The endoplasmic reticulum (ER) is a dynamic sub-cellular compartment that is essential to eukaryotic lifebecause it contributes significantly to the synthesis offundamental building blocks of the cell, includingproteins and lipids, and it acts as an important archi-tectural scaffold to maintain a well-organized spatialdistribution of the other endomembrane organelles.Recent analyses with live cell imaging coupled withgenetics studies have brought to light the incredibledynamism of this organelle and the underlying driversas well as the impact of the ER organization on thegeneral cellular homeostasis and plant growth. In thisreview, we highlight the most recent advances inthe understanding of the mechanisms that enable themorphological integrity of the plant ER in relation to theother organelles and the cytoskeleton.
The endomembrane system comprises endocytic andbiosynthetic cellular processes that are closely inte-grated. At the core of the endomembrane system liesthe ER, an essential and largely pleiotropic organelle.With its network of interconnected tubules and flat-tened cisternae, the ER represents the organelle with thelargestmembrane surface area and can be considered asthe gatekeeper of the secretory pathway that controlsmultiple checkpoints in protein biosynthesis: folding,quality control, signaling, and degradation. In additionto proteins such as receptors, ion channels, and en-zymes, the ER synthesizes a wide variety of cargomolecules that control a large spectrum of physiologicaland essential processes, and are eventually shippedfrom the ER or retained in this organelle (Aridor andHannan, 2000; Kim and Brandizzi, 2016; Brandizzi,2017). Furthermore, with its function in controllingprotein synthesis and folding, the ER has an importantrole in abiotic and biotic stress resistance throughthe unfolded protein response signaling (Angeloset al., 2017). The ER also is an important cellular
compartment for calcium storage and carbohydratemetabolism (Vitale and Denecke, 1999; Vitale and Galili,2001).
At a submicron level, the ER network is organizedin domains that are morphologically distinct andthat assume specific functions (Staehelin, 1997). These
1 This work was supported by the Chemical Sciences, Geosciences,and Biosciences Division, Office of Basic Energy Sciences, Officeof Science, U.S. Department of Energy (award no. DE-FG02-91ER20021), for infrastructure, and the National Science Foundation(MCB 1714561) and AgBioResearch, Michigan State University, toF.B.
2 Address correspondence to [email protected]. and F.B. wrote the article.[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.17.01261
178 Plant Physiology�, January 2018, Vol. 176, pp. 178–186, www.plantphysiol.org � 2018 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon September 18, 2020 - Published by Downloaded from
characteristics make the ER a morphologically con-tinuous cellular compartment that is nonuniform atthe functional and structural level. In humans, altera-tions in ER-mediated processes cause disease pheno-types that have been classified into three groups: (1)cargo retention and degradation, (2) cargo accumula-tion and ER stress, and (3) ER transport machinerydiseases (Aridor and Hannan, 2000). Also in plantcells, defects in ER functionality lead to various de-velopmental defects (Tamura et al., 2005; Conger et al.,2011; Stefano et al., 2012; Renna et al., 2013), sup-porting a critical role of the ER for organism biology atlarge.Morphologically, the ER is able to reorganize, en-
large, and contract its highly dynamic polygonal tu-bular network both spatially and temporally (Ridgeet al., 1999; Sparkes et al., 2009a; Stefano et al., 2014).The integrity of the ER network structure is importantto maintain an efficient unfolded protein response(UPR), as demonstrated by the evidence that loss ofER-shaping proteins leads to attenuation of the UPRsignaling in conditions of accrual of unfolded secre-tory proteins in the ER (Lai et al., 2014). Therefore,there is a strong connection between the morphologyand the functional integrity of the ER. The plant ERalso entertains strong functional connections withother organelles, including the Golgi apparatus andthe vacuole to which newly synthesized proteins canbe exported (Brandizzi et al., 2002; Shimada et al.,2003; Brandizzi, 2017), but also with chloroplasts withwhich the ER synthesizes essential lipids (Hurlocket al., 2014; Block and Jouhet, 2015). Possibly via directconnections, the ER movement influences the move-ment of other organelles, supporting an emergingmodel in cell biology that the ER interactionwith otherorganelles is important not only for the exchange ofconstituents with other cellular compartments butalso for their spatial distribution and function (Stefanoet al., 2014, 2015) In this review, we highlight some ofthe recent and exciting literature addressing the fun-damental questions on how the ER morphology anddynamics are controlled and how the ER interactswith the cytoskeleton and other organelles in plantcells.
