Endoplasmic Reticulum Tubules Research Articles

Simultaneous Two-Angle Axial Ratiometry for Fast Live and Long-Term Three-Dimensional Super-Resolution Fluorescence Imaging.

The application of optical microscopy in four-dimensional (spatial and temporal) super-resolution imaging poses challenges because of the requirement of a long acquisition time or high illumination intensity. In this paper, we introduce simultaneous two-angle axial ratiometry (STARII) for <20 nm axial super-resolution imaging and for fast and long-term imaging of live cells up to hundreds of frames per second. This method involves recording two raw images in two incident angle channels in the context of evanescent wave illumination and obtaining the corresponding intensity ratio. Furthermore, we demonstrate the combination of STARII with the lateral super-resolution method to resolve three-dimensional nanoscale structures of microtubules and to visualize the long-term dynamical plasma membrane curvature and fast remodeling of endoplasmic reticulum tubule meshwork and three-way junctions. These demonstrations indicate an important potential application of STARII in investigating nanoscale cellular complex processes in the native state.

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

TMCC3 localizes at the three-way junctions for the proper tubular network of the endoplasmic reticulum.

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.

NeurodegenERation: The Central Role for ER Contacts in Neuronal Function and Axonopathy, Lessons From Hereditary Spastic Paraplegias and Related Diseases.

The hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative conditions whose characteristic feature is degeneration of the longest axons within the corticospinal tract which leads to progressive spasticity and weakness of the lower limbs. Though highly genetically heterogeneous, the majority of HSP cases are caused by mutations in genes encoding proteins that are responsible for generating and organizing the tubular endoplasmic reticulum (ER). Despite this, the role of the ER within neurons, particularly the long axons affected in HSP, is not well understood. Throughout axons, ER tubules make extensive contacts with other organelles, the cytoskeleton and the plasma membrane. At these ER contacts, protein complexes work in concert to perform specialized functions including organelle shaping, calcium homeostasis and lipid biogenesis, all of which are vital for neuronal survival and may be disrupted by HSP-causing mutations. In this article we summarize the proteins which mediate ER contacts, review the functions these contacts are known to carry out within neurons, and discuss the potential contribution of disruption of ER contacts to axonopathy in HSP.

CAND1 regulates lunapark for the proper tubular network of the endoplasmic reticulum

Endoplasmic reticulum (ER) tubules connect each other by three-way junctions, resulting in a tubular ER network. Oligomerization of three-way junction protein lunapark (Lnp) is important for its localization and the three-way junction stability. On the other hand, Lnp has an N-terminal ubiquitin ligase activity domain, which is also important for the three-way junction localization. To understand the mode of action of Lnp, we isolated Cullin-associated and neddylation-dissociated 1 (CAND1), a regulator of Skp1-Cul1-F-box (SCF) ubiquitin ligase, as a Lnp-binding protein by affinity chromatography. CAND1 and Lnp form a higher-molecular-weight complex in vitro, while they do not co-localize at the three-way junctions. CAND1 reduces the auto-ubiquitination activity of Lnp. CAND1 knockdown enhances proteasomal degradation of Lnp and reduces the tubular ER network in mammalian cells. These results suggest that CAND1 has the potency to promote the formation of the higher-molecular-weight complex with Lnp and reduce the auto-ubiquitination activity of Lnp, thereby regulating the three-way junction stability of the tubular ER network.

Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61βGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules.

Making and breaking contacts

Cell Biology Within our cells, mitochondria frequently make contact and undergo fission and fusion events to regulate the mitochondrial network. Mitochondrial contact is important for normal cellular metabolism. Wong et al. used super-resolution live-cell imaging to examine intermitochondrial contacts sites in human cell lines. They found that intermitochondrial contact restricts the motility of individual mitochondria within the cell but can be modulated. Lysosome contact promotes mitochondrial untethering by RAB7 guanosine triphosphate (GTP) hydrolysis. By contrast, contact with endoplasmic reticulum (ER) does not prompt decoupling, although ER tubules marked intermitochondrial untethering events. Multiple lysosomal and mitochondrial GTPases appear to coordinate to regulate mitochondrial contact dynamics. If these dynamics are disrupted by mutation, for example, in cells from patients with the hereditary neuropathy Charcot-Marie-Tooth disease, mitochondrial contacts are slower to disassemble. This results in dysfunctional mitochondrial dynamics, which culminates in axonal degradation. Dev. Cell 50 , 339 (2019).

Reticulon 4a promotes exocytosis in mammalian cells.

