ER-PM Contact Sites Research Articles

The endoplasmic reticulum-plasma membrane tethering protein Ist2 regulates endoplasmic reticulum stress response in <italic>Candida albicans</italic>

The endoplasmic reticulum-plasma membrane contact (ER-PM contact) is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans . The measurement of growth curves, fluorescence detection of the unfolded protein response (UPR) reporting system, and the measurement of the cytosolic Ca2+ level revealed that the IST2 deletion strain ist2 Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca2+ levels under ER stress caused by tunicamycin decreased. In addition, the Ca2+ chelating agent EGTA could eliminate the difference between ist2 Δ/Δ and WT in ER stress tolerance. Therefore, Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca2+ level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.

Sterol Flow between the Plasma Membrane and the Endosome is Regulated by the LAM Family Protein Ltc1

Sterols are crucial components of biological membranes that help maintain membrane integrity and regulate various processes such as endocytosis, protein oligomerization and signaling. Although synthetized in the ER, sterols are at highest concentrations at the plasma membrane (PM) in all eukaryotic organisms. Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. While D4H is detected at the PM during steady-state growth, inhibition of Arp2/3-dependent F-actin assembly unexpectedly promotes the reversible re-localization of the probe to internal sterol rich compartments (STRIC), as shown by correlative light-electron microscopy. Time-lapse imaging identifies STRIC as a late secretory, endosomal compartment labelled by the synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi. Instead, it depends on Ltc1, a LAM/StARkin-family protein that localizes to ER-PM contact sites. In ltc1Δ, sterols over-accumulate at the PM, which forms extended ER-interacting invaginations, indicating that sterol transfer by Ltc1 contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking components but requires Arp2/3 activity, suggesting a novel physiological role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the retrograde and anterograde flow of sterols in the cell.

Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes.

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.

Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation.

The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.

Remodeling of ER–plasma membrane contact sites but not STIM1 phosphorylation inhibits Ca2+ influx in mitosis

Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.

E-syt1 Re-arranges STIM1 Clusters to Stabilize Ring-shaped ER-PM Contact Sites and Accelerate Ca2+ Store Replenishment

In many non-excitable cells, the depletion of endoplasmic reticulum (ER) Ca2+ stores leads to the dynamic formation of membrane contact sites (MCSs) between the ER and the plasma membrane (PM), which activates the store-operated Ca2+ entry (SOCE) to refill the ER store. Two different Ca2+-sensitive proteins, STIM1 and extended synaptotagmin-1 (E-syt1), are activated during this process. Due to the lack of live cell super-resolution imaging, how MCSs are dynamically regulated by STIM1 and E-syt1 coordinately during ER Ca2+ store depletion and replenishment remain unknown. With home-built super-resolution microscopes that provide superior axial and lateral resolution in live cells, we revealed that extracellular Ca2+ influx via SOCE activated E-syt1s to move towards the PM by ~12 nm. Unexpectedly, activated E-syt1s did not constitute the MCSs per se, but re-arranged neighboring ER structures into ring-shaped MCSs (230~280 nm in diameter) enclosing E-syt1 puncta, which helped to stabilize MCSs and accelerate local ER Ca2+ replenishment. Overall, we have demonstrated different roles of STIM1 and E-syt1 in MCS formation regulation, SOCE activation and ER Ca2+ store replenishment.

Role of membrane shape in regulating the phosphatidylinositol cycle at contact sites.

The extensive number of metabolic processes regulated by phosphatidylinositol (PI) phospholipids highlights their physiological importance. The major metabolic pathway for their biosynthesis in cells is the PI-cycle. Contrary to most metabolic cycles, reactions of the PI-cycle occur in two different locations; those are, the plasma membrane (PM) and endoplasmic reticulum (ER). Lipid movement between the two organelles is, therefore, a requirement of the cyclical process. Moreover, in mammals the PI-cycle yield PI molecular species enriched in specific acyl chains, namely 1-stearoyl-2-arachidonoyl acyl chains. Hence, to ensure cycle efficiency and specificity it should take place in specialized regions of PM and ER rather than being randomly distributed among those membranes. Along these lines, ER-PM contact sites have emerged as the location where a number of proteins related to the PI-cycle have been reported to localize. Of importance to this review is the presence of the epsilon isoform of diacylglycerol kinase (DGKε) at ER-PM contact sites. In the PI-cycle DGKε is in part responsible for the acyl chain enrichment of the PI molecular species. However, it has recently been shown that the enzyme can only engage in the PI-cycle upon membrane morphological changes. In this review we will discuss the PI-cycle at ER-PM contact sites and how the generation of membrane negative Gaussian curvature nearby those regions could regulate the cycle. We will focus our discussion on the hypothesis that actin polymerization provides the mechanical force needed to change membrane shape nearby ER-PM contact sites, which will transiently trigger DGKε and, therefore, link enzymatic catalysis and lipid transfer in the PI-cycle.