THE ER IS A PLEOMORPHIC ORGANELLE WHOSEMORPHOLOGY AND DYNAMICS CHANGE DURINGTHE LIFE OF THE CELL
In general, the plant ER assumes the shape of amembrane network resembling the arrangement of aspider web with interconnected tubules and cisternaewithin the cell. Although the bulk of the ER is restrictedat the cell cortex where it is sandwiched between thetonoplast and the plasma membrane (PM), long ERtubular strands characterized by a high streaming ve-locity cross along the central vacuole (Ueda et al., 2010;Sparkes et al., 2011). Electron microscopy studies haverevealed the presence of smooth ER, rough ER, and
nuclear envelope regions (Hawes et al., 1981; Craig andStaehelin, 1988; Staehelin, 1997). The rough and smoothER regions are subdomains with associated ribosomesor ribosome-free regions, respectively. The nuclear en-velope is enwrapped by the ER, resulting in a doublemembrane delimiting the nucleus. Additionally, the ERpasses through the plasmodesmata, which are tinychannels that protrude into the cell wall and intercon-nect the cytoplasm of neighboring cells (Carr, 1976;Wright and Oparka, 2006). These structures are uniqueto plants and are crossed by a narrow tube-like struc-ture, named desmotubule, which is derived from theER (Quader and Zachariadis, 2006; Knox et al., 2015;Nicolas et al., 2017). As a result, the ER of each cell isinterconnected to the neighboring cells through thesechannels, forming a virtually unique organelle whoseextension is not delimited by the cell’s boundaries. TheER also is attached to the PM through ER-PM contactsites (EPCSs), which are largely immotile subdomainsof the ER that underlie the PM and whose number anddensity at the cell cortex diminish as cells expand(McFarlane et al., 2017). Fusion profiles of the ERmembrane with the PM at the EPCSs have not beenobserved; however, proteins such as VAP27 proteinsand Synaptotagmin1 (SYT1) have been shown to ac-cumulate at the EPCSs (Wang et al., 2014; Levy et al.,2015; Pérez-Sancho et al., 2015; Siao et al., 2016;McFarlane et al., 2017). The VAP proteins are conservedacross kingdoms and possess three main regions, aC-terminal transmembrane domain, an N-terminalmajor sperm domain, and a coiled-coil domain (Wanget al., 2014). The VAP subfamily of VAP27 proteinsmaycontribute to ER anchoring to the cell surface via theplasmodesmata as well as bridging the ERwith the PM,through yet unknown mechanisms that depend on cellwall integrity (Wang P. et al., 2016). The plant SYT1 is aclose ortholog of the yeast tricalbins and metazoansynaptotagmins that serve as ER-PM anchors in thesenonplant cell systems. Similar to VAP27, SYT1 is lo-calized to the bulk ER, and the EPCSs and SYT1 hasbeen localized at EPCSs demarcated by VAP27 (Pérez-Sancho et al., 2015). Based on the ability of VAP27 tointeract with the plant-specific actin-binding proteinNET3c and microtubules (MTs), it appears that theEPCSs marked by VAP27 may serve as ER-PM hubswhere the two major cytoskeletal components of plantcells converge (Wang et al., 2014). While roles for non-plant EPCSs are emerging, including lipid homeo-stasis and Ca2+ influx (van der Kant and Neefjes, 2014;Wakana et al., 2015), a physiological role for the plantEPCSs has yet to be defined. It has been demonstratedthat SYT1 interacts with phospholipids possiblythrough electrostatic interactions and may thereforebridge the ER membrane with the PM. A loss of SYT1reduces the ability of cells to withstand mechanicalpressure (Pérez-Sancho et al., 2015), which may un-derlie a function of these sites in the plant adaptationto abiotic stresses present in the natural environment.Themolecular mechanisms underlying such a role haveyet to be explored, but the evidence that the EPCSs
demarcated by SYT1 largely overlapwith VAP27-EPCSssuggests a functional connection between mechano-sensing and cytoskeletal organization at these enigmaticsites. The identification of SYT1 and VAP27 as com-ponents of the EPCS proteome opens up the excitingopportunity to further define the EPCS constituents,which may provide additional tools to understand thecellular role(s) of plant EPCSs.