Endoplasmic reticulum (ER) tubules and sheets conventionally correspond to smooth and rough ER, respectively. The ratio of ER tubules-to-sheets varies in different cell types and changes in response to cellular conditions, potentially impacting the functional output of the ER. To directly test whether ER morphology impacts vesicular trafficking, we increased the tubule-to-sheet ratio in three different ways, by overexpressing Rtn4a, Rtn4b, or REEP5. Only Rtn4a overexpression increased exocytosis, but not overall levels, of several cell surface and secreted proteins. Furthermore, Rtn4a depletion reduced cell surface trafficking without affecting ER morphology. Similar results were observed in three different mammalian cell lines, suggesting that Rtn4a generally enhances exocytosis independently of changes in ER morphology. Finally, we show that Rtn4a levels modulate cell adhesion, possibly by regulating trafficking of integrins to the cell surface. Taking the results together, we find that altering ER morphology does not necessarily affect protein trafficking, but that Rtn4a specifically enhances exocytosis.

Apogossypol-mediated reorganisation of the endoplasmic reticulum antagonises mitochondrial fission and apoptosis

The endoplasmic reticulum (ER) with its elaborate network of highly curved tubules and flat sheets interacts with several other organelles, including mitochondria, peroxisomes and endosomes, to play vital roles in their membrane dynamics and functions. Previously, we identified structurally diverse chemicals from different pharmacological classes, which induce a reversible reorganisation of ER membranes. Using apogossypol as a prototypic tool compound, we now show that ER membrane reorganisation occurs at the level of ER tubules but does not involve ER sheets. Reorganisation of ER membranes prevents DRP-1-mediated mitochondrial fission, thereby antagonising the functions of several mitochondrial fission-inducing agents. Previous reports have suggested that ER membranes mark the constriction sites of mitochondria by localising DRP-1, as well as BAX on mitochondrial membranes to facilitate both mitochondrial fission and outer membrane permeabilisation. Following ER membrane reorganisation and subsequent exposure to an apoptotic stimulus (BH3 mimetics), DRP-1 still colocalises with the reorganised ER membranes but BAX translocation and activation, cytochrome c release and phosphatidylserine externalisation are all inhibited, thereby diminishing the ability of BH3 mimetics to induce the intrinsic apoptotic pathway. Strikingly, both ER membrane reorganisation and its resulting inhibition of apoptosis could be reversed by inhibitors of dihydroorotate dehydrogenase (DHODH), namely teriflunomide and its active metabolite, leflunomide. However, neither genetic inhibition of DHODH using RNA interference nor metabolic supplementation with orotate or uridine to circumvent the consequences of a loss of DHODH activity rescued the effects of DHODH inhibitors, suggesting that the effects of these inhibitors in preventing ER membrane reorganisation is most likely independent of their ability to antagonise DHODH activity. Our results strengthen the hypothesis that ER is fundamental for key mitochondrial functions, such as fusion-fission dynamics and apoptosis.

Lysosomal Regulation of Inter-mitochondrial Contact Fate and Motility in Charcot-Marie-Tooth Type 2

Properly regulated mitochondrial networks are essential for cellular function and implicated in multiple diseases. Mitochondria undergo fission and fusion events, but the dynamics and regulation of a third event of inter-mitochondrial contact formation remain unclear. Using super-resolution imaging, we demonstrate that inter-mitochondrial contacts frequently form and play a fundamental role in mitochondrial networks by restricting mitochondrial motility. Inter-mitochondrial contact untethering events are marked and regulated by mitochondria-lysosome contacts, which are modulated by RAB7 GTP hydrolysis. Moreover, inter-mitochondrial contact formation and untethering are further regulated by Mfn1/2 and Drp1 GTP hydrolysis, respectively. Surprisingly, endoplasmic reticulum tubules are also present at inter-mitochondrial contact untethering events, in addition to mitochondrial fission and fusion events. Importantly, we find that multiple Charcot-Marie-Tooth type 2 disease-linked mutations in Mfn2 (CMT2A), RAB7 (CMT2B), and TRPV4 (CMT2C) converge on prolonged inter-mitochondrial contacts and defective mitochondrial motility, highlighting a role for inter-mitochondrial contacts in mitochondrial network regulation and disease.

