vivo not be able to undergo efficient membrane fusion because Lnp

(Friedman et al., 2010). Under normal conditions, the disassembly could prevent ATL from entering the junctional sheets or because Journal of Cell Science

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A B Further studies are required to fully understand the molecular mechanism of network formation. A major goal is to obtain a P Zn structure of the Rtn or REEP proteins, which will shed light on how these proteins induce high membrane curvature. The possible role of oligomers formed by these proteins and the significance of their TM hairpins and amphipathic helices also need to be clarified. Further studies are also needed on whether a physical interaction between the curvature-stabilizing proteins and the fusion-mediating GTPases is required for network formation. It remains puzzling that there are so many different Rtn and P Zn REEP proteins, even within a single cell. Several members of these P Zn protein families have large cytosolic domains of unknown function. Presumably, these domains bind other proteins and might diversify the function of the Rtns and REEPs. In addition, several Rtn-like proteins have been discovered, including Tts1 in S. pombe (Zhang and Oliferenko, 2014; Zhang et al., 2010) and its homologs in other organisms, the ER autophagy receptor FAM134 (also known as RETREG1) (Khaminets et al., 2015) and ARL6IP1, which recruits

P Zn an inositol 5-phosphatase to the ER (Dong et al., 2018). Whether these proteins induce high membrane curvature and whether such an effect is required for their functions remains to be investigated. Another unresolved issue concerns the interaction between the Fig. 4. Bicelle formation by reconstituted lunapark protein. (A) Purified ER and the cytoskeleton. In mammalian cells, ER tubules are often lunapark from Xenopus laevis was mixed with lipids (molar ratio 1:5) and the detergent was removed. The sample was analyzed by negative-stain EM. generated by the force generated by either microtubule-associated The figure is reproduced from Wang et al. (2018) where it was published under a molecular motors or by polymerizing microtubule tips (Lee and CC BY-NC-SA 3.0 licence (https://creativecommons.org/licenses/by-nc-sa/3.0/). Chen, 1988; Prinz et al., 2000; Terasaki et al., 1986; Waterman- (B) Model for lunapark-induced bicelle formation. Lunapark is assumed to sit in the Storer and Salmon, 1998). The proteins that mediate the interaction membrane discs in both orientations. The edges of the membrane discs might with motors or microtubules have not yet been identified. In be stabilized by residual detergent. Stacking of the discs is mediated by both S. cerevisiae, ER tubules are formed along actin filaments (Estrada the phosphorylation (P) and Zn2+-finger (Zn) domains of lunapark. et al., 2003). Although the cytoskeleton is required for the generation and distribution of the ER network, ultimately the membranes at these regions become less deformable. In addition, alignment of ER tubules with the cytoskeleton is not perfect. In Lnp inhibits ATL-mediated membrane fusion (Zhou et al., 2018). A addition, the ER network does not collapse upon depolymerization mechanism in which Lnp counteracts ATL is consistent with the of actin filaments in yeast (Prinz et al., 2000), and it retracts only observations that overexpression of Lnp in mammalian tissue with some delay upon depolymerization of microtubules in culture cells leads to expanded junctional sheets with ATL located at mammalian cells (Terasaki et al., 1986). In vitro, an ER network the edges and that co-overexpression of ATL restores a normal ER can be generated in the absence of an intact cytoskeleton (Dreier and network (Wang et al., 2016). It is also consistent with the Rapoport, 2000). Interestingly, p180 and CLIMP-63, the membrane observation that in S. cerevisiae, Lnp and Sey1 have opposing proteins implicated in the generation of peripheral ER sheets effects on ER morphology (Chen et al., 2012). The postulated in mammalian cells, contain microtubule-binding domains function of Lnp would be important in the center of mammalian (Klopfenstein et al., 1998; Ogawa-Goto et al., 2007), but their cells, where the larger volume allows the ER network to be dense in precise function remains unclear. all three directions. In contrast, at the periphery of the cell, the ER Peripheral ER sheets seem to be present in all cells, although network is essentially two dimensional and Lnp would not be some may actually be fenestrated (Puhka et al., 2012) or composed required to prevent excessive fusion; this also explains why in of dense clusters of three-way tubular junctions (Nixon-Abell et al., mammalian cells lacking Lnp, the residual network localizes to the 2016). The abundance of peripheral ER sheets varies among cells periphery of the cells (Wang et al., 2016). and changes during the cell cycle. Although controversial (Puhka et al., 2012), in some mammalian tissue culture cells (Lu et al., Future perspectives 2009) and in Xenopus extracts (Wang et al., 2013), ER tubules Significant progress has been made in understanding how the convert into sheets during mitosis. Sheets are generally thought to morphology of the ER is achieved, providing a paradigm for how correspond to rough ER (Shibata et al., 2006), that is, regions other membrane-bound organelles may be shaped. The recent containing membrane-bound ribosomes. The membranes of the reconstitution experiments discussed here show that the minimum rough and smooth ER are continuous, and yet the ribosome-studded system required to form a reticular ER network consists of a area is kept segregated. How such a distinct rough ER domain is curvature-stabilizing protein, that is, a member of the Rtn or REEP formed remains unclear. The concentration of sheets in one area of families, and a membrane-fusing GTPase, either ATL or Sey1 the cell requires intact polysomes and the presence of the sheet- (Powers et al., 2017). The resulting network represents a dynamic promoting membrane proteins p180, kinectin and CLIMP-63 state that requires continuous GTP hydrolysis by ATL/Sey1. (Shibata et al., 2010). Perhaps, these membrane proteins link Although in vivo the ER is not formed de novo, but rather from different polysomes to form a rough ER domain. How peripheral ER pre-existing membranes by addition of newly synthesized lipids and sheets are stacked on top of each other in highly secretory cells also proteins, the reconstitution experiments allow mechanistic studies remains to be investigated. Serial sectioning EM has demonstrated on how the final membrane structure is attained. that the sheets are connected by twisted membrane surfaces, Journal of Cell Science

5 REVIEW Journal of Cell Science (2019) 132, jcs227611. doi:10.1242/jcs.227611 resembling a parking garage in which the different levels are Estrada, P., Kim, J., Coleman, J., Walker, L., Dunn, B., Takizawa, P., Novick, P. connected by helicoidal ramps (Terasaki et al., 2013). However, the and Ferro-Novick, S. (2003). Myo4p and She3p are required for cortical ER inheritance in . J. Cell Biol. 163, 1255-1266. proteins required for sheet stacking are currently unknown. Friedman, J. R., Webster, B. M., Mastronarde, D. N., Verhey, K. J. and Voeltz, Finally, the mechanism of nuclear envelope formation remains G. K. (2010). ER sliding dynamics and ER-mitochondrial contacts occur on one of the most challenging unresolved issues. Experiments with acetylated microtubules. J. Cell Biol. 190, 363-375. Ge, H., Martinez, E., Chiang, C.-M. and Roeder, R. G. (1996). 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