With electron microscopy analyses, in addition to theEPCSs and plasmodesmata, the plant ER also has beenfound in contact with other membranes, includingGolgi, mitochondria, vacuole, and plastids (Juniperet al., 1982; Staehelin, 1997). Indeed, using laser traptechnology, it has been shown that the ER physicallycontacts the chloroplasts obtained from ruptured pro-toplasts expressing a fluorescent ER marker. Morespecifically, it was shown that the released chloroplastsremained attached to ER fragments that could bestretched out by optical tweezers. The applied force of400 pN, which is a magnitude compatible with protein-protein interactions, could not drag a chloroplast freefrom its attached ER (Andersson et al., 2007). Usingconfocal imaging of a fluorescent fusion, the Brassicanapus CLIP1 lipase/acylhydrolase (BnCLIP1) has beendetected recently in transient expression in tobacco(Nicotiana tabacum) at the ER-chloroplast contact sites(Tan et al., 2011), also known as PLAMs. BnCLIP1 en-zyme exhibited a discrete localization on the outer en-velope membrane at the junction between the ER andplastids. The subcellular distribution of a protein suchas BnCLIP1 at the PLAM is consistent with a potentialrole of these sites in the coordinated synthesis of lipidsbetween the ER and plastids (Tan et al., 2011). Yet, it isunknown whether proteins of this kind have only abiosynthetic role at the PLAMs, such as lipid synthesisand transport, or have a scaffolding role to tether thetwo organelles together.
Using an optical trapping and tweezer system,physical contacts of the ER with the Golgi also havebeen established. In plant cells, the Golgi apparatus isdispersed into polarized mini-stacks that are motile(Boevink et al., 1998; Nebenführ et al., 1999). By trapping
and pullingGolgi stacks in cells coexpressing fluorescentreporters for the ER and for the Golgi, it was shown thatthe pulling of Golgi stacks in cells where the movementof the ER and the Golgi was chemically inhibited wasfollowed by a movement of an ER tubule in associationwith the Golgi (Sparkes et al., 2009b). Recently, using asimilar approach, it was shown that overexpression of atruncated membrane-anchored Golgi matrix proteinAtCASP lacking the coiled-coil domain in the cytosolicregion could weaken the ER-Golgi connections, sup-porting the presence of proteinaceous scaffolding thattethers the ER and the Golgi together (Osterrieder et al.,2017). Although the identification of AtCASP as a pu-tative ER-Golgi tether is a landmark in plant cell biology,it will be important to pursue further the identification ofthe proteins responsible for the tethering of differentmembranes with the plant ER not only at the Golgi butalso with the PM and the other organelles in the plantcell. This is a field that is considerably lagging behindcompared with nonplant cell systems (Rocha et al.,2009; Eden et al., 2010; Kornmann et al., 2011; Stefanet al., 2011; Michel and Kornmann, 2012; Murley et al.,2015).
THE ER CHANGES SHAPE DURINGCELL DEVELOPMENT
One of the most noticeable features visible throughmicroscopy analyses of live cells expressing bio-reporters of the ER is the high dynamicity of thisorganelle; indeed, the ER tubules and cisternae con-tinually move and rearrange, evolving the overallarchitecture during time and cell development. Curi-ously, in plant cells, the ER does not have only oneform. Indeed, during the life cycle of a plant cell, theER undergoes considerable reorganization of themorphology and dynamics (Fig. 1; Ridge et al., 1999;Stefano et al., 2014; McFarlane et al., 2017). Initial re-ports that the ER assumes different shapes were pro-vided more than 2 decades ago in analyses using afluorescent probe for lipids (DiOC6) as well as a
Figure 1. In plant cells, ER architecture is correlated to cell expansion. Shown are confocal images of wild-typeArabidopsis Col-0cotyledon epidermal cells expressing an ER lumen marker ERYK (Nelson et al., 2007) at different phases of cell expansion. Notethe change in the morphology of the ER network, which from a most cisternal appearance in cells 3 d after germination (DAG)assumes progressively a more reticulated organization as cells expand. Scale bar = 5 mm.