141-OR: Regulation of Mitochondrial Dynamics in Brown Adipocytes by the Endoplasmic Reticulum-Associated Degradation

Recent studies have implicated the endoplasmic reticulum (ER) in organelle crosstalk including mitochondria; however, the underlying molecular mechanism and physiological significance remain largely unclear. Here we show that the Sel1L-Hrd1 protein complex of the most conserved ER-associated protein degradation (ERAD) in brown adipocytes is essential for cold-induced thermogenesis in part by managing mitochondrial dynamics. While largely indistinguishable from their wild type littermates, adipocyte or brown adipocyte-specific Sel1L-deficient mice are cold sensitive. Mechanistically, we found that mitochondria in Sel1L-deficient brown adipocytes are dysfunctional with a large portion of them being enlarged and becoming irregularly shaped, i.e., “megamitochondria,” as a result of attenuated fission. Moreover, Sel1L deficiency in cold-stimulated brown adipocytes alters ER-mitochondrial contact and consequently, mitochondria grow around ER tubules leading to the formation of unique intramitochondrial tubule(s). Lastly, brown adipocyte-specific Hrd1-deficient mice exhibit similar phenotypes in terms of cold sensitivity, mitochondrial morphology and dynamics, but not Ire1a- and β-adrenergic receptors-deficient mice. We conclude that Sel1L-Hrd1 ERAD in brown adipocytes is intimately involved in the regulation of mitochondrial dynamics in thermogenesis and provides exciting novel insights into the emerging field of organelle crosstalk. Disclosure Z. Zhou: None. M. Torres: None. L. Qi: None. Funding American Diabetes Association (1-19-PDF-093 to Z.Z.); National Institutes of Health

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

TEX264 Is an Endoplasmic Reticulum-Resident ATG8-Interacting Protein Critical for ER Remodeling during Nutrient Stress.

Cells respond to nutrient stress by trafficking cytosolic contents to lysosomes for degradation via macroautophagy. The endoplasmic reticulum (ER) serves as an initiation site for autophagosomes and is also remodeled in response to nutrient stress through ER-phagy, a form of selective autophagy. Quantitative proteome analysis during nutrient stress identified an unstudied single-pass transmembrane ER protein, TEX264, as an ER-phagy receptor. TEX264 uses an LC3-interacting region (LIR) to traffic into ATG8-positive puncta that often initiate from three-way ER tubule junctions and subsequently fuse with lysosomes. Interaction and proximity biotinylation proteomics identified a cohort of autophagy regulatory proteins and cargo adaptors located near TEX264 in an LIR-dependent manner. Global proteomics and ER-phagy flux analysis revealed the stabilization of a cohort of ER proteins in TEX264-/- cells during nutrient stress. This work reveals TEX264 as an unrecognized ER-phagy receptor that acts independently of other candidate ER-phagy receptors to remodel the ER during nutrient stress.

REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis.

The endoplasmic reticulum (ER) is extensively remodeled during metazoan open mitosis. However, whether the ER becomes more tubular or more cisternal during mitosis is controversial, and dedicated factors governing the morphology of the mitotic ER have remained elusive. Here, we describe the ER membrane proteins REEP3 and REEP4 as major determinants of ER morphology in metaphase cells. REEP3/4 are specifically required for generating the high-curvature morphology of mitotic ER and promote ER tubulation through their reticulon homology domains (RHDs). This ER-shaping activity of REEP3/4 is distinct from their previously described function to clear ER from metaphase chromatin. We further show that related REEP proteins do not contribute to mitotic ER shaping and provide evidence that the REEP3/4 carboxyterminus mediates regulation of the proteins. These findings confirm that ER converts to higher curvature during mitosis, identify REEP3/4 as specific and crucial morphogenic factors mediating ER tubulation during mitosis, and define the first cell cycle-specific role for RHD proteins.

Quantitative analysis of plant ER architecture and dynamics

The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.

Feedback-Driven Mechanisms between Microtubules and the Endoplasmic Reticulum Instruct Neuronal Polarity

Establishment of neuronal polarity depends on local microtubule (MT) reorganization. The endoplasmic reticulum (ER) consists of cisternae and tubules and, like MTs, forms an extensive network throughout the entire cell. How the two networks interact and control neuronal development is an outstanding question. Here we show that the interplay between MTs and the ER is essential for neuronal polarity. ER tubules localize within the axon, whereas ER cisternae are retained in the somatodendritic domain. MTs are essential for axonal ER tubule stabilization, and, reciprocally, the ER is required for stabilizing and organizing axonal MTs. Recruitment of ER tubules into one minor neurite initiates axon formation, whereas ER retention in the perinuclear area or disruption of ER tubules prevent neuronal polarization. The ER-shaping protein P180, present in axonal ER tubules, controls axon specification by regulating local MT remodeling. We propose a model in which feedback-driven regulation between the ER and MTs instructs neuronal polarity.