Plant ER-PM Contact Sites in Endocytosis and Autophagy: Does the Local Composition of Membrane Phospholipid Play a Role?

Plant ER-PM Contact Sites in Endocytosis and Autophagy: Does the Local Composition of Membrane Phospholipid Play a Role?

Ionic stress enhances ER–PM connectivity via phosphoinositide-associated SYT1 contact site expansion in Arabidopsis

The interorganelle communication mediated by membrane contact sites (MCSs) is an evolutionary hallmark of eukaryotic cells. MCS connections enable the nonvesicular exchange of information between organelles and allow them to coordinate responses to changing cellular environments. In plants, the importance of MCS components in the responses to environmental stress has been widely established, but the molecular mechanisms regulating interorganelle connectivity during stress still remain opaque. In this report, we use the model plant Arabidopsis thaliana to show that ionic stress increases endoplasmic reticulum (ER)-plasma membrane (PM) connectivity by promoting the cortical expansion of synaptotagmin 1 (SYT1)-enriched ER-PM contact sites (S-EPCSs). We define differential roles for the cortical cytoskeleton in the regulation of S-EPCS dynamics and ER-PM connectivity, and we identify the accumulation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the PM as a molecular signal associated with the ER-PM connectivity changes. Our study highlights the functional conservation of EPCS components and PM phosphoinositides as modulators of ER-PM connectivity in eukaryotes, and uncovers unique aspects of the spatiotemporal regulation of ER-PM connectivity in plants.

Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion

ABSTRACTMembrane contact sites (MCS) have increasingly received attention because of their general role in a number of important cellular processes. SYNAPTOTAGMIN 1 (SYT1) is a tethering factor connecting the endoplasmic reticulum (ER) and the plasma membrane (PM) in plant cells. Confocal microscopy using fluorescent protein fusion is an indispensable tool for studying protein localisation and functions. However, several studies have reported that fluorescent protein dimerisation affects the subcellular localisation of proteins tagged by the fluorescent protein. Here, we investigate the effects of fluorescent protein dimerisation by comparing the subcellular localisation of SYT1 fused with a synthetic GFP (SYT1-sGFP) and SYT1 fused with a monomeric GFP (SYT1-mGFP). SYT1-mGFP was confined to specific domains in the ER, whereas SYT1-sGFP spread along the ER when transiently overexpressed. SYT1-localised regions were suggested to correspond to ER-PM contact sites because of its immobility. Similar results were obtained in the transgenic Arabidopsis, even though SYT1-sGFP and SYT1-mGFP were expressed at comparable levels. It is suggested that SYT1-mGFP more accurately reproduced SYT1 localisation in intact cells because the proportion of persistent area in the ER was more similar between the wild type and the plant expressing SYT1-mGFP than between the wild type and the plant expressing SYT1-sGFP. Taken together, these results suggest that the fusion of sGFP makes SYT1-sGFP form excessive ER-PM contact sites in the ER.

Synaptotagmin-Associated Endoplasmic Reticulum-Plasma Membrane Contact Sites Are Localized to Immobile ER Tubules.