fluorescent protein targeted to the bulk ER (Hepleret al., 1990; Ridge et al., 1999). The DiOC6 dye was usedto explore the structure of the ER in moss during budformation. Noticeable changes in ER architecture wereestablished, starting from a dense meshwork of mem-branes that was reorganized into an open reticularnetwork as the cell underwent growth. Through anal-yses performed using a fluorescent protein tagged tothe ER on root and hypocotyl cells of Arabidopsis(Arabidopsis thaliana), it was possible to distinguish anER characterized by lamellar sheets in early phases ofcell expansion followed by a change toward a reticulatetubular structure, which is typical of the ER of fullyexpanded cells (Ridge et al., 1999). It also was noticedthat in fully expanded root epidermal cells that giveorigin to root hairs, the reticular ER network was con-densed at the sites where the root hairs are formed(Ridge et al., 1999). The organization of the ER mor-phology becomes evenmore puzzling in dividing Pinusroot cells in which the ER at preprophase and prophaseis spatially rearranged to overlay the MTs (Quader andZachariadis, 2006). Indeed, treatment with oryzalin, aMT inhibitor, affects the formation of the tubular ER(tER)-preprophase band, tER-metaphase spindle, andtER phragmoplast, suggesting that at least in Pinus rootcells the ER network organization may depend on MTsduring mitosis and cytokinesis. Although togetherthese results support that the ER assumes differentmorphology during cell growth, it is yet to be shownwhether there may be a functional link between theshape of the ER and the ER function at specific stages ofcell growth and development. It has been shown re-cently that mutations in the ER-shaping protein RootHair Defective3 (RHD3) compromise not only theoverall organization of the ER and the transition fromextensive sheet-like form to a reticulated pattern, butalso cell elongation (Stefano et al., 2014). These resultstherefore support the existence of a close connectionbetween the ER shape and cell elongation, although theunderlying mechanisms have yet to be defined. Basedon the evidence that the loss of RHD3 alters the distri-bution of auxin in roots (Stefano et al., 2015), increasesthe cell’s phospholipid content, and leads to an at-tenuation of the UPR of the ER (Lai et al., 2014;Maneta-Peyret et al., 2014), the relationship betweenER morphology and cell growth may be the sum ofseveral processes that are affected by the alteration ofER architecture.
ER DYNAMICS DEPEND ON THE CYTOSKELETONAND ER-SHAPING PROTEINS
In fully expanded cells, the ER network is highlypleiotropic. Despite an anchoring to stable EPCSs, thenetwork undergoes profound rearrangements throughtubule interconversion into other tubules as well asfusion of tubules with cisternae. While processes offission/fragmentation have been verified in nonplantcells systems, such as sea urchin, starfish, lacrimal cells,
and nonneuronal cell lines (Terasaki and Jaffe, 1991;Jaffe and Terasaki, 1993; Subramanian andMeyer, 1997;Dayel et al., 1999; Ribeiro et al., 2000; Harmer et al.,2002; Kucharz et al., 2009), the common profiles forrearrangement of the plant ER are homotypic mem-brane fusion and tubule emergence from other tubules.These rearrangements are guided primarily by the actincytoskeleton (Sparkes et al., 2009a), with the MTs of-fering a minor yet significant contribution. Specifically,MTs guide ER tubule extension with an almost 20-foldslower rate compared with actin-based extension, andMTs appear to provide anchoring for the formation ofmultiway junctions (Hamada et al., 2014). While theidentity of the proteins connecting the plant ER to theMTs are yet unknown, recently two proteins have beenshown to link ER to actin. The first protein identified,SYP73, belongs to the three-member SYP7 family ofplant-specific SNAREs (Sanderfoot et al., 2000). Theseproteins, named SYP71, SYP72, and SYP73, share a highdegree of sequence similarity (Sanderfoot et al., 2000;Cao et al., 2016).
SYP73 binds actin directly and possesses a short lu-minal domain, a putative transmembrane domain, anda cytosolic region, which contains an actin-bindingmotif (Cao et al., 2016). SYP73 is primarily localized tothe ER. In conditions of overexpression of SYP73, thearchitecture of the ER changes dramatically with a re-duction of the cisternal profiles and a network patternthat overlaps the actin filaments. Conversely, a loss ofSYP73 causes enlargement of the ER, reduces itsstreaming, and compromises cell elongation (Cao et al.,2016). These results pose that SYP73 is an ER-actinlinker. SYP73 is only one of the components of the SYPfamily; therefore, other SYP proteins might share sim-ilar functions that may be important in different de-velopmental stages. The other protein implicated inER-actin connection is NET3B (for NETWORKED 3B),which is a plant-specific protein belonging to a super-family containing 13 members (Hawkins et al., 2014).All NET proteins contain two important domains. Inthe C-terminal region, a predicted coiled-coil domainmay be important for protein-protein interactions, andthe second important domain that characterizes thisfamily is the NET actin-binding domain, which may actas an adapter to link membranes to the actin cables. Inparticular, NET3B has been shown to associate in vivowith the ER. When the protein fused with a fluorescentmarker is overexpressed, similarly to conditions ofSYP73 overexpression, the architecture of the ER rear-ranges into resembling the actin cytoskeleton (Wangand Hussey, 2017). Although differently from SYP73, adirect interaction of NET3B with actin has not beenestablished yet, these results suggest that NET3B mayfunction as a linker between the ER and actin. Analysisof a NET3B knockout did not show any significantdefects when compared with the wild type (Wang andHussey, 2017), suggesting that there may be functionalredundancy among the NET proteins.