Atlastin-1 regulates morphology and function of endoplasmic reticulum in dendrites

Endoplasmic reticulum (ER) is characterized by interconnected tubules and sheets. Neuronal ER adopts specific morphology in axons, dendrites and soma. Here we study mechanisms underlying ER morphogenesis in a C. elegans sensory neuron PVD. In PVD soma and dendrite branch points, ER tubules connect to form networks. ER tubules fill primary dendrites but only extend to some but not all dendritic branches. We find that the Atlastin-1 ortholog, atln-1 is required for neuronal ER morphology. In atln-1 mutants with impaired GTPase activity, ER networks in soma and dendrite branch points are reduced and replaced by tubules, and ER tubules retracted from high-order dendritic branches, causing destabilized microtubule in these branches. The abnormal ER morphology likely causes defects in mitochondria fission at dendritic branch points. Mutant alleles of Atlastin-1 found in Hereditary Spastic Paraplegia (HSP) patients show similar ER phenotypes, suggesting that neuronal ER impairment contributes to HSP disease pathogenesis.

Dynamic nanoscale morphology of the ER surveyed by STED microscopy.

The endoplasmic reticulum (ER) is composed of interconnected membrane sheets and tubules. Superresolution microscopy recently revealed densely packed, rapidly moving ER tubules mistaken for sheets by conventional light microscopy, highlighting the importance of revisiting classical views of ER structure with high spatiotemporal resolution in living cells. In this study, we use live-cell stimulated emission depletion (STED) microscopy to survey the architecture of the ER at 50-nm resolution. We determine the nanoscale dimensions of ER tubules and sheets for the first time in living cells. We demonstrate that ER sheets contain highly dynamic, subdiffraction-sized holes, which we call nanoholes, that coexist with uniform sheet regions. Reticulon family members localize to curved edges of holes within sheets and are required for their formation. The luminal tether Climp63 and microtubule cytoskeleton modulate their nanoscale dynamics and organization. Thus, by providing the first quantitative analysis of ER membrane structure and dynamics at the nanoscale, our work reveals that the ER in living cells is not limited to uniform sheets and tubules; instead, we suggest the ER contains a continuum of membrane structures that includes dynamic nanoholes in sheets as well as clustered tubules.

The atlastin membrane anchor forms an intramembrane hairpin that does not span the phospholipid bilayer

The endoplasmic reticulum (ER) is composed of flattened sheets and interconnected tubules that extend throughout the cytosol and makes physical contact with all other cytoplasmic organelles. This cytoplasmic distribution requires continuous remodeling. These discrete ER morphologies require specialized proteins that drive and maintain membrane curvature. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal cytosolic tail. Here, we examined several Drosophila-human atlastin chimeras to identify functional domains of human atlastin-1 in vitro Although all chimeras could hydrolyze GTP, only chimeras containing the human C-terminal tail, hydrophobic segments, or both could fuse membranes in vitro We also determined that co-reconstitution of atlastin with reticulon does not influence GTPase activity or membrane fusion. Finally, we found that both human and Drosophila atlastin hydrophobic membrane anchors do not span the membrane, but rather form two intramembrane hairpin loops. The topology of these hairpins remains static during membrane fusion and does not appear to play an active role in lipid mixing.

Diffusion of Exit Sites on the Endoplasmic Reticulum: A Random Walk on a Shivering Backbone

Major parts of the endoplasmic reticulum (ER) in eukaryotic cells are organized as a dynamic network of membrane tubules connected by three-way junctions. On this network, self-assembled membrane domains, called ER exit sites (ERES), provide platforms at which nascent cargo proteins are packaged into vesicular carriers for subsequent transport along the secretory pathway. Although ERES appear stationary and spatially confined on long timescales, we show here via single-particle tracking that they exhibit a microtubule-dependent and heterogeneous anomalous diffusion behavior on short and intermediate timescales. By quantifying key parameters of their random walk, we show that the subdiffusive motion of ERES is distinct from that of ER junctions, i.e., ERES are not tied to junctions but rather are mobile on ER tubules. We complement and corroborate our experimental findings with model simulations that also indicate that ERES are not actively moved by microtubules. Altogether, our study shows that ERES perform a random walk on the shivering ER backbone, indirectly powered by microtubular activity. Similar phenomena can be expected for other domains on subcellular structures, setting a caveat for the interpretation of domain-tracking data.