The plant endoplasmic reticulum (ER), which is morphologically divided into tubules and sheets, seems to flow continuously as a whole, but locally, mobile and immobile regions exist. In eukaryotes, the ER physically and functionally interacts with the plasma membrane (PM) at domains called ER-PM contact sites (EPCSs). Extended synaptotagmin family proteins are concentrated in the cortical ER to form one type of EPCS; however, it is unclear whether the localization of extended synaptotagmin corresponds to the EPCS and where in the cortical ER the EPCSs are formed. Here, we analyzed the spatiotemporal localization of SYNAPTOTAGMIN1 (SYT1), a synaptotagmin in Arabidopsis (Arabidopsis thaliana), to investigate the precise distribution of SYT1-associated EPCSs in the cortical ER. Three-dimensional imaging using superresolution confocal live imaging microscopy demonstrated that SYT1 was specifically localized to the ER-PM boundary. Time-lapse imaging revealed that SYT1 was distributed to immobile ER tubules, but not to mobile tubules. Moreover, SYT1 was frequently localized to the edges of ER sheets that were transformed into immobile ER tubules over time. A lower intracellular calcium ion concentration resulted in an increased EPCS area and disrupted the ER network. Finally, SYT1 deficiency caused a reduction of the immobile tubules and enlargement of the ER meshes. Taken together, our findings show that SYT1-associated EPCS are distributed to immobile tubules and play an important role in the formation of the tubular ER network. This provides important insight into the relationship between the function and dynamics/morphology of the cortical ER.

Oxysterol-binding protein-related protein (ORP) 6 localizes to the ER and ER-plasma membrane contact sites and is involved in the turnover of PI4P in cerebellar granule neurons

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER–plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER–PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER–PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Plant Endocytosis Requires the ER Membrane-Anchored Proteins VAP27-1 and VAP27-3

Through yet-undefined mechanisms, the plant endoplasmic reticulum (ER) has a critical role in endocytosis. The plant ER establishes a close association with endosomes and contacts the plasma membrane (PM) at ER-PM contact sites (EPCSs) demarcated bythe ER membrane-associated VAMP-associated-proteins (VAP). Here, we investigated two plant VAPs, VAP27-1 and VAP27-3, and found an interaction with clathrin and a requirement for the homeostasis of clathrin dynamics at endocytic membranes and endocytosis. We also demonstrated direct interaction of VAP27-proteins with phosphatidylinositol-phosphate lipids (PIPs) that populate endocytic membranes. These results support that, through interaction with PIPs, VAP27-proteins bridge the ER with endocytic membranes and maintain endocytic traffic, likely through their interaction with clathrin.

GRAM marks the spot for STIM. Commentary on “GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells”

GRAM domain proteins were reported as novel ER-PM tethers defining specific membrane contact sites (MCS) subdomains. GRAMD2a pre-marks the sites occupied by STIM1 at MCS and its ablation impairs STIM1 translocation, but not store-operated Ca2+ entry. We discuss these apparently counterintuitive findings in the context of STIM/ORAI signaling at MCS.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

GRAM domain proteins specialize functionally distinct ER-PM contact sites in human cells.

Endoplasmic reticulum (ER) membrane contact sites (MCSs) are crucial regulatory hubs in cells, playing roles in signaling, organelle dynamics, and ion and lipid homeostasis. Previous work demonstrated that the highly conserved yeast Ltc/Lam sterol transporters localize and function at ER MCSs. Our analysis of the human family members, GRAMD1a and GRAMD2a, demonstrates that they are ER-PM MCS proteins, which mark separate regions of the plasma membrane (PM) and perform distinct functions in vivo. GRAMD2a, but not GRAMD1a, co-localizes with the E-Syt2/3 tethers at ER-PM contacts in a PIP lipid-dependent manner and pre-marks the subset of PI(4,5)P2-enriched ER-PM MCSs utilized for STIM1 recruitment. Data from an analysis of cells lacking GRAMD2a suggest that it is an organizer of ER-PM MCSs with pleiotropic functions including calcium homeostasis. Thus, our data demonstrate the existence of multiple ER-PM domains in human cells that are functionally specialized by GRAM-domain containing proteins.

Characterization of Proteins Localized to Plant ER-PM Contact Sites.

Like in most eukaryotic cells, the plant endoplasmic reticulum (ER) network is physically linked to the plasma membrane (PM), forming ER-PM contact sites (EPCS). The protein complex required for maintaining the EPCS is composed of ER integral membrane proteins (e.g., VAP27, synaptotagmins), PM-associated proteins (e.g., NET3C), and the cytoskeleton. Here, we describe methods for identifying possible EPCS-associated proteins. These include GFP-tagged protein expression followed by image analysis, and immuno-gold labeling at the ultrastructural level. In combination, these methods can be used to identify the localization of putative EPCS proteins as well as used to postulate their subcellular function.

Dual targeting of a virus movement protein to ER and plasma membrane subdomains is essential for plasmodesmata localization.

Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.