While the cytoskeleton provides a dynamic frame-work for the overall ER architecture, ER proteins such
as reticulons and RHD3 have an important role in theshaping of the network. Reticulons are membrane in-tegral proteins that assume a wedge-like topology withtheir transmembrane regions (Voeltz et al., 2006;Nziengui et al., 2007; Sparkes et al., 2010). By insertinginto the membrane, the reticulons form low-mobilityoligomers and induce high curvature of the ER mem-brane, which results in the formation and stabilizationof tubules (Shibata et al., 2008; Hu et al., 2011). Con-sistent with this function, overexpression of Arabi-dopsis RTN13, a reticulon that localizes at the ERtubules and the edges of ER cisternae, causes constric-tions of the ER lumen and reduces the diffusion of lu-men markers in the ER (Tolley et al., 2008, 2010). Thefunction of RTN13 depends on a small conserved do-main at the C-terminal region that contains a putativeamphipathic helix (APH; Breeze et al., 2016). Deletionof APH did not impair oligomer formation but dis-rupted the membrane-shaping function of RTN13in vivo (Breeze et al., 2016). These results are importantas they support that the membrane-shaping functionof reticulons may not be linked to their ability tooligomerize but to the presence of the APH domain.The plant family of reticulons contains 21 members(Nziengui et al., 2007; Sparkes et al., 2010). Given thelarge size of this family and the possibility that retic-ulons may share overlapping functions, no phenotypeof the ER network in knock-outs has been reported.Nonetheless, it would be interesting to carry out com-plementation tests with reticulons lacking the APH topinpoint the molecular role of this domain in the con-text of ER shaping.
In addition to the reticulons, RHD3 has importantshaping activity at the ER. RHD3 is an ER integralprotein with two putative transmembrane domains. Adefective allele of RHD3 was first identified in a screenfor root hair defects (Schiefelbein and Somerville, 1990).Loss of function of RHD3 leads to a reduced elongationof cells of the primary root (Stefano et al., 2012). At asubcellular level, the loss of RHD3 also reduces theformation of three-way junctions owing to the capacityof the protein to fuse membranes in a GTP-dependentfashion (Ueda et al., 2016). In this context, RHD3functions analogously to similar membrane-associateddynamin-like GTPases, such as metazoan atlastin andyeast Sey1p (Chen et al., 2011; Zhang et al., 2013; Yanet al., 2015). Nonetheless, it has been shown thatthe functional regulation of RHD3 may depend onplant-unique features. In particular, overexpression ofthe C-terminal region of RHD3 (RHD3 amino acids677–802) disrupts the ER network integrity; conversely,overexpression of the analogous Sey1p region (Sey1pamino acids 682–776) does not (Stefano and Brandizzi,2014). Intriguingly, the C-terminal domain of RHD3 isphosphorylated, and it has been shown that kinasetreatment of RHD3 induces oligomerization of thisprotein, which in turn may modulate its ER-shapingfunction (Ueda et al., 2016).
One puzzling question about RHD3 in relation to ERshape in general concerns the physiological role of ER
membrane fusion. RHD3 belongs to a three-memberfamily of proteins composed of RHD3, RHD3-like 1, andRHD3-like 2. The evidence that a double deletion ofRHD3 and RHD3-like 1 is lethal and that the combinedloss of RHD3 and RHD3-like 2 causes pollen lethality(Zhang et al., 2013) suggests that the formation of thetubular ER network is extremely important for the cell.Nonetheless, the evidence that in very young cells the ERdoes not have a reticulated form raises the question onhow the shape of the ERmay influence the physiology ofcells at certain stages of growth compared with others.
In addition to reticulons and RHD3 proteins, theremay be other proteins involved in ER shape. For ex-ample, in nonplant cells, the DP1/YOP1 proteins workas ER shapers in synergy with the reticulons. In Ara-bidopsis, five Yop1 homologs have been identified inthe HVA22 family of proteins (Brands and Ho, 2002).One HVA22 protein, fused with a fluorescent protein,is localized at the ER with RHD3 (Chen et al., 2011).A functional characterization of HVA22 proteins inthe context of ER shape is lacking, and it cannotbe excluded that additional proteins may be involved inplant ER architecture. In metazoans and yeast, it has beenshown that lunapark (Lnp1), a two-transmembranedomain protein, is required for ER shaping. In partic-ular, Lnp1 has been implicated in contributing to thetubule-to-sheet conversion, most likely by stabilizingthe three-way ER junctions (Chen et al., 2015; Wang S.et al., 2016). A functional ortholog of Lnp1 has yet tobe identified in Arabidopsis, and it cannot be yet ex-cluded that other proteins may have analogous func-tions to Lnp1 or that the stabilization of the plant ERjunctions depends on different mechanisms comparedwith nonplant cell systems.
ER MOVEMENT AND CYTOPLASMIC STREAMING:MORE THAN MOLECULAR TRACKS AND MOTORS?
While in animal cells the rearrangement of the ERnetwork as well as transport of vesicles largely dependon MTs, in plant cells this role is served primarily byactin and myosin motor proteins, like the plant-specificmyosin XI-K (Li and Nebenführ, 2007; Prokhnevskyet al., 2008; Peremyslov et al., 2010; Ueda et al., 2010).Indeed, the loss of myosin compromises the movementof ER, Golgi stacks, peroxisomes, and mitochondria(Peremyslov et al., 2008; Ueda et al., 2010), which col-lectively is called cytoplasmic streaming (Woodhouseand Goldstein, 2013; Stefano et al., 2014). The biologicalrole of cytoplasmic streaming has not been established ata molecular level, but it is plausible to hypothesize that,owing to the presence of a large central vacuole that canoccupy up to 90% of the total cell volume, themovementof the cytoplasmic content may facilitate the delivery ofnutrients as well as communication between distal sitesin the cells. The ER network is a pervasive organellethat contacts heterotypic membranes (Andersson et al.,2007; Mehrshahi et al., 2013; Stefano et al., 2015), andit therefore may have a bearing on their movement.
Indeed, as plant cells expand, concomitantly with thechanges of the ER architecture from cisternal to tubularmorphology, the velocity of ER streaming as well asoverall cytoplasmic streaming increases (Stefano et al.,2014). Based on this evidence and the findings that theloss of RHD3 compromises the spatial distribution aswell as the streaming not only of the ER but also of otherorganelles, such as the Golgi, peroxisomes, mitochon-dria, and endosomes, during cell expansion, it has beensuggested that the ER movement may contribute to thegeneral cytoplasmic streaming through the physicalconnections that it establishes with heterotypic mem-branes (Stefano et al., 2014, 2015). Intriguingly, a dis-ruption of endocytosis has been verified in connectionwith the loss of ER streaming and disruption of ERmorphology in an RHD3 mutant (Stefano et al., 2015).These results support the hypothesis that the dynamicpositioning of organelles such as endosomes is impor-tant to ensure their function andmaintain the overall cellhomeostasis.
CONCLUDING REMARKS
We are witnessing an exciting era for the under-standing of the plant ER. Although in recent years
enormous advances have been made toward the un-derstanding of how the structure and dynamic archi-tecture of the ER are maintained, it is yet unknown howother fundamental aspects of the ER are established,including how ER subdomains attain and maintaintheir identity and what their cellular role may be. SomeER subdomains have been investigated recently, andthe existence of an association between the ER and en-dosomes and the identity of the molecular players in-volved in the association of the ER with the PM or actinhave been established. The proteins and regulatoryprocesses underlying homotypic tubule fusion also areemerging (Fig. 2). One of the challenges for upcomingyears will be to gain higher resolution of the three-dimensional architecture of the ER in relation to otherorganelles and the cytoskeleton. This likely will beachievable with electron tomography, which allowsvisualizing structures at a high resolution (6–8 nm;Donohoe et al., 2006). An example of the power of thismethod is provided by a recent study using cryo-electron tomography showing at molecular resolutionthe three-dimensional architecture of EPCSs in non-plant cells (Fernández-Busnadiego et al., 2015). Thisapproach is likely to lead to more insights on the recentlydiscovered linkage of the ER with the cytoskeleton and
Figure 2. Diagram showing the association between the ER and other organelles in a plant cell. Abbreviations not defined in thetext: CW, cell wall; PD, plasmodesma; V, vacuole; N, nucleus; Px, peroxisome;Mt, mitochondrion; GA, Golgi stack; En, endosome.Black numbered square regions indicate the ER-organelle associations or ER rearrangement properties identified in plant cells.
other components of the endomembrane system inplant cells.Received September 5, 2017; accepted October 1, 2017; published October 6,2017